WorldWideScience

Sample records for small rna discovery

  1. RNA as a small molecule druggable target.

    Science.gov (United States)

    Rizvi, Noreen F; Smith, Graham F

    2017-12-01

    Small molecule drugs have readily been developed against many proteins in the human proteome, but RNA has remained an elusive target for drug discovery. Increasingly, we see that RNA, and to a lesser extent DNA elements, show a persistent tertiary structure responsible for many diverse and complex cellular functions. In this digest, we have summarized recent advances in screening approaches for RNA targets and outlined the discovery of novel, drug-like small molecules against RNA targets from various classes and therapeutic areas. The link of structure, function, and small-molecule Druggability validates now for the first time that RNA can be the targets of therapeutic agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Discovery of replicating circular RNAs by RNA-seq and computational algorithms.

    Directory of Open Access Journals (Sweden)

    Zhixiang Zhang

    2014-12-01

    Full Text Available Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR. However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd and Apple hammerhead viroid-like RNA (AHVd-like RNA, respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small

  3. Species-independent MicroRNA Gene Discovery

    KAUST Repository

    Kamanu, Timothy K.

    2012-12-01

    MicroRNA (miRNA) are a class of small endogenous non-coding RNA that are mainly negative transcriptional and post-transcriptional regulators in both plants and animals. Recent studies have shown that miRNA are involved in different types of cancer and other incurable diseases such as autism and Alzheimer’s. Functional miRNAs are excised from hairpin-like sequences that are known as miRNA genes. There are about 21,000 known miRNA genes, most of which have been determined using experimental methods. miRNA genes are classified into different groups (miRNA families). This study reports about 19,000 unknown miRNA genes in nine species whereby approximately 15,300 predictions were computationally validated to contain at least one experimentally verified functional miRNA product. The predictions are based on a novel computational strategy which relies on miRNA family groupings and exploits the physics and geometry of miRNA genes to unveil the hidden palindromic signals and symmetries in miRNA gene sequences. Unlike conventional computational miRNA gene discovery methods, the algorithm developed here is species-independent: it allows prediction at higher accuracy and resolution from arbitrary RNA/DNA sequences in any species and thus enables examination of repeat-prone genomic regions which are thought to be non-informative or ’junk’ sequences. The information non-redundancy of uni-directional RNA sequences compared to information redundancy of bi-directional DNA is demonstrated, a fact that is overlooked by most pattern discovery algorithms. A novel method for computing upstream and downstream miRNA gene boundaries based on mathematical/statistical functions is suggested, as well as cutoffs for annotation of miRNA genes in different miRNA families. Another tool is proposed to allow hypotheses generation and visualization of data matrices, intra- and inter-species chromosomal distribution of miRNA genes or miRNA families. Our results indicate that: miRNA and miRNA

  4. Pulmonary administration of small interfering RNA : The route to go?

    NARCIS (Netherlands)

    Ruigrok, Mitchel; Frijlink, Henderik W.; Hinrichs, Wouter

    2016-01-01

    Ever since the discovery of RNA interference (RNAi), which is a post-transcriptional gene silencing mechanism, researchers have been studying the therapeutic potential of using small interfering RNA (siRNA) to treat diseases that are characterized by excessive gene expression. Excessive gene

  5. Argonaute: The executor of small RNA function.

    Science.gov (United States)

    Azlan, Azali; Dzaki, Najat; Azzam, Ghows

    2016-08-20

    The discovery of small non-coding RNAs - microRNA (miRNA), short interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) - represents one of the most exciting frontiers in biology specifically on the mechanism of gene regulation. In order to execute their functions, these small RNAs require physical interactions with their protein partners, the Argonaute (AGO) family proteins. Over the years, numerous studies have made tremendous progress on understanding the roles of AGO in gene silencing in various organisms. In this review, we summarize recent progress of AGO-mediated gene silencing and other cellular processes in which AGO proteins have been implicated with a particular focus on progress made in flies, humans and other model organisms as compliment. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  6. High-throughput platform assay technology for the discovery of pre-microrna-selective small molecule probes.

    Science.gov (United States)

    Lorenz, Daniel A; Song, James M; Garner, Amanda L

    2015-01-21

    MicroRNAs (miRNA) play critical roles in human development and disease. As such, the targeting of miRNAs is considered attractive as a novel therapeutic strategy. A major bottleneck toward this goal, however, has been the identification of small molecule probes that are specific for select RNAs and methods that will facilitate such discovery efforts. Using pre-microRNAs as proof-of-concept, herein we report a conceptually new and innovative approach for assaying RNA-small molecule interactions. Through this platform assay technology, which we term catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a method that can be implemented in high throughput, is virtually free of false readouts, and is general for all nucleic acids. Through cat-ELCCA, we envision the discovery of selective small molecule ligands for disease-relevant miRNAs to promote the field of RNA-targeted drug discovery and further our understanding of the role of miRNAs in cellular biology.

  7. SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data.

    Science.gov (United States)

    de Andrade, Roberto R S; Vaslin, Maite F S

    2014-03-07

    Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/.

  8. A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

    Directory of Open Access Journals (Sweden)

    Chen Yun-Ru

    2012-09-01

    Full Text Available Abstract Background Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available. Results We demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum using the T4 RNA ligase 1 adenylated adapter. Conclusion We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.

  9. Discovery of centrosomal RNA and centrosomal hypothesis of cellular ageing and differentiation.

    Science.gov (United States)

    Chichinadze, Konstantin; Tkemaladze, Jaba; Lazarashvili, Ann

    2012-01-01

    In 2006, a group of scientists studying centrosomes of Spisula solidissima mollusc oocytes under the leadership of Alliegro (Alliegro, M.C.; Alliegro, M.A.; Palazzo, R.E. Centrosome-associated RNA in surf clam oocytes. Proc. Natl. Acad. Sci. USA 2006, 103(24), 9034-9038) reliably demonstrated the existence of specific RNA in centrosome, called centrosomal RNA (cnRNA). In their first article, five different RNAs (cnRNAs 11, 102, 113, 170, and 184) were described. During the process of full sequencing of the first transcript (cnRNA 11), it was discovered that the transcript contained a conserved structure-a reverse transcriptase domain located together with the most important centrosomal protein, γ-tubulin. In an article published in 2005, we made assumptions about several possible mechanisms for determining the most important functions of centrosomal structures and referred to one of them as a "RNA-dependent mechanism." This idea about participation of hypothetic centrosomal small interference RNA and/or microRNA in the process was made one year prior to the discovery of cnRNA by Alliegro's group. The discovery of specific RNA in a centrosome is indirect evidence of a centrosomal hypothesis of cellular ageing and differentiation. The presence of a reverse transcriptase domain in this type of RNA, together with its uniqueness and specificity, makes the centrosome a place of information storage and reproduction.

  10. RNA Editing and Drug Discovery for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Wei-Hsuan Huang

    2013-01-01

    Full Text Available RNA editing is vital to provide the RNA and protein complexity to regulate the gene expression. Correct RNA editing maintains the cell function and organism development. Imbalance of the RNA editing machinery may lead to diseases and cancers. Recently, RNA editing has been recognized as a target for drug discovery although few studies targeting RNA editing for disease and cancer therapy were reported in the field of natural products. Therefore, RNA editing may be a potential target for therapeutic natural products. In this review, we provide a literature overview of the biological functions of RNA editing on gene expression, diseases, cancers, and drugs. The bioinformatics resources of RNA editing were also summarized.

  11. miRDis: a Web tool for endogenous and exogenous microRNA discovery based on deep-sequencing data analysis.

    Science.gov (United States)

    Zhang, Hanyuan; Vieira Resende E Silva, Bruno; Cui, Juan

    2018-05-01

    Small RNA sequencing is the most widely used tool for microRNA (miRNA) discovery, and shows great potential for the efficient study of miRNA cross-species transport, i.e., by detecting the presence of exogenous miRNA sequences in the host species. Because of the increased appreciation of dietary miRNAs and their far-reaching implication in human health, research interests are currently growing with regard to exogenous miRNAs bioavailability, mechanisms of cross-species transport and miRNA function in cellular biological processes. In this article, we present microRNA Discovery (miRDis), a new small RNA sequencing data analysis pipeline for both endogenous and exogenous miRNA detection. Specifically, we developed and deployed a Web service that supports the annotation and expression profiling data of known host miRNAs and the detection of novel miRNAs, other noncoding RNAs, and the exogenous miRNAs from dietary species. As a proof-of-concept, we analyzed a set of human plasma sequencing data from a milk-feeding study where 225 human miRNAs were detected in the plasma samples and 44 show elevated expression after milk intake. By examining the bovine-specific sequences, data indicate that three bovine miRNAs (bta-miR-378, -181* and -150) are present in human plasma possibly because of the dietary uptake. Further evaluation based on different sets of public data demonstrates that miRDis outperforms other state-of-the-art tools in both detection and quantification of miRNA from either animal or plant sources. The miRDis Web server is available at: http://sbbi.unl.edu/miRDis/index.php.

  12. iSRAP - a one-touch research tool for rapid profiling of small RNA-seq data.

    Science.gov (United States)

    Quek, Camelia; Jung, Chol-Hee; Bellingham, Shayne A; Lonie, Andrew; Hill, Andrew F

    2015-01-01

    Small non-coding RNAs have been significantly recognized as the key modulators in many biological processes, and are emerging as promising biomarkers for several diseases. These RNA species are transcribed in cells and can be packaged in extracellular vesicles, which are small vesicles released from many biotypes, and are involved in intercellular communication. Currently, the advent of next-generation sequencing (NGS) technology for high-throughput profiling has further advanced the biological insights of non-coding RNA on a genome-wide scale and has become the preferred approach for the discovery and quantification of non-coding RNA species. Despite the routine practice of NGS, the processing of large data sets poses difficulty for analysis before conducting downstream experiments. Often, the current analysis tools are designed for specific RNA species, such as microRNA, and are limited in flexibility for modifying parameters for optimization. An analysis tool that allows for maximum control of different software is essential for drawing concrete conclusions for differentially expressed transcripts. Here, we developed a one-touch integrated small RNA analysis pipeline (iSRAP) research tool that is composed of widely used tools for rapid profiling of small RNAs. The performance test of iSRAP using publicly and in-house available data sets shows its ability of comprehensive profiling of small RNAs of various classes, and analysis of differentially expressed small RNAs. iSRAP offers comprehensive analysis of small RNA sequencing data that leverage informed decisions on the downstream analyses of small RNA studies, including extracellular vesicles such as exosomes.

  13. iSRAP – a one-touch research tool for rapid profiling of small RNA-seq data

    Science.gov (United States)

    Quek, Camelia; Jung, Chol-hee; Bellingham, Shayne A.; Lonie, Andrew; Hill, Andrew F.

    2015-01-01

    Small non-coding RNAs have been significantly recognized as the key modulators in many biological processes, and are emerging as promising biomarkers for several diseases. These RNA species are transcribed in cells and can be packaged in extracellular vesicles, which are small vesicles released from many biotypes, and are involved in intercellular communication. Currently, the advent of next-generation sequencing (NGS) technology for high-throughput profiling has further advanced the biological insights of non-coding RNA on a genome-wide scale and has become the preferred approach for the discovery and quantification of non-coding RNA species. Despite the routine practice of NGS, the processing of large data sets poses difficulty for analysis before conducting downstream experiments. Often, the current analysis tools are designed for specific RNA species, such as microRNA, and are limited in flexibility for modifying parameters for optimization. An analysis tool that allows for maximum control of different software is essential for drawing concrete conclusions for differentially expressed transcripts. Here, we developed a one-touch integrated small RNA analysis pipeline (iSRAP) research tool that is composed of widely used tools for rapid profiling of small RNAs. The performance test of iSRAP using publicly and in-house available data sets shows its ability of comprehensive profiling of small RNAs of various classes, and analysis of differentially expressed small RNAs. iSRAP offers comprehensive analysis of small RNA sequencing data that leverage informed decisions on the downstream analyses of small RNA studies, including extracellular vesicles such as exosomes. PMID:26561006

  14. SNP discovery in the bovine milk transcriptome using RNA-Seq technology.

    Science.gov (United States)

    Cánovas, Angela; Rincon, Gonzalo; Islas-Trejo, Alma; Wickramasinghe, Saumya; Medrano, Juan F

    2010-12-01

    High-throughput sequencing of RNA (RNA-Seq) was developed primarily to analyze global gene expression in different tissues. However, it also is an efficient way to discover coding SNPs. The objective of this study was to perform a SNP discovery analysis in the milk transcriptome using RNA-Seq. Seven milk samples from Holstein cows were analyzed by sequencing cDNAs using the Illumina Genome Analyzer system. We detected 19,175 genes expressed in milk samples corresponding to approximately 70% of the total number of genes analyzed. The SNP detection analysis revealed 100,734 SNPs in Holstein samples, and a large number of those corresponded to differences between the Holstein breed and the Hereford bovine genome assembly Btau4.0. The number of polymorphic SNPs within Holstein cows was 33,045. The accuracy of RNA-Seq SNP discovery was tested by comparing SNPs detected in a set of 42 candidate genes expressed in milk that had been resequenced earlier using Sanger sequencing technology. Seventy of 86 SNPs were detected using both RNA-Seq and Sanger sequencing technologies. The KASPar Genotyping System was used to validate unique SNPs found by RNA-Seq but not observed by Sanger technology. Our results confirm that analyzing the transcriptome using RNA-Seq technology is an efficient and cost-effective method to identify SNPs in transcribed regions. This study creates guidelines to maximize the accuracy of SNP discovery and prevention of false-positive SNP detection, and provides more than 33,000 SNPs located in coding regions of genes expressed during lactation that can be used to develop genotyping platforms to perform marker-trait association studies in Holstein cattle.

  15. Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy

    Directory of Open Access Journals (Sweden)

    Miele E

    2012-07-01

    Full Text Available Evelina Miele,1,* Gian Paolo Spinelli,2,* Ermanno Miele,3 Enzo Di Fabrizio,3,6 Elisabetta Ferretti,4 Silverio Tomao,2 Alberto Gulino,1,5 1Department of Molecular Medicine, 2Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, 3Nanostructures, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova, 4Department of Experimental Medicine, Sapienza University of Rome, Rome, 5Center for Life Nanoscience, Istituto Italiano di Tecnologia, Rome, Italy, 6BIONEM lab, University of Magna Graecia, Campus S. Venuta, Viale Europa 88100 Catanzaro, Italy *These authors contributed equally to this workAbstract: During recent decades there have been remarkable advances and profound changes in cancer therapy. Many therapeutic strategies learned at the bench, including monoclonal antibodies and small molecule inhibitors, have been used at the bedside, leading to important successes. One of the most important advances in biology has been the discovery that small interfering RNA (siRNA is able to regulate the expression of genes, by a phenomenon known as RNA interference (RNAi. RNAi is one of the most rapidly growing fields of research in biology and therapeutics. Much research effort has gone into the application of this new discovery in the treatment of various diseases, including cancer. However, even though these molecules may have potential and strong utility, some limitations make their clinical application difficult, including delivery problems, side effects due to off-target actions, disturbance of physiological functions of the cellular machinery involved in gene silencing, and induction of the innate immune response. Many researchers have attempted to overcome these limitations and to improve the safety of potential RNAi-based therapeutics. Nanoparticles, which are nanostructured entities with tunable size, shape, and surface, as well as biological behavior, provide an ideal opportunity to modify current

  16. De Novo Discovery of Structured ncRNA Motifs in Genomic Sequences

    DEFF Research Database (Denmark)

    Ruzzo, Walter L; Gorodkin, Jan

    2014-01-01

    De novo discovery of "motifs" capturing the commonalities among related noncoding ncRNA structured RNAs is among the most difficult problems in computational biology. This chapter outlines the challenges presented by this problem, together with some approaches towards solving them, with an emphas...... on an approach based on the CMfinder CMfinder program as a case study. Applications to genomic screens for novel de novo structured ncRNA ncRNA s, including structured RNA elements in untranslated portions of protein-coding genes, are presented.......De novo discovery of "motifs" capturing the commonalities among related noncoding ncRNA structured RNAs is among the most difficult problems in computational biology. This chapter outlines the challenges presented by this problem, together with some approaches towards solving them, with an emphasis...

  17. Discovery and small RNA profile of Pecan mosaic-associated virus, a novel potyvirus of pecan trees.

    Science.gov (United States)

    Su, Xiu; Fu, Shuai; Qian, Yajuan; Zhang, Liqin; Xu, Yi; Zhou, Xueping

    2016-05-26

    A novel potyvirus was discovered in pecan (Carya illinoensis) showing leaf mosaic symptom through the use of deep sequencing of small RNAs. The complete genome of this virus was determined to comprise of 9,310 nucleotides (nt), and shared 24.0% to 58.9% nucleotide similarities with that of other Potyviridae viruses. The genome was deduced to encode a single open reading frame (polyprotein) on the plus strand. Phylogenetic analysis based on the whole genome sequence and coat protein amino acid sequence showed that this virus is most closely related to Lettuce mosaic virus. Using electron microscopy, the typical Potyvirus filamentous particles were identified in infected pecan leaves with mosaic symptoms. Our results clearly show that this virus is a new member of the genus Potyvirus in the family Potyviridae. The virus is tentatively named Pecan mosaic-associated virus (PMaV). Additionally, profiling of the PMaV-derived small RNA (PMaV-sRNA) showed that the most abundant PMaV-sRNAs were 21-nt in length. There are several hotspots for small RNA production along the PMaV genome; two 21-nt PMaV-sRNAs starting at 811 nt and 610 nt of the minus-strand genome were highly repeated.

  18. Selective small-molecule inhibition of an RNA structural element

    Energy Technology Data Exchange (ETDEWEB)

    Howe, John A.; Wang, Hao; Fischmann, Thierry O.; Balibar, Carl J.; Xiao, Li; Galgoci, Andrew M.; Malinverni, Juliana C.; Mayhood, Todd; Villafania, Artjohn; Nahvi, Ali; Murgolo, Nicholas; Barbieri, Christopher M.; Mann, Paul A.; Carr, Donna; Xia, Ellen; Zuck, Paul; Riley, Dan; Painter, Ronald E.; Walker, Scott S.; Sherborne, Brad; de Jesus, Reynalda; Pan, Weidong; Plotkin, Michael A.; Wu, Jin; Rindgen, Diane; Cummings, John; Garlisi, Charles G.; Zhang, Rumin; Sheth, Payal R.; Gill, Charles J.; Tang, Haifeng; Roemer , Terry (Merck)

    2015-09-30

    Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

  19. Switching off small RNA regulation with trap-mRNA

    DEFF Research Database (Denmark)

    Overgaard, Martin; Johansen, Jesper; Møller-Jensen, Jakob

    2009-01-01

    to operate at the level of transcription initiation. By employing a highly sensitive genetic screen we uncovered a novel RNA-based regulatory principle in which induction of a trap-mRNA leads to selective degradation of a small regulatory RNA molecule, thereby abolishing the sRNA-based silencing of its...

  20. Hierarchical virtual screening approaches in small molecule drug discovery.

    Science.gov (United States)

    Kumar, Ashutosh; Zhang, Kam Y J

    2015-01-01

    Virtual screening has played a significant role in the discovery of small molecule inhibitors of therapeutic targets in last two decades. Various ligand and structure-based virtual screening approaches are employed to identify small molecule ligands for proteins of interest. These approaches are often combined in either hierarchical or parallel manner to take advantage of the strength and avoid the limitations associated with individual methods. Hierarchical combination of ligand and structure-based virtual screening approaches has received noteworthy success in numerous drug discovery campaigns. In hierarchical virtual screening, several filters using ligand and structure-based approaches are sequentially applied to reduce a large screening library to a number small enough for experimental testing. In this review, we focus on different hierarchical virtual screening strategies and their application in the discovery of small molecule modulators of important drug targets. Several virtual screening studies are discussed to demonstrate the successful application of hierarchical virtual screening in small molecule drug discovery. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Small Molecule Modifiers of the microRNA and RNA Interference Pathway

    OpenAIRE

    Deiters, Alexander

    2009-01-01

    Recently, the RNA interference (RNAi) pathway has become the target of small molecule inhibitors and activators. RNAi has been well established as a research tool in the sequence-specific silencing of genes in eukaryotic cells and organisms by using exogenous, small, double-stranded RNA molecules of approximately 20 nucleotides. Moreover, a recently discovered post-transcriptional gene regulatory mechanism employs microRNAs (miRNAs), a class of endogenously expressed small RNA molecules, whic...

  2. Modeling bias and variation in the stochastic processes of small RNA sequencing.

    Science.gov (United States)

    Argyropoulos, Christos; Etheridge, Alton; Sakhanenko, Nikita; Galas, David

    2017-06-20

    The use of RNA-seq as the preferred method for the discovery and validation of small RNA biomarkers has been hindered by high quantitative variability and biased sequence counts. In this paper we develop a statistical model for sequence counts that accounts for ligase bias and stochastic variation in sequence counts. This model implies a linear quadratic relation between the mean and variance of sequence counts. Using a large number of sequencing datasets, we demonstrate how one can use the generalized additive models for location, scale and shape (GAMLSS) distributional regression framework to calculate and apply empirical correction factors for ligase bias. Bias correction could remove more than 40% of the bias for miRNAs. Empirical bias correction factors appear to be nearly constant over at least one and up to four orders of magnitude of total RNA input and independent of sample composition. Using synthetic mixes of known composition, we show that the GAMLSS approach can analyze differential expression with greater accuracy, higher sensitivity and specificity than six existing algorithms (DESeq2, edgeR, EBSeq, limma, DSS, voom) for the analysis of small RNA-seq data. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

    Science.gov (United States)

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

    2013-11-13

    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

  4. Discovery and Development of Therapeutic Drugs against Lethal Human RNA Viruses: a Multidisciplinary Assault.

    Science.gov (United States)

    1991-07-16

    AD-A239 742 AD GRANT NO: DAMD17-89-Z-9021 TITLE: DISCOVERY AND DEVELOPMENT OF THERAPEUTIC DRUGS AGAINST LETHAL HUMAN RNA VIRUSES: A MULTIDISCIPLINARY...62787A871 AB WrJDA317987 11. TITLE (Include Securty Classification) DISCOVERY AND DEVELOPMENT OF THERAPEUTIC DRUGS AGAINST LETHAL HUMAN RNA VIRUSES: A...G. R. Pettit, III, D.-S. Huang, and G. R. Pettit, 23rd Int’l. Horticulture Congress, Italy, 8/27 - 9/1/90. "Bryostatins Define the Role of Protein

  5. Defining RNA-Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA.

    Science.gov (United States)

    Velagapudi, Sai Pradeep; Luo, Yiling; Tran, Tuan; Haniff, Hafeez S; Nakai, Yoshio; Fallahi, Mohammad; Martinez, Gustavo J; Childs-Disney, Jessica L; Disney, Matthew D

    2017-03-22

    RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif-small molecule interactions identified via selection. Named High Throughput Structure-Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif-small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule-RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs.

  6. Recent advances in developing small molecules targeting RNA.

    Science.gov (United States)

    Guan, Lirui; Disney, Matthew D

    2012-01-20

    RNAs are underexploited targets for small molecule drugs or chemical probes of function. This may be due, in part, to a fundamental lack of understanding of the types of small molecules that bind RNA specifically and the types of RNA motifs that specifically bind small molecules. In this review, we describe recent advances in the development and design of small molecules that bind to RNA and modulate function that aim to fill this void.

  7. Repertoire of bovine miRNA and miRNA-like small regulatory RNAs expressed upon viral infection.

    Directory of Open Access Journals (Sweden)

    Evgeny A Glazov

    Full Text Available MicroRNA (miRNA and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small RNAs characterized in mammals and to examine relationship between host miRNA expression and viral infection we used Illumina's ultrahigh throughput sequencing approach. We sequenced three small RNA libraries prepared from cell line derived from the adult bovine kidney under normal conditions and upon infection of the cell line with Bovine herpesvirus 1. We used a bioinformatics approach to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected 219 out of 356 known bovine miRNAs and 115 respective miRNA* sequences. In addition we identified five new bovine orthologs of known mammalian miRNAs and discovered 268 new cow miRNAs many of which are not identifiable in other mammalian genomes and thus might be specific to the ruminant lineage. In addition we found seven new bovine mirtron candidates. We also discovered 10 small nucleolar RNA (snoRNA loci that give rise to small RNA with possible miRNA-like function. Results presented in this study extend our knowledge of the biology and evolution of small regulatory RNAs in mammals and illuminate mechanisms of small RNA biogenesis and function. New miRNA sequences and the original sequencing data have been submitted to miRNA repository (miRBase and NCBI GEO archive respectively. We envisage that these resources will facilitate functional annotation of the bovine genome and promote further functional and comparative genomics studies of small regulatory RNA in mammals.

  8. Small silencing RNAs: an expanding universe.

    Science.gov (United States)

    Ghildiyal, Megha; Zamore, Phillip D

    2009-02-01

    Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, their modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected, and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats.

  9. Chimira: analysis of small RNA sequencing data and microRNA modifications.

    Science.gov (United States)

    Vitsios, Dimitrios M; Enright, Anton J

    2015-10-15

    Chimira is a web-based system for microRNA (miRNA) analysis from small RNA-Seq data. Sequences are automatically cleaned, trimmed, size selected and mapped directly to miRNA hairpin sequences. This generates count-based miRNA expression data for subsequent statistical analysis. Moreover, it is capable of identifying epi-transcriptomic modifications in the input sequences. Supported modification types include multiple types of 3'-modifications (e.g. uridylation, adenylation), 5'-modifications and also internal modifications or variation (ADAR editing or single nucleotide polymorphisms). Besides cleaning and mapping of input sequences to miRNAs, Chimira provides a simple and intuitive set of tools for the analysis and interpretation of the results (see also Supplementary Material). These allow the visual study of the differential expression between two specific samples or sets of samples, the identification of the most highly expressed miRNAs within sample pairs (or sets of samples) and also the projection of the modification profile for specific miRNAs across all samples. Other tools have already been published in the past for various types of small RNA-Seq analysis, such as UEA workbench, seqBuster, MAGI, OASIS and CAP-miRSeq, CPSS for modifications identification. A comprehensive comparison of Chimira with each of these tools is provided in the Supplementary Material. Chimira outperforms all of these tools in total execution speed and aims to facilitate simple, fast and reliable analysis of small RNA-Seq data allowing also, for the first time, identification of global microRNA modification profiles in a simple intuitive interface. Chimira has been developed as a web application and it is accessible here: http://www.ebi.ac.uk/research/enright/software/chimira. aje@ebi.ac.uk Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

  10. A 3' UTR-Derived Small RNA Provides the Regulatory Noncoding Arm of the Inner Membrane Stress Response.

    Science.gov (United States)

    Chao, Yanjie; Vogel, Jörg

    2016-02-04

    Small RNAs (sRNAs) from conserved noncoding genes are crucial regulators in bacterial signaling pathways but have remained elusive in the Cpx response to inner membrane stress. Here we report that an alternative biogenesis pathway releasing the conserved mRNA 3' UTR of stress chaperone CpxP as an ∼60-nt sRNA provides the noncoding arm of the Cpx response. This so-called CpxQ sRNA, generated by general mRNA decay through RNase E, acts as an Hfq-dependent repressor of multiple mRNAs encoding extracytoplasmic proteins. Both CpxQ and the Cpx pathway are required for cell survival under conditions of dissipation of membrane potential. Our discovery of CpxQ illustrates how the conversion of a transcribed 3' UTR into an sRNA doubles the output of a single mRNA to produce two factors with spatially segregated functions during inner membrane stress: a chaperone that targets problematic proteins in the periplasm and a regulatory RNA that dampens their synthesis in the cytosol. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. An efficient genetic algorithm for structural RNA pairwise alignment and its application to non-coding RNA discovery in yeast

    Directory of Open Access Journals (Sweden)

    Taneda Akito

    2008-12-01

    Full Text Available Abstract Background Aligning RNA sequences with low sequence identity has been a challenging problem since such a computation essentially needs an algorithm with high complexities for taking structural conservation into account. Although many sophisticated algorithms for the purpose have been proposed to date, further improvement in efficiency is necessary to accelerate its large-scale applications including non-coding RNA (ncRNA discovery. Results We developed a new genetic algorithm, Cofolga2, for simultaneously computing pairwise RNA sequence alignment and consensus folding, and benchmarked it using BRAliBase 2.1. The benchmark results showed that our new algorithm is accurate and efficient in both time and memory usage. Then, combining with the originally trained SVM, we applied the new algorithm to novel ncRNA discovery where we compared S. cerevisiae genome with six related genomes in a pairwise manner. By focusing our search to the relatively short regions (50 bp to 2,000 bp sandwiched by conserved sequences, we successfully predict 714 intergenic and 1,311 sense or antisense ncRNA candidates, which were found in the pairwise alignments with stable consensus secondary structure and low sequence identity (≤ 50%. By comparing with the previous predictions, we found that > 92% of the candidates is novel candidates. The estimated rate of false positives in the predicted candidates is 51%. Twenty-five percent of the intergenic candidates has supports for expression in cell, i.e. their genomic positions overlap those of the experimentally determined transcripts in literature. By manual inspection of the results, moreover, we obtained four multiple alignments with low sequence identity which reveal consensus structures shared by three species/sequences. Conclusion The present method gives an efficient tool complementary to sequence-alignment-based ncRNA finders.

  12. (AAV)-mediated expression of small interfering RNA

    African Journals Online (AJOL)

    Effective inhibition of specific gene by adenoassociated virus (AAV)-mediated expression of small interfering RNA. ... To perform functional tests on siRNA, which was expressed by the viral vector, recombinant AAVs, coding for siRNA against exogenous gene, EGFP, and endogenous gene, p53, were established and ...

  13. Conifers have a unique small RNA silencing signature.

    Science.gov (United States)

    Dolgosheina, Elena V; Morin, Ryan D; Aksay, Gozde; Sahinalp, S Cenk; Magrini, Vincent; Mardis, Elaine R; Mattsson, Jim; Unrau, Peter J

    2008-08-01

    Plants produce small RNAs to negatively regulate genes, viral nucleic acids, and repetitive elements at either the transcriptional or post-transcriptional level in a process that is referred to as RNA silencing. While RNA silencing has been extensively studied across the different phyla of the animal kingdom (e.g., mouse, fly, worm), similar studies in the plant kingdom have focused primarily on angiosperms, thus limiting evolutionary studies of RNA silencing in plants. Here we report on an unexpected phylogenetic difference in the size distribution of small RNAs among the vascular plants. By extracting total RNA from freshly growing shoot tissue, we conducted a survey of small RNAs in 24 vascular plant species. We find that conifers, which radiated from the other seed-bearing plants approximately 260 million years ago, fail to produce significant amounts of 24-nucleotide (nt) RNAs that are known to guide DNA methylation and heterochromatin formation in angiosperms. Instead, they synthesize a diverse population of small RNAs that are exactly 21-nt long. This finding was confirmed by high-throughput sequencing of the small RNA sequences from a conifer, Pinus contorta. A conifer EST search revealed the presence of a novel Dicer-like (DCL) family, which may be responsible for the observed change in small RNA expression. No evidence for DCL3, an enzyme that matures 24-nt RNAs in angiosperms, was found. We hypothesize that the diverse class of 21-nt RNAs found in conifers may help to maintain organization of their unusually large genomes.

  14. BEAM web server: a tool for structural RNA motif discovery.

    Science.gov (United States)

    Pietrosanto, Marco; Adinolfi, Marta; Casula, Riccardo; Ausiello, Gabriele; Ferrè, Fabrizio; Helmer-Citterich, Manuela

    2018-03-15

    RNA structural motif finding is a relevant problem that becomes computationally hard when working on high-throughput data (e.g. eCLIP, PAR-CLIP), often represented by thousands of RNA molecules. Currently, the BEAM server is the only web tool capable to handle tens of thousands of RNA in input with a motif discovery procedure that is only limited by the current secondary structure prediction accuracies. The recently developed method BEAM (BEAr Motifs finder) can analyze tens of thousands of RNA molecules and identify RNA secondary structure motifs associated to a measure of their statistical significance. BEAM is extremely fast thanks to the BEAR encoding that transforms each RNA secondary structure in a string of characters. BEAM also exploits the evolutionary knowledge contained in a substitution matrix of secondary structure elements, extracted from the RFAM database of families of homologous RNAs. The BEAM web server has been designed to streamline data pre-processing by automatically handling folding and encoding of RNA sequences, giving users a choice for the preferred folding program. The server provides an intuitive and informative results page with the list of secondary structure motifs identified, the logo of each motif, its significance, graphic representation and information about its position in the RNA molecules sharing it. The web server is freely available at http://beam.uniroma2.it/ and it is implemented in NodeJS and Python with all major browsers supported. marco.pietrosanto@uniroma2.it. Supplementary data are available at Bioinformatics online.

  15. TargetRNA: a tool for predicting targets of small RNA action in bacteria

    OpenAIRE

    Tjaden, Brian

    2008-01-01

    Many small RNA (sRNA) genes in bacteria act as posttranscriptional regulators of target messenger RNAs. Here, we present TargetRNA, a web tool for predicting mRNA targets of sRNA action in bacteria. TargetRNA takes as input a genomic sequence that may correspond to an sRNA gene. TargetRNA then uses a dynamic programming algorithm to search each annotated message in a specified genome for mRNAs that evince basepair-binding potential to the input sRNA sequence. Based on the calculated basepair-...

  16. Sample size calculation while controlling false discovery rate for differential expression analysis with RNA-sequencing experiments.

    Science.gov (United States)

    Bi, Ran; Liu, Peng

    2016-03-31

    RNA-Sequencing (RNA-seq) experiments have been popularly applied to transcriptome studies in recent years. Such experiments are still relatively costly. As a result, RNA-seq experiments often employ a small number of replicates. Power analysis and sample size calculation are challenging in the context of differential expression analysis with RNA-seq data. One challenge is that there are no closed-form formulae to calculate power for the popularly applied tests for differential expression analysis. In addition, false discovery rate (FDR), instead of family-wise type I error rate, is controlled for the multiple testing error in RNA-seq data analysis. So far, there are very few proposals on sample size calculation for RNA-seq experiments. In this paper, we propose a procedure for sample size calculation while controlling FDR for RNA-seq experimental design. Our procedure is based on the weighted linear model analysis facilitated by the voom method which has been shown to have competitive performance in terms of power and FDR control for RNA-seq differential expression analysis. We derive a method that approximates the average power across the differentially expressed genes, and then calculate the sample size to achieve a desired average power while controlling FDR. Simulation results demonstrate that the actual power of several popularly applied tests for differential expression is achieved and is close to the desired power for RNA-seq data with sample size calculated based on our method. Our proposed method provides an efficient algorithm to calculate sample size while controlling FDR for RNA-seq experimental design. We also provide an R package ssizeRNA that implements our proposed method and can be downloaded from the Comprehensive R Archive Network ( http://cran.r-project.org ).

  17. Hidden layers of human small RNAs

    DEFF Research Database (Denmark)

    Kawaji, Hideya; Nakamura, Mari; Takahashi, Yukari

    2008-01-01

    small RNA have focused on miRNA and/or siRNA rather than on the exploration of additional classes of RNAs. RESULTS: Here, we explored human small RNAs by unbiased sequencing of RNAs with sizes of 19-40 nt. We provide substantial evidences for the existence of independent classes of small RNAs. Our data......BACKGROUND: Small RNA attracts increasing interest based on the discovery of RNA silencing and the rapid progress of our understanding of these phenomena. Although recent studies suggest the possible existence of yet undiscovered types of small RNAs in higher organisms, many studies to profile...... shows that well-characterized non-coding RNA, such as tRNA, snoRNA, and snRNA are cleaved at sites specific to the class of ncRNA. In particular, tRNA cleavage is regulated depending on tRNA type and tissue expression. We also found small RNAs mapped to genomic regions that are transcribed in both...

  18. The UEA Small RNA Workbench: A Suite of Computational Tools for Small RNA Analysis.

    Science.gov (United States)

    Mohorianu, Irina; Stocks, Matthew Benedict; Applegate, Christopher Steven; Folkes, Leighton; Moulton, Vincent

    2017-01-01

    RNA silencing (RNA interference, RNAi) is a complex, highly conserved mechanism mediated by short, typically 20-24 nt in length, noncoding RNAs known as small RNAs (sRNAs). They act as guides for the sequence-specific transcriptional and posttranscriptional regulation of target mRNAs and play a key role in the fine-tuning of biological processes such as growth, response to stresses, or defense mechanism.High-throughput sequencing (HTS) technologies are employed to capture the expression levels of sRNA populations. The processing of the resulting big data sets facilitated the computational analysis of the sRNA patterns of variation within biological samples such as time point experiments, tissue series or various treatments. Rapid technological advances enable larger experiments, often with biological replicates leading to a vast amount of raw data. As a result, in this fast-evolving field, the existing methods for sequence characterization and prediction of interaction (regulatory) networks periodically require adapting or in extreme cases, a complete redesign to cope with the data deluge. In addition, the presence of numerous tools focused only on particular steps of HTS analysis hinders the systematic parsing of the results and their interpretation.The UEA small RNA Workbench (v1-4), described in this chapter, provides a user-friendly, modular, interactive analysis in the form of a suite of computational tools designed to process and mine sRNA datasets for interesting characteristics that can be linked back to the observed phenotypes. First, we show how to preprocess the raw sequencing output and prepare it for downstream analysis. Then we review some quality checks that can be used as a first indication of sources of variability between samples. Next we show how the Workbench can provide a comparison of the effects of different normalization approaches on the distributions of expression, enhanced methods for the identification of differentially expressed

  19. miRge - A Multiplexed Method of Processing Small RNA-Seq Data to Determine MicroRNA Entropy.

    Directory of Open Access Journals (Sweden)

    Alexander S Baras

    Full Text Available Small RNA RNA-seq for microRNAs (miRNAs is a rapidly developing field where opportunities still exist to create better bioinformatics tools to process these large datasets and generate new, useful analyses. We built miRge to be a fast, smart small RNA-seq solution to process samples in a highly multiplexed fashion. miRge employs a Bayesian alignment approach, whereby reads are sequentially aligned against customized mature miRNA, hairpin miRNA, noncoding RNA and mRNA sequence libraries. miRNAs are summarized at the level of raw reads in addition to reads per million (RPM. Reads for all other RNA species (tRNA, rRNA, snoRNA, mRNA are provided, which is useful for identifying potential contaminants and optimizing small RNA purification strategies. miRge was designed to optimally identify miRNA isomiRs and employs an entropy based statistical measurement to identify differential production of isomiRs. This allowed us to identify decreasing entropy in isomiRs as stem cells mature into retinal pigment epithelial cells. Conversely, we show that pancreatic tumor miRNAs have similar entropy to matched normal pancreatic tissues. In a head-to-head comparison with other miRNA analysis tools (miRExpress 2.0, sRNAbench, omiRAs, miRDeep2, Chimira, UEA small RNA Workbench, miRge was faster (4 to 32-fold and was among the top-two methods in maximally aligning miRNAs reads per sample. Moreover, miRge has no inherent limits to its multiplexing. miRge was capable of simultaneously analyzing 100 small RNA-Seq samples in 52 minutes, providing an integrated analysis of miRNA expression across all samples. As miRge was designed for analysis of single as well as multiple samples, miRge is an ideal tool for high and low-throughput users. miRge is freely available at http://atlas.pathology.jhu.edu/baras/miRge.html.

  20. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA).

    Science.gov (United States)

    Li, Huinan; Wu, Cheng; Aramayo, Rodolfo; Sachs, Matthew S; Harlow, Mark L

    2015-10-08

    Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission.

  1. Re-inspection of small RNA sequence datasets reveals several novel human miRNA genes.

    Directory of Open Access Journals (Sweden)

    Thomas Birkballe Hansen

    Full Text Available BACKGROUND: miRNAs are key players in gene expression regulation. To fully understand the complex nature of cellular differentiation or initiation and progression of disease, it is important to assess the expression patterns of as many miRNAs as possible. Thereby, identifying novel miRNAs is an essential prerequisite to make possible a comprehensive and coherent understanding of cellular biology. METHODOLOGY/PRINCIPAL FINDINGS: Based on two extensive, but previously published, small RNA sequence datasets from human embryonic stem cells and human embroid bodies, respectively [1], we identified 112 novel miRNA-like structures and were able to validate miRNA processing in 12 out of 17 investigated cases. Several miRNA candidates were furthermore substantiated by including additional available small RNA datasets, thereby demonstrating the power of combining datasets to identify miRNAs that otherwise may be assigned as experimental noise. CONCLUSIONS/SIGNIFICANCE: Our analysis highlights that existing datasets are not yet exhaustedly studied and continuous re-analysis of the available data is important to uncover all features of small RNA sequencing.

  2. microRNA Biomarker Discovery and High-Throughput DNA Sequencing Are Possible Using Long-term Archived Serum Samples.

    Science.gov (United States)

    Rounge, Trine B; Lauritzen, Marianne; Langseth, Hilde; Enerly, Espen; Lyle, Robert; Gislefoss, Randi E

    2015-09-01

    The impacts of long-term storage and varying preanalytical factors on the quality and quantity of DNA and miRNA from archived serum have not been fully assessed. Preanalytical and analytical variations and degradation may introduce bias in representation of DNA and miRNA and may result in loss or corruption of quantitative data. We have evaluated DNA and miRNA quantity, quality, and variability in samples stored up to 40 years using one of the oldest prospective serum collections in the world, the Janus Serumbank, a biorepository dedicated to cancer research. miRNAs are present and stable in archived serum samples frozen at -25°C for at least 40 years. Long-time storage did not reduce miRNA yields; however, varying preanalytical conditions had a significant effect and should be taken into consideration during project design. Of note, 500 μL serum yielded sufficient miRNA for qPCR and small RNA sequencing and on average 650 unique miRNAs were detected in samples from presumably healthy donors. Of note, 500 μL serum yielded sufficient DNA for whole-genome sequencing and subsequent SNP calling, giving a uniform representation of the genomes. DNA and miRNA are stable during long-term storage, making large prospectively collected serum repositories an invaluable source for miRNA and DNA biomarker discovery. Large-scale biomarker studies with long follow-up time are possible utilizing biorepositories with archived serum and state-of-the-art technology. ©2015 American Association for Cancer Research.

  3. Methods to enable the design of bioactive small molecules targeting RNA.

    Science.gov (United States)

    Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

    2014-02-21

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome.

  4. iMir: an integrated pipeline for high-throughput analysis of small non-coding RNA data obtained by smallRNA-Seq.

    Science.gov (United States)

    Giurato, Giorgio; De Filippo, Maria Rosaria; Rinaldi, Antonio; Hashim, Adnan; Nassa, Giovanni; Ravo, Maria; Rizzo, Francesca; Tarallo, Roberta; Weisz, Alessandro

    2013-12-13

    Qualitative and quantitative analysis of small non-coding RNAs by next generation sequencing (smallRNA-Seq) represents a novel technology increasingly used to investigate with high sensitivity and specificity RNA population comprising microRNAs and other regulatory small transcripts. Analysis of smallRNA-Seq data to gather biologically relevant information, i.e. detection and differential expression analysis of known and novel non-coding RNAs, target prediction, etc., requires implementation of multiple statistical and bioinformatics tools from different sources, each focusing on a specific step of the analysis pipeline. As a consequence, the analytical workflow is slowed down by the need for continuous interventions by the operator, a critical factor when large numbers of datasets need to be analyzed at once. We designed a novel modular pipeline (iMir) for comprehensive analysis of smallRNA-Seq data, comprising specific tools for adapter trimming, quality filtering, differential expression analysis, biological target prediction and other useful options by integrating multiple open source modules and resources in an automated workflow. As statistics is crucial in deep-sequencing data analysis, we devised and integrated in iMir tools based on different statistical approaches to allow the operator to analyze data rigorously. The pipeline created here proved to be efficient and time-saving than currently available methods and, in addition, flexible enough to allow the user to select the preferred combination of analytical steps. We present here the results obtained by applying this pipeline to analyze simultaneously 6 smallRNA-Seq datasets from either exponentially growing or growth-arrested human breast cancer MCF-7 cells, that led to the rapid and accurate identification, quantitation and differential expression analysis of ~450 miRNAs, including several novel miRNAs and isomiRs, as well as identification of the putative mRNA targets of differentially expressed mi

  5. The potential of circulating extracellular small RNAs (smexRNA) in veterinary diagnostics-Identifying biomarker signatures by multivariate data analysis.

    Science.gov (United States)

    Melanie, Spornraft; Benedikt, Kirchner; Pfaffl, Michael W; Irmgard, Riedmaier

    2015-09-01

    Worldwide growth and performance-enhancing substances are used in cattle husbandry to increase productivity. In certain countries however e.g., in the EU, these practices are forbidden to prevent the consumers from potential health risks of substance residues in food. To maximize economic profit, 'black sheep' among farmers might circumvent the detection methods used in routine controls, which highlights the need for an innovative and reliable detection method. Transcriptomics is a promising new approach in the discovery of veterinary medicine biomarkers and also a missing puzzle piece, as up to date, metabolomics and proteomics are paramount. Due to increased stability and easy sampling, circulating extracellular small RNAs (smexRNAs) in bovine plasma were small RNA-sequenced and their potential to serve as biomarker candidates was evaluated using multivariate data analysis tools. After running the data evaluation pipeline, the proportion of miRNAs (microRNAs) and piRNAs (PIWI-interacting small non-coding RNAs) on the total sequenced reads was calculated. Additionally, top 10 signatures were compared which revealed that the readcount data sets were highly affected by the most abundant miRNA and piRNA profiles. To evaluate the discriminative power of multivariate data analyses to identify animals after veterinary drug application on the basis of smexRNAs, OPLS-DA was performed. In summary, the quality of miRNA models using all mapped reads for both treatment groups (animals treated with steroid hormones or the β-agonist clenbuterol) is predominant to those generated with combined data sets or piRNAs alone. Using multivariate projection methodologies like OPLS-DA have proven the best potential to generate discriminative miRNA models, supported by small RNA-Seq data. Based on the presented comparative OPLS-DA, miRNAs are the favorable smexRNA biomarker candidates in the research field of veterinary drug abuse.

  6. Computational prediction of miRNA genes from small RNA sequencing data

    Directory of Open Access Journals (Sweden)

    Wenjing eKang

    2015-01-01

    Full Text Available Next-generation sequencing now for the first time allows researchers to gauge the depth and variation of entire transcriptomes. However, now as rare transcripts can be detected that are present in cells at single copies, more advanced computational tools are needed to accurately annotate and profile them. miRNAs are 22 nucleotide small RNAs (sRNAs that post-transcriptionally reduce the output of protein coding genes. They have established roles in numerous biological processes, including cancers and other diseases. During miRNA biogenesis, the sRNAs are sequentially cleaved from precursor molecules that have a characteristic hairpin RNA structure. The vast majority of new miRNA genes that are discovered are mined from small RNA sequencing (sRNA-seq, which can detect more than a billion RNAs in a single run. However, given that many of the detected RNAs are degradation products from all types of transcripts, the accurate identification of miRNAs remain a non-trivial computational problem. Here we review the tools available to predict animal miRNAs from sRNA sequencing data. We present tools for generalist and specialist use cases, including prediction from massively pooled data or in species without reference genome. We also present wet-lab methods used to validate predicted miRNAs, and approaches to computationally benchmark prediction accuracy. For each tool, we reference validation experiments and benchmarking efforts. Last, we discuss the future of the field.

  7. Differentially expressed microRNA in multiple sclerosis: A window into pathogenesis?

    DEFF Research Database (Denmark)

    Martin, Nellie Anne; Illés, Zsolt

    2014-01-01

    MicroRNA are small non-coding RNA that mediate mRNA translation repression or mRNA degradation, and thereby refine protein expression levels. More than 30–60% of all genes are regulated by microRNA. Exploring disease-related microRNA signatures is an emerging tool in biomarker discovery, and sile......MicroRNA are small non-coding RNA that mediate mRNA translation repression or mRNA degradation, and thereby refine protein expression levels. More than 30–60% of all genes are regulated by microRNA. Exploring disease-related microRNA signatures is an emerging tool in biomarker discovery......RNA related to multiple sclerosis has increased significantly in recent years. Differentially expressed microRNA have been identified in the whole blood, serum, plasma, cerebrospinal fluid, peripheral blood mononuclear cells, blood-derived cell subsets and brain lesions of patients with multiple sclerosis....... Most studies applied a non-candidate approach of screening by microarray and validation by quantitative polymerase chain reaction or next generation sequencing; others used a candidate-driven approach. Despite a relatively high number of multiple sclerosis-associated microRNA, just a few could...

  8. Evaluation of tools for highly variable gene discovery from single-cell RNA-seq data.

    Science.gov (United States)

    Yip, Shun H; Sham, Pak Chung; Wang, Junwen

    2018-02-21

    Traditional RNA sequencing (RNA-seq) allows the detection of gene expression variations between two or more cell populations through differentially expressed gene (DEG) analysis. However, genes that contribute to cell-to-cell differences are not discoverable with RNA-seq because RNA-seq samples are obtained from a mixture of cells. Single-cell RNA-seq (scRNA-seq) allows the detection of gene expression in each cell. With scRNA-seq, highly variable gene (HVG) discovery allows the detection of genes that contribute strongly to cell-to-cell variation within a homogeneous cell population, such as a population of embryonic stem cells. This analysis is implemented in many software packages. In this study, we compare seven HVG methods from six software packages, including BASiCS, Brennecke, scLVM, scran, scVEGs and Seurat. Our results demonstrate that reproducibility in HVG analysis requires a larger sample size than DEG analysis. Discrepancies between methods and potential issues in these tools are discussed and recommendations are made.

  9. DETECTION OF BACTERIAL SMALL TRANSCRIPTS FROM RNA-SEQ DATA: A COMPARATIVE ASSESSMENT.

    Science.gov (United States)

    Peña-Castillo, Lourdes; Grüell, Marc; Mulligan, Martin E; Lang, Andrew S

    2016-01-01

    Small non-coding RNAs (sRNAs) are regulatory RNA molecules that have been identified in a multitude of bacterial species and shown to control numerous cellular processes through various regulatory mechanisms. In the last decade, next generation RNA sequencing (RNA-seq) has been used for the genome-wide detection of bacterial sRNAs. Here we describe sRNA-Detect, a novel approach to identify expressed small transcripts from prokaryotic RNA-seq data. Using RNA-seq data from three bacterial species and two sequencing platforms, we performed a comparative assessment of five computational approaches for the detection of small transcripts. We demonstrate that sRNA-Detect improves upon current standalone computational approaches for identifying novel small transcripts in bacteria.

  10. Characterization and comparative analysis of small RNAs in three small RNA libraries of the brown planthopper (Nilaparvata lugens).

    Science.gov (United States)

    Chen, Qiuhong; Lu, Lin; Hua, Hongxia; Zhou, Fei; Lu, Liaoxun; Lin, Yongjun

    2012-01-01

    The brown planthopper (BPH), Nilaparvata lugens (Stå;l), which belongs to Homopteran, Delphacidae, is one of the most serious and destructive pests of rice. Feeding BPH with homologous dsRNA in vitro can lead to the death of BPH, which gives a valuable clue to the prevention and control of this pest, however, we know little about its small RNA world. Small RNA libraries for three developmental stages of BPH (CX-male adult, CC-female adult, CY-last instar female nymph) had been constructed and sequenced. It revealed a prolific small RNA world of BPH. We obtained a final list of 452 (CX), 430 (CC), and 381 (CY) conserved microRNAs (miRNAs), respectively, as well as a total of 71 new miRNAs in the three libraries. All the miRNAs had their own expression profiles in the three libraries. The phylogenic evolution of the miRNA families in BPH was consistent with other species. The new miRNA sequences demonstrated some base biases. Our study discovered a large number of small RNAs through deep sequencing of three small RNA libraries of BPH. Many animal-conserved miRNA families as well as some novel miRNAs have been detected in our libraries. This is the first achievement to discover the small RNA world of BPH. A lot of new valuable information about BPH small RNAs has been revealed which was helpful for studying insect molecular biology and insect resistant research.

  11. Application of small RNA sequencing to identify microRNAs in acute kidney injury and fibrosis

    Energy Technology Data Exchange (ETDEWEB)

    Pellegrini, Kathryn L. [Department of Medicine, Renal Division, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Gerlach, Cory V. [Department of Medicine, Renal Division, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA (United States); Laboratory of Systems Pharmacology, Harvard Program in Therapeutic Sciences, Harvard Medical School, Boston, MA (United States); Craciun, Florin L.; Ramachandran, Krithika [Department of Medicine, Renal Division, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Bijol, Vanesa [Department of Pathology, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Kissick, Haydn T. [Department of Surgery, Urology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States); Vaidya, Vishal S., E-mail: vvaidya@bwh.harvard.edu [Department of Medicine, Renal Division, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA (United States); Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA (United States); Laboratory of Systems Pharmacology, Harvard Program in Therapeutic Sciences, Harvard Medical School, Boston, MA (United States)

    2016-12-01

    Establishing a microRNA (miRNA) expression profile in affected tissues provides an important foundation for the discovery of miRNAs involved in the development or progression of pathologic conditions. We conducted small RNA sequencing to generate a temporal profile of miRNA expression in the kidneys using a mouse model of folic acid-induced (250 mg/kg i.p.) kidney injury and fibrosis. From the 103 miRNAs that were differentially expressed over the time course (> 2-fold, p < 0.05), we chose to further investigate miR-18a-5p, which is expressed during the acute stage of the injury; miR-132-3p, which is upregulated during transition between acute and fibrotic injury; and miR-146b-5p, which is highly expressed at the peak of fibrosis. Using qRT-PCR, we confirmed the increased expression of these candidate miRNAs in the folic acid model as well as in other established mouse models of acute injury (ischemia/reperfusion injury) and fibrosis (unilateral ureteral obstruction). In situ hybridization confirmed high expression of miR-18a-5p, miR-132-3p and miR-146b-5p throughout the kidney cortex in mice and humans with severe kidney injury or fibrosis. When primary human proximal tubular epithelial cells were treated with model nephrotoxicants such as cadmium chloride (CdCl{sub 2}), arsenic trioxide, aristolochic acid (AA), potassium dichromate (K{sub 2}Cr{sub 2}O{sub 7}) and cisplatin, miRNA-132-3p was upregulated 4.3-fold after AA treatment and 1.5-fold after K{sub 2}Cr{sub 2}O{sub 7} and CdCl{sub 2} treatment. These results demonstrate the application of temporal small RNA sequencing to identify miR-18a, miR-132 and miR-146b as differentially expressed miRNAs during distinct phases of kidney injury and fibrosis progression. - Highlights: • We used small RNA sequencing to identify differentially expressed miRNAs in kidney. • Distinct patterns were found for acute injury and fibrotic stages in the kidney. • Upregulation of miR-18a, -132 and -146b was confirmed in mice

  12. Primary and secondary structure of U8 small nuclear RNA

    International Nuclear Information System (INIS)

    Reddy, R.; Henning, D.; Busch, H.

    1985-01-01

    U8 small nuclear RNA is a new, capped, 140 nucleotides long RNA species found in Novikoff hepatoma cells. Its sequence is: m3GpppAmUmCGUCAGGA GGUUAAUCCU UACCUGUCCC UCCUUUCGGA GGGCAGAUAG AAAAUGAUGA UUGGAGCUUG CAUGAUCUGC UGAUUAUAGC AUUUCCGUGU AAUCAGGACC UGACAACAUC CUGAUUGCUU CUAUCUGAUUOH. This RNA is present in approximately 25,000 copies/cell, and it is enriched in nucleolar preparations. Like U1, U2, U4/U6, and U5 RNAs, U8 RNA was also present as a ribonucleoprotein associated with the Sm antigen. The rat U8 RNA was highly homologous (greater than 90%) to a recently characterized 5.4 S RNA from mouse cells infected with spleen focus-forming virus. In addition to the U8 RNA, three other U small nuclear RNAs were found in anti-Sm antibody immunoprecipitates from labeled rat and HeLa cells. Each of these contained a m3GpppAm cap structure; their apparent chain lengths were 60, 130, and 65 nucleotides. These U small nuclear RNAs are designated U7, U9, and U10 RNAs, respectively

  13. A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool.

    Science.gov (United States)

    Manterola, Lorea; Guruceaga, Elizabeth; Gállego Pérez-Larraya, Jaime; González-Huarriz, Marisol; Jauregui, Patricia; Tejada, Sonia; Diez-Valle, Ricardo; Segura, Victor; Samprón, Nicolás; Barrena, Cristina; Ruiz, Irune; Agirre, Amaia; Ayuso, Angel; Rodríguez, Javier; González, Alvaro; Xipell, Enric; Matheu, Ander; López de Munain, Adolfo; Tuñón, Teresa; Zazpe, Idoya; García-Foncillas, Jesús; Paris, Sophie; Delattre, Jean Yves; Alonso, Marta M

    2014-04-01

    Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and its prognosis remains dismal despite intensive research and therapeutic advances. Diagnostic biomarkers would be clinically meaningful to allow for early detection of the tumor and for those cases in which surgery is contraindicated or biopsy results are inconclusive. Recent findings show that GBM cells release microvesicles that contain a select subset of cellular proteins and RNA. The aim of this hypothesis-generating study was to assess the diagnostic potential of miRNAs found in microvesicles isolated from the serum of GBM patients. To control disease heterogeneity, we used patients with newly diagnosed GBM. In the discovery stage, PCR-based TaqMan Low Density Arrays followed by individual quantitative reverse transcriptase polymerase chain reaction were used to test the differences in the miRNA expression levels of serum microvesicles among 25 GBM patients and healthy controls paired by age and sex. The detected noncoding RNAs were then validated in another 50 GBM patients. We found that the expression levels of 1 small noncoding RNA (RNU6-1) and 2 microRNAs (miR-320 and miR-574-3p) were significantly associated with a GBM diagnosis. In addition, RNU6-1 was consistently an independent predictor of a GBM diagnosis. Altogether our results uncovered a small noncoding RNA signature in microvesicles isolated from GBM patient serum that could be used as a fast and reliable differential diagnostic biomarker.

  14. Characterization and comparative analysis of small RNAs in three small RNA libraries of the brown planthopper (Nilaparvata lugens.

    Directory of Open Access Journals (Sweden)

    Qiuhong Chen

    Full Text Available BACKGROUND: The brown planthopper (BPH, Nilaparvata lugens (Stå;l, which belongs to Homopteran, Delphacidae, is one of the most serious and destructive pests of rice. Feeding BPH with homologous dsRNA in vitro can lead to the death of BPH, which gives a valuable clue to the prevention and control of this pest, however, we know little about its small RNA world. METHODOLOGY/PRINCIPAL FINDINGS: Small RNA libraries for three developmental stages of BPH (CX-male adult, CC-female adult, CY-last instar female nymph had been constructed and sequenced. It revealed a prolific small RNA world of BPH. We obtained a final list of 452 (CX, 430 (CC, and 381 (CY conserved microRNAs (miRNAs, respectively, as well as a total of 71 new miRNAs in the three libraries. All the miRNAs had their own expression profiles in the three libraries. The phylogenic evolution of the miRNA families in BPH was consistent with other species. The new miRNA sequences demonstrated some base biases. CONCLUSION: Our study discovered a large number of small RNAs through deep sequencing of three small RNA libraries of BPH. Many animal-conserved miRNA families as well as some novel miRNAs have been detected in our libraries. This is the first achievement to discover the small RNA world of BPH. A lot of new valuable information about BPH small RNAs has been revealed which was helpful for studying insect molecular biology and insect resistant research.

  15. Unique small RNA signatures uncovered in the tammar wallaby genome

    Directory of Open Access Journals (Sweden)

    Lindsay James

    2012-10-01

    Full Text Available Abstract Background Small RNAs have proven to be essential regulatory molecules encoded within eukaryotic genomes. These short RNAs participate in a diverse array of cellular processes including gene regulation, chromatin dynamics and genome defense. The tammar wallaby, a marsupial mammal, is a powerful comparative model for studying the evolution of regulatory networks. As part of the genome sequencing initiative for the tammar, we have explored the evolution of each of the major classes of mammalian small RNAs in an Australian marsupial for the first time, including the first genome-scale analysis of the newest class of small RNAs, centromere repeat associated short interacting RNAs (crasiRNAs. Results Using next generation sequencing, we have characterized the major classes of small RNAs, micro (mi RNAs, piwi interacting (pi RNAs, and the centromere repeat associated short interacting (crasi RNAs in the tammar. We examined each of these small RNA classes with respect to the newly assembled tammar wallaby genome for gene and repeat features, salient features that define their canonical sequences, and the constitution of both highly conserved and species-specific members. Using a combination of miRNA hairpin predictions and co-mapping with miRBase entries, we identified a highly conserved cluster of miRNA genes on the X chromosome in the tammar and a total of 94 other predicted miRNA producing genes. Mapping all miRNAs to the tammar genome and comparing target genes among tammar, mouse and human, we identified 163 conserved target genes. An additional nine genes were identified in tammar that do not have an orthologous miRNA target in human and likely represent novel miRNA-regulated genes in the tammar. A survey of the tammar gonadal piRNAs shows that these small RNAs are enriched in retroelements and carry members from both marsupial and tammar-specific repeat classes. Lastly, this study includes the first in-depth analyses of the newly

  16. Discovery and annotation of small proteins using genomics, proteomics and computational approaches

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xiaohan; Tschaplinski, Timothy J.; Hurst, Gregory B.; Jawdy, Sara; Abraham, Paul E.; Lankford, Patricia K.; Adams, Rachel M.; Shah, Manesh B.; Hettich, Robert L.; Lindquist, Erika; Kalluri, Udaya C.; Gunter, Lee E.; Pennacchio, Christa; Tuskan, Gerald A.

    2011-03-02

    Small proteins (10 200 amino acids aa in length) encoded by short open reading frames (sORF) play important regulatory roles in various biological processes, including tumor progression, stress response, flowering, and hormone signaling. However, ab initio discovery of small proteins has been relatively overlooked. Recent advances in deep transcriptome sequencing make it possible to efficiently identify sORFs at the genome level. In this study, we obtained 2.6 million expressed sequence tag (EST) reads from Populus deltoides leaf transcriptome and reconstructed full-length transcripts from the EST sequences. We identified an initial set of 12,852 sORFs encoding proteins of 10 200 aa in length. Three computational approaches were then used to enrich for bona fide protein-coding sORFs from the initial sORF set: (1) codingpotential prediction, (2) evolutionary conservation between P. deltoides and other plant species, and (3) gene family clustering within P. deltoides. As a result, a high-confidence sORF candidate set containing 1469 genes was obtained. Analysis of the protein domains, non-protein-coding RNA motifs, sequence length distribution, and protein mass spectrometry data supported this high-confidence sORF set. In the high-confidence sORF candidate set, known protein domains were identified in 1282 genes (higher-confidence sORF candidate set), out of which 611 genes, designated as highest-confidence candidate sORF set, were supported by proteomics data. Of the 611 highest-confidence candidate sORF genes, 56 were new to the current Populus genome annotation. This study not only demonstrates that there are potential sORF candidates to be annotated in sequenced genomes, but also presents an efficient strategy for discovery of sORFs in species with no genome annotation yet available.

  17. Small molecule alteration of RNA sequence in cells and animals.

    Science.gov (United States)

    Guan, Lirui; Luo, Yiling; Ja, William W; Disney, Matthew D

    2017-10-18

    RNA regulation and maintenance are critical for proper cell function. Small molecules that specifically alter RNA sequence would be exceptionally useful as probes of RNA structure and function or as potential therapeutics. Here, we demonstrate a photochemical approach for altering the trinucleotide expanded repeat causative of myotonic muscular dystrophy type 1 (DM1), r(CUG) exp . The small molecule, 2H-4-Ru, binds to r(CUG) exp and converts guanosine residues to 8-oxo-7,8-dihydroguanosine upon photochemical irradiation. We demonstrate targeted modification upon irradiation in cell culture and in Drosophila larvae provided a diet containing 2H-4-Ru. Our results highlight a general chemical biology approach for altering RNA sequence in vivo by using small molecules and photochemistry. Furthermore, these studies show that addition of 8-oxo-G lesions into RNA 3' untranslated regions does not affect its steady state levels. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

    Directory of Open Access Journals (Sweden)

    Jonathan A Kopechek

    Full Text Available RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14. Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9 or control RNA (n = 8 during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3 confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively. Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.

  19. Structural insights into mechanisms of the small RNA methyltransferase HEN1

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Ying; Ji, Lijuan; Huang, Qichen; Vassylyev, Dmitry G.; Chen, Xuemei; Ma, Jin-Biao; (UAB); (UCR)

    2010-02-22

    RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of {approx}20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-L-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 {angstrom} crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-L-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg{sup 2+}-dependent 2'-O-methylation mechanism.

  20. Examining the intersection between splicing, nuclear export and small RNA pathways.

    Science.gov (United States)

    Nabih, Amena; Sobotka, Julia A; Wu, Monica Z; Wedeles, Christopher J; Claycomb, Julie M

    2017-11-01

    Nuclear Argonaute/small RNA pathways in a variety of eukaryotic species are generally known to regulate gene expression via chromatin modulation and transcription attenuation in a process known as transcriptional gene silencing (TGS). However, recent data, including genetic screens, phylogenetic profiling, and molecular mechanistic studies, also point to a novel and emerging intersection between the splicing and nuclear export machinery with nuclear Argonaute/small RNA pathways in many organisms. In this review, we summarize the field's current understanding regarding the relationship between splicing, export and small RNA pathways, and consider the biological implications for coordinated regulation of transcripts by these pathways. We also address the importance and available approaches for understanding the RNA regulatory logic generated by the intersection of these particular pathways in the context of synthetic biology. The interactions between various eukaryotic RNA regulatory pathways, particularly splicing, nuclear export and small RNA pathways provide a type of combinatorial code that informs the identity ("self" versus "non-self") and dictates the fate of each transcript in a cell. Although the molecular mechanisms for how splicing and nuclear export impact small RNA pathways are not entirely clear at this early stage, the links between these pathways are widespread across eukaryotic phyla. The link between splicing, nuclear export, and small RNA pathways is emerging and establishes a new frontier for understanding the combinatorial logic of gene regulation across species that could someday be harnessed for therapeutic, biotechnology and agricultural applications. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Oasis 2: improved online analysis of small RNA-seq data.

    Science.gov (United States)

    Rahman, Raza-Ur; Gautam, Abhivyakti; Bethune, Jörn; Sattar, Abdul; Fiosins, Maksims; Magruder, Daniel Sumner; Capece, Vincenzo; Shomroni, Orr; Bonn, Stefan

    2018-02-14

    Small RNA molecules play important roles in many biological processes and their dysregulation or dysfunction can cause disease. The current method of choice for genome-wide sRNA expression profiling is deep sequencing. Here we present Oasis 2, which is a new main release of the Oasis web application for the detection, differential expression, and classification of small RNAs in deep sequencing data. Compared to its predecessor Oasis, Oasis 2 features a novel and speed-optimized sRNA detection module that supports the identification of small RNAs in any organism with higher accuracy. Next to the improved detection of small RNAs in a target organism, the software now also recognizes potential cross-species miRNAs and viral and bacterial sRNAs in infected samples. In addition, novel miRNAs can now be queried and visualized interactively, providing essential information for over 700 high-quality miRNA predictions across 14 organisms. Robust biomarker signatures can now be obtained using the novel enhanced classification module. Oasis 2 enables biologists and medical researchers to rapidly analyze and query small RNA deep sequencing data with improved precision, recall, and speed, in an interactive and user-friendly environment. Oasis 2 is implemented in Java, J2EE, mysql, Python, R, PHP and JavaScript. It is freely available at https://oasis.dzne.de.

  2. Preparation of Small RNA NGS Libraries from Biofluids.

    Science.gov (United States)

    Etheridge, Alton; Wang, Kai; Baxter, David; Galas, David

    2018-01-01

    Next generation sequencing (NGS) is a powerful method for transcriptome analysis. Unlike other gene expression profiling methods, such as microarrays, NGS provides additional information such as splicing variants, sequence polymorphisms, and novel transcripts. For this reason, NGS is well suited for comprehensive profiling of the wide range of extracellular RNAs (exRNAs) in biofluids. ExRNAs are of great interest because of their possible biological role in cell-to-cell communication and for their potential use as biomarkers or for therapeutic purposes. Here, we describe a modified protocol for preparation of small RNA libraries for NGS analysis. This protocol has been optimized for use with low-input exRNA-containing samples, such as plasma or serum, and has modifications designed to reduce the sequence-specific bias typically encountered with commercial small RNA library construction kits.

  3. piRNA analysis framework from small RNA-Seq data by a novel cluster prediction tool - PILFER.

    Science.gov (United States)

    Ray, Rishav; Pandey, Priyanka

    2017-12-19

    With the increasing number of studies focusing on PIWI-interacting RNA (piRNAs), it is now pertinent to develop efficient tools dedicated towards piRNA analysis. We have developed a novel cluster prediction tool called PILFER (PIrna cLuster FindER), which can accurately predict piRNA clusters from small RNA sequencing data. PILFER is an open source, easy to use tool, and can be executed even on a personal computer with minimum resources. It uses a sliding-window mechanism by integrating the expression of the reads along with the spatial information to predict the piRNA clusters. We have additionally defined a piRNA analysis pipeline incorporating PILFER to detect and annotate piRNAs and their clusters from raw small RNA sequencing data and implemented it on publicly available data from healthy germline and somatic tissues. We compared PILFER with other existing piRNA cluster prediction tools and found it to be statistically more accurate and superior in many aspects such as the robustness of PILFER clusters is higher and memory efficiency is more. Overall, PILFER provides a fast and accurate solution to piRNA cluster prediction. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Modulation of RNA function by aminoglycoside antibiotics.

    Science.gov (United States)

    Schroeder, R; Waldsich, C; Wank, H

    2000-01-04

    One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.

  5. Seeing the forest for the trees: annotating small RNA producing genes in plants.

    Science.gov (United States)

    Coruh, Ceyda; Shahid, Saima; Axtell, Michael J

    2014-04-01

    A key goal in genomics is the complete annotation of the expressed regions of the genome. In plants, substantial portions of the genome make regulatory small RNAs produced by Dicer-Like (DCL) proteins and utilized by Argonaute (AGO) proteins. These include miRNAs and various types of endogenous siRNAs. Small RNA-seq, enabled by cheap and fast DNA sequencing, has produced an enormous volume of data on plant miRNA and siRNA expression in recent years. In this review, we discuss recent progress in using small RNA-seq data to produce stable and reliable annotations of miRNA and siRNA genes in plants. In addition, we highlight key goals for the future of small RNA gene annotation in plants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Conifers have a unique small RNA silencing signature

    OpenAIRE

    Dolgosheina, Elena V.; Morin, Ryan D.; Aksay, Gozde; Sahinalp, S. Cenk; Magrini, Vincent; Mardis, Elaine R.; Mattsson, Jim; Unrau, Peter J.

    2008-01-01

    Plants produce small RNAs to negatively regulate genes, viral nucleic acids, and repetitive elements at either the transcriptional or post-transcriptional level in a process that is referred to as RNA silencing. While RNA silencing has been extensively studied across the different phyla of the animal kingdom (e.g., mouse, fly, worm), similar studies in the plant kingdom have focused primarily on angiosperms, thus limiting evolutionary studies of RNA silencing in plants. Here we report on an u...

  7. Cell and small animal models for phenotypic drug discovery

    Directory of Open Access Journals (Sweden)

    Szabo M

    2017-06-01

    Full Text Available Mihaly Szabo,1 Sara Svensson Akusjärvi,1 Ankur Saxena,1 Jianping Liu,2 Gayathri Chandrasekar,1 Satish S Kitambi1 1Department of Microbiology Tumor, and Cell Biology, 2Department of Biochemistry and Biophysics, Karolinska Institutet, Solna, Sweden Abstract: The phenotype-based drug discovery (PDD approach is re-emerging as an alternative platform for drug discovery. This review provides an overview of the various model systems and technical advances in imaging and image analyses that strengthen the PDD platform. In PDD screens, compounds of therapeutic value are identified based on the phenotypic perturbations produced irrespective of target(s or mechanism of action. In this article, examples of phenotypic changes that can be detected and quantified with relative ease in a cell-based setup are discussed. In addition, a higher order of PDD screening setup using small animal models is also explored. As PDD screens integrate physiology and multiple signaling mechanisms during the screening process, the identified hits have higher biomedical applicability. Taken together, this review highlights the advantages gained by adopting a PDD approach in drug discovery. Such a PDD platform can complement target-based systems that are currently in practice to accelerate drug discovery. Keywords: phenotype, screening, PDD, discovery, zebrafish, drug

  8. Systems Pharmacology in Small Molecular Drug Discovery

    Directory of Open Access Journals (Sweden)

    Wei Zhou

    2016-02-01

    Full Text Available Drug discovery is a risky, costly and time-consuming process depending on multidisciplinary methods to create safe and effective medicines. Although considerable progress has been made by high-throughput screening methods in drug design, the cost of developing contemporary approved drugs did not match that in the past decade. The major reason is the late-stage clinical failures in Phases II and III because of the complicated interactions between drug-specific, human body and environmental aspects affecting the safety and efficacy of a drug. There is a growing hope that systems-level consideration may provide a new perspective to overcome such current difficulties of drug discovery and development. The systems pharmacology method emerged as a holistic approach and has attracted more and more attention recently. The applications of systems pharmacology not only provide the pharmacodynamic evaluation and target identification of drug molecules, but also give a systems-level of understanding the interaction mechanism between drugs and complex disease. Therefore, the present review is an attempt to introduce how holistic systems pharmacology that integrated in silico ADME/T (i.e., absorption, distribution, metabolism, excretion and toxicity, target fishing and network pharmacology facilitates the discovery of small molecular drugs at the system level.

  9. DNApi: A De Novo Adapter Prediction Algorithm for Small RNA Sequencing Data.

    Science.gov (United States)

    Tsuji, Junko; Weng, Zhiping

    2016-01-01

    With the rapid accumulation of publicly available small RNA sequencing datasets, third-party meta-analysis across many datasets is becoming increasingly powerful. Although removing the 3´ adapter is an essential step for small RNA sequencing analysis, the adapter sequence information is not always available in the metadata. The information can be also erroneous even when it is available. In this study, we developed DNApi, a lightweight Python software package that predicts the 3´ adapter sequence de novo and provides the user with cleansed small RNA sequences ready for down stream analysis. Tested on 539 publicly available small RNA libraries accompanied with 3´ adapter sequences in their metadata, DNApi shows near-perfect accuracy (98.5%) with fast runtime (~2.85 seconds per library) and efficient memory usage (~43 MB on average). In addition to 3´ adapter prediction, it is also important to classify whether the input small RNA libraries were already processed, i.e. the 3´ adapters were removed. DNApi perfectly judged that given another batch of datasets, 192 publicly available processed libraries were "ready-to-map" small RNA sequence. DNApi is compatible with Python 2 and 3, and is available at https://github.com/jnktsj/DNApi. The 731 small RNA libraries used for DNApi evaluation were from human tissues and were carefully and manually collected. This study also provides readers with the curated datasets that can be integrated into their studies.

  10. Telomeric trans-silencing: an epigenetic repression combining RNA silencing and heterochromatin formation.

    Directory of Open Access Journals (Sweden)

    Thibaut Josse

    2007-09-01

    Full Text Available The study of P-element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE, a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequence or TAS has the capacity to repress in trans in the female germline, a homologous transposon, or transgene located in euchromatin. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. Here, we report that TSE displays an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted factor. We show that this silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var205 encoding Heterochromatin Protein 1 and Su(var3-7 and the repeat-associated small interfering RNA (or rasiRNA silencing pathway (aubergine, homeless, armitage, and piwi. In contrast, TSE is not sensitive to mutations affecting r2d2, which is involved in the small interfering RNA (or siRNA silencing pathway, nor is it sensitive to a mutation in loquacious, which is involved in the micro RNA (or miRNA silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a repeat-associated small interfering RNA pathway linked to heterochromatin formation, which was co-opted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway.

  11. The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism

    Science.gov (United States)

    Bækkedal, Cecilie; Haugen, Peik

    2015-01-01

    The Spot 42 RNA is a 109 nucleotide long (in Escherichia coli) noncoding small regulatory RNA (sRNA) encoded by the spf (spot fourty-two) gene. spf is found in gamma-proteobacteria and the majority of experimental work on Spot 42 RNA has been performed using E. coli, and recently Aliivibrio salmonicida. In the cell Spot 42 RNA plays essential roles as a regulator in carbohydrate metabolism and uptake, and its expression is activated by glucose, and inhibited by the cAMP-CRP complex. Here we summarize the current knowledge on Spot 42, and present the natural distribution of spf, show family-specific secondary structural features of Spot 42, and link highly conserved structural regions to mRNA target binding. PMID:26327359

  12. The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism.

    Science.gov (United States)

    Bækkedal, Cecilie; Haugen, Peik

    2015-01-01

    The Spot 42 RNA is a 109 nucleotide long (in Escherichia coli) noncoding small regulatory RNA (sRNA) encoded by the spf (spot fourty-two) gene. spf is found in gamma-proteobacteria and the majority of experimental work on Spot 42 RNA has been performed using E. coli, and recently Aliivibrio salmonicida. In the cell Spot 42 RNA plays essential roles as a regulator in carbohydrate metabolism and uptake, and its expression is activated by glucose, and inhibited by the cAMP-CRP complex. Here we summarize the current knowledge on Spot 42, and present the natural distribution of spf, show family-specific secondary structural features of Spot 42, and link highly conserved structural regions to mRNA target binding.

  13. Positive Bioluminescence Imaging of MicroRNA Expression in Small Animal Models Using an Engineered Genetic-Switch Expression System, RILES.

    Science.gov (United States)

    Baril, Patrick; Pichon, Chantal

    2016-01-01

    MicroRNAs (miRNAs) are a class of small, noncoding RNAs which regulate gene expression by directing their target mRNA for degradation or translational repression. Since their discovery in the early 1990s, miRNAs have emerged as key components in the posttranscriptional regulation of gene networks, shaping many biological processes from development, morphogenesis, differentiation, proliferation and apoptosis. Although understanding of the molecular basis of miRNA biology is improving, methods to monitor the dynamic and the spatiotemporal aspects of miRNA expression under physiopathological conditions are required. However, monitoring of miRNAs is difficult due to their small size, low abundance, high degree of sequence similarity, and their dynamic expression pattern which is subjected to tight transcriptional and post-transcriptional controls. Recently, we developed a miRNA monitoring system called RILES, standing for RNAi-inducible expression system, which relies on an engineered regulatable expression system, to switch on the expression of the luciferase gene when the targeted miRNA is expressed in cells. We demonstrated that RILES is a specific, sensitive, and robust method to determine the fine-tuning of miRNA expression during the development of an experimental pathological process in mice. Because RILES offers the possibility for longitudinal studies on individual subjects, sharper insights into miRNA regulation can be generated, with applications in physiology, pathophysiology and development of RNAi-based therapies. This chapter describes methods and protocols to monitor the expression of myomiR-206, -1, and -133 in the tibialis anterior muscle of mice. These protocols can be used and adapted to monitor the expression of other miRNAs in other biological processes.

  14. Alterations in messenger RNA and small nuclear RNA metabolism resulting from fluorouracil incorporation

    International Nuclear Information System (INIS)

    Armstrong, R.D.; Cadman, E.C.

    1985-01-01

    Studies were completed to examine the effect of 5-fluorouracil (FUra) incorporation on messenger RNA (mRNA) and small molecular weight nuclear RNA (SnRNA) metabolism. Studies of mRNA were completed using cDNA-mRNA hybridization methods to specifically examine dihydrofolate reductase (DHFR) mRNA. C 3 -L5178Y murine leukemia cells which are gene-amplified for DHFR, were exposed to FUra for 6, 12 or 24 hr, and the nuclear and cytoplasmic levels of DHFR-mRNA determined by hybridization with 32 P-DHFR-cDNA. FUra produced a dose-dependent increase in nuclear DHFR-mRNA levels, while total cytoplasmic DHFR-mRNA levels appeared to be unchanged. To examine only mRNA synthesized during FUra exposure, cells were also treated concurrently with [ 3 H] cytidine, and the [ 3 H]mRNA-cDNA hybrids measured following S 1 -nuclease treatment. FUra produced a concentration-dependent increase in nascent nuclear DHFR-mRNA levels, and a decrease in nascent cytoplasmic DHFR-mRNAs levels. These results suggest that FUra produces either an inhibition of mRNA processing, or an inhibition of nuclear-cytoplasmic transport. Preliminary experiments to examine ATP-dependent mRNA transport were completed with isolated nuclei from cells treated with FUra for 1 or 24 hr and then pulse-labeled for 1 hr with [ 3 H] cytidine. The results demonstrate a FUra-concentration and time-dependent inhibition of ATP-mediated mRNA efflux

  15. CPSS: a computational platform for the analysis of small RNA deep sequencing data.

    Science.gov (United States)

    Zhang, Yuanwei; Xu, Bo; Yang, Yifan; Ban, Rongjun; Zhang, Huan; Jiang, Xiaohua; Cooke, Howard J; Xue, Yu; Shi, Qinghua

    2012-07-15

    Next generation sequencing (NGS) techniques have been widely used to document the small ribonucleic acids (RNAs) implicated in a variety of biological, physiological and pathological processes. An integrated computational tool is needed for handling and analysing the enormous datasets from small RNA deep sequencing approach. Herein, we present a novel web server, CPSS (a computational platform for the analysis of small RNA deep sequencing data), designed to completely annotate and functionally analyse microRNAs (miRNAs) from NGS data on one platform with a single data submission. Small RNA NGS data can be submitted to this server with analysis results being returned in two parts: (i) annotation analysis, which provides the most comprehensive analysis for small RNA transcriptome, including length distribution and genome mapping of sequencing reads, small RNA quantification, prediction of novel miRNAs, identification of differentially expressed miRNAs, piwi-interacting RNAs and other non-coding small RNAs between paired samples and detection of miRNA editing and modifications and (ii) functional analysis, including prediction of miRNA targeted genes by multiple tools, enrichment of gene ontology terms, signalling pathway involvement and protein-protein interaction analysis for the predicted genes. CPSS, a ready-to-use web server that integrates most functions of currently available bioinformatics tools, provides all the information wanted by the majority of users from small RNA deep sequencing datasets. CPSS is implemented in PHP/PERL+MySQL+R and can be freely accessed at http://mcg.ustc.edu.cn/db/cpss/index.html or http://mcg.ustc.edu.cn/sdap1/cpss/index.html.

  16. sRNAnalyzer-a flexible and customizable small RNA sequencing data analysis pipeline.

    Science.gov (United States)

    Wu, Xiaogang; Kim, Taek-Kyun; Baxter, David; Scherler, Kelsey; Gordon, Aaron; Fong, Olivia; Etheridge, Alton; Galas, David J; Wang, Kai

    2017-12-01

    Although many tools have been developed to analyze small RNA sequencing (sRNA-Seq) data, it remains challenging to accurately analyze the small RNA population, mainly due to multiple sequence ID assignment caused by short read length. Additional issues in small RNA analysis include low consistency of microRNA (miRNA) measurement results across different platforms, miRNA mapping associated with miRNA sequence variation (isomiR) and RNA editing, and the origin of those unmapped reads after screening against all endogenous reference sequence databases. To address these issues, we built a comprehensive and customizable sRNA-Seq data analysis pipeline-sRNAnalyzer, which enables: (i) comprehensive miRNA profiling strategies to better handle isomiRs and summarization based on each nucleotide position to detect potential SNPs in miRNAs, (ii) different sequence mapping result assignment approaches to simulate results from microarray/qRT-PCR platforms and a local probabilistic model to assign mapping results to the most-likely IDs, (iii) comprehensive ribosomal RNA filtering for accurate mapping of exogenous RNAs and summarization based on taxonomy annotation. We evaluated our pipeline on both artificial samples (including synthetic miRNA and Escherichia coli cultures) and biological samples (human tissue and plasma). sRNAnalyzer is implemented in Perl and available at: http://srnanalyzer.systemsbiology.net/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. sRNAnalyzer—a flexible and customizable small RNA sequencing data analysis pipeline

    Science.gov (United States)

    Kim, Taek-Kyun; Baxter, David; Scherler, Kelsey; Gordon, Aaron; Fong, Olivia; Etheridge, Alton; Galas, David J.

    2017-01-01

    Abstract Although many tools have been developed to analyze small RNA sequencing (sRNA-Seq) data, it remains challenging to accurately analyze the small RNA population, mainly due to multiple sequence ID assignment caused by short read length. Additional issues in small RNA analysis include low consistency of microRNA (miRNA) measurement results across different platforms, miRNA mapping associated with miRNA sequence variation (isomiR) and RNA editing, and the origin of those unmapped reads after screening against all endogenous reference sequence databases. To address these issues, we built a comprehensive and customizable sRNA-Seq data analysis pipeline—sRNAnalyzer, which enables: (i) comprehensive miRNA profiling strategies to better handle isomiRs and summarization based on each nucleotide position to detect potential SNPs in miRNAs, (ii) different sequence mapping result assignment approaches to simulate results from microarray/qRT-PCR platforms and a local probabilistic model to assign mapping results to the most-likely IDs, (iii) comprehensive ribosomal RNA filtering for accurate mapping of exogenous RNAs and summarization based on taxonomy annotation. We evaluated our pipeline on both artificial samples (including synthetic miRNA and Escherichia coli cultures) and biological samples (human tissue and plasma). sRNAnalyzer is implemented in Perl and available at: http://srnanalyzer.systemsbiology.net/. PMID:29069500

  18. Exploration of small RNA-seq data for small non-coding RNAs in Human Colorectal Cancer.

    Science.gov (United States)

    Koduru, Srinivas V; Tiwari, Amit K; Hazard, Sprague W; Mahajan, Milind; Ravnic, Dino J

    2017-01-01

    Background: Improved healthcare and recent breakthroughs in technology have substantially reduced cancer mortality rates worldwide. Recent advancements in next-generation sequencing (NGS) have allowed genomic analysis of the human transcriptome. Now, using NGS we can further look into small non-coding regions of RNAs (sncRNAs) such as microRNAs (miRNAs), Piwi-interacting-RNAs (piRNAs), long non-coding RNAs (lncRNAs), and small nuclear/nucleolar RNAs (sn/snoRNAs) among others. Recent studies looking at sncRNAs indicate their role in important biological processes such as cancer progression and predict their role as biomarkers for disease diagnosis, prognosis, and therapy. Results: In the present study, we data mined publically available small RNA sequencing data from colorectal tissue samples of eight matched patients (benign, tumor, and metastasis) and remapped the data for various small RNA annotations. We identified aberrant expression of 13 miRNAs in tumor and metastasis specimens [tumor vs benign group (19 miRNAs) and metastasis vs benign group (38 miRNAs)] of which five were upregulated, and eight were downregulated, during disease progression. Pathway analysis of aberrantly expressed miRNAs showed that the majority of miRNAs involved in colon cancer were also involved in other cancers. Analysis of piRNAs revealed six to be over-expressed in the tumor vs benign cohort and 24 in the metastasis vs benign group. Only two piRNAs were shared between the two cohorts. Examining other types of small RNAs [sn/snoRNAs, mt_rRNA, miscRNA, nonsense mediated decay (NMD), and rRNAs] identified 15 sncRNAs in the tumor vs benign group and 104 in the metastasis vs benign group, with only four others being commonly expressed. Conclusion: In summary, our comprehensive analysis on publicly available small RNA-seq data identified multiple differentially expressed sncRNAs during colorectal cancer progression at different stages compared to normal colon tissue. We speculate that

  19. High-Level Accumulation of Exogenous Small RNAs Not Affecting Endogenous Small RNA Biogenesis and Function in Plants

    Institute of Scientific and Technical Information of China (English)

    SHEN Wan-xia; Neil A Smith; ZHOU Chang-yong; WANG Ming-bo

    2014-01-01

    RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide (nt) small interfering RNA (siRNA) and microRNA (miRNA). Infection of plants with viral pathogens or transformation of plants with RNA interference (RNAi) constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA (satRNA) infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the speciifc yellowing symptoms generally associated with infection by Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat). We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA (hpRNA)-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not signiifcantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no signiifcant impact on normal plant development.

  20. Towards annotating the plant epigenome: the Arabidopsis thaliana small RNA locus map.

    Science.gov (United States)

    Hardcastle, Thomas J; Müller, Sebastian Y; Baulcombe, David C

    2018-04-20

    Based on 98 public and internal small RNA high throughput sequencing libraries, we mapped small RNAs to the genome of the model organism Arabidopsis thaliana and defined loci based on their expression using an empirical Bayesian approach. The resulting loci were subsequently classified based on their genetic and epigenetic context as well as their expression properties. We present the results of this classification, which broadly conforms to previously reported divisions between transcriptional and post-transcriptional gene silencing small RNAs, and to PolIV and PolV dependencies. However, we are able to demonstrate the existence of further subdivisions in the small RNA population of functional significance. Moreover, we present a framework for similar analyses of small RNA populations in all species.

  1. Intratracheal Administration of Small Interfering RNA Targeting Fas Reduces Lung Ischemia-Reperfusion Injury.

    Science.gov (United States)

    Del Sorbo, Lorenzo; Costamagna, Andrea; Muraca, Giuseppe; Rotondo, Giuseppe; Civiletti, Federica; Vizio, Barbara; Bosco, Ornella; Martin Conte, Erica L; Frati, Giacomo; Delsedime, Luisa; Lupia, Enrico; Fanelli, Vito; Ranieri, V Marco

    2016-08-01

    Lung ischemia-reperfusion injury is the main cause of primary graft dysfunction after lung transplantation and results in increased morbidity and mortality. Fas-mediated apoptosis is one of the pathologic mechanisms involved in the development of ischemia-reperfusion injury. We hypothesized that the inhibition of Fas gene expression in lungs by intratracheal administration of small interfering RNA could reduce lung ischemia-reperfusion injury in an ex vivo model reproducing the procedural sequence of lung transplantation. Prospective, randomized, controlled experimental study. University research laboratory. C57/BL6 mice weighing 28-30 g. Ischemia-reperfusion injury was induced in lungs isolated from mice, 48 hours after treatment with intratracheal small interfering RNA targeting Fas, control small interfering RNA, or vehicle. Isolated lungs were exposed to 6 hours of cold ischemia (4°C), followed by 2 hours of warm (37°C) reperfusion with a solution containing 10% of fresh whole blood and mechanical ventilation with constant low driving pressure. Fas gene expression was significantly silenced at the level of messenger RNA and protein after ischemia-reperfusion in lungs treated with small interfering RNA targeting Fas compared with lungs treated with control small interfering RNA or vehicle. Silencing of Fas gene expression resulted in reduced edema formation (bronchoalveolar lavage protein concentration and lung histology) and improvement in lung compliance. These effects were associated with a significant reduction of pulmonary cell apoptosis of lungs treated with small interfering RNA targeting Fas, which did not affect cytokine release and neutrophil infiltration. Fas expression silencing in the lung by small interfering RNA is effective against ischemia-reperfusion injury. This approach represents a potential innovative strategy of organ preservation before lung transplantation.

  2. Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

    Science.gov (United States)

    Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

    2007-03-13

    The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

  3. Discovery and validation of Barrett's esophagus microRNA transcriptome by next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Ajay Bansal

    Full Text Available Barrett's esophagus (BE is transition from squamous to columnar mucosa as a result of gastroesophageal reflux disease (GERD. The role of microRNA during this transition has not been systematically studied.For initial screening, total RNA from 5 GERD and 6 BE patients was size fractionated. RNA <70 nucleotides was subjected to SOLiD 3 library preparation and next generation sequencing (NGS. Bioinformatics analysis was performed using R package "DEseq". A p value<0.05 adjusted for a false discovery rate of 5% was considered significant. NGS-identified miRNA were validated using qRT-PCR in an independent group of 40 GERD and 27 BE patients. MicroRNA expression of human BE tissues was also compared with three BE cell lines.NGS detected 19.6 million raw reads per sample. 53.1% of filtered reads mapped to miRBase version 18. NGS analysis followed by qRT-PCR validation found 10 differentially expressed miRNA; several are novel (-708-5p, -944, -224-5p and -3065-5p. Up- or down- regulation predicted by NGS was matched by qRT-PCR in every case. Human BE tissues and BE cell lines showed a high degree of concordance (70-80% in miRNA expression. Prediction analysis identified targets that mapped to developmental signaling pathways such as TGFβ and Notch and inflammatory pathways such as toll-like receptor signaling and TGFβ. Cluster analysis found similarly regulated (up or down miRNA to share common targets suggesting coordination between miRNA.Using highly sensitive next-generation sequencing, we have performed a comprehensive genome wide analysis of microRNA in BE and GERD patients. Differentially expressed miRNA between BE and GERD have been further validated. Expression of miRNA between BE human tissues and BE cell lines are highly correlated. These miRNA should be studied in biological models to further understand BE development.

  4. RNA-Seq of the nucleolus reveals abundant SNORD44-derived small RNAs.

    Directory of Open Access Journals (Sweden)

    Baoyan Bai

    Full Text Available Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA. Sequences from 47 sdRNAs were identified, which mapped to both 5' and 3' ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha-mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.

  5. Systemic delivery of siRNA in pumpkin by a plant PHLOEM SMALL RNA-BINDING PROTEIN 1-ribonucleoprotein complex.

    Science.gov (United States)

    Ham, Byung-Kook; Li, Gang; Jia, Weitao; Leary, Julie A; Lucas, William J

    2014-11-01

    In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense-related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL-RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem-localized protein kinase, PSRPK1. During long-distance transport, PSRP1-sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1-sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  6. The origin and effect of small RNA signaling in plants

    Directory of Open Access Journals (Sweden)

    Jean-Sébastien eParent

    2012-08-01

    Full Text Available Given their sessile condition, land plants need to integrate environmental cues rapidly and send signal throughout the organism to modify their metabolism accordingly. Small RNA (sRNA molecules are among the messengers that plant cells use to carry such signals. These molecules originate from fold-back stem-loops transcribed from endogenous loci or from perfect double-stranded RNA produced through the action of RNA-dependent RNA polymerases. Once produced, sRNAs associate with Argonaute and other proteins to form the RNA-induced silencing complex (RISC that executes silencing of complementary RNA molecules. Depending on the nature of the RNA target and the Argonaute protein involved, RISC triggers either DNA methylation and chromatin modification (leading to transcriptional gene silencing, TGS or RNA cleavage or translational inhibition (leading to post-transcriptional gene silencing, PTGS. In some cases, sRNAs move to neighboring cells and/or to the vascular tissues for long-distance trafficking. Many genes are involved in the biogenesis of sRNAs and recent studies have shown that both their origin and their protein partners have great influence on their activity and range. Here we summarize the work done to uncover the mode of action of the different classes of small RNA with special emphasis on their movement and how plants can take advantage of their mobility. We also review the various genetic requirements needed for production, movement and perception of the silencing signal.

  7. smallWig: parallel compression of RNA-seq WIG files.

    Science.gov (United States)

    Wang, Zhiying; Weissman, Tsachy; Milenkovic, Olgica

    2016-01-15

    We developed a new lossless compression method for WIG data, named smallWig, offering the best known compression rates for RNA-seq data and featuring random access functionalities that enable visualization, summary statistics analysis and fast queries from the compressed files. Our approach results in order of magnitude improvements compared with bigWig and ensures compression rates only a fraction of those produced by cWig. The key features of the smallWig algorithm are statistical data analysis and a combination of source coding methods that ensure high flexibility and make the algorithm suitable for different applications. Furthermore, for general-purpose file compression, the compression rate of smallWig approaches the empirical entropy of the tested WIG data. For compression with random query features, smallWig uses a simple block-based compression scheme that introduces only a minor overhead in the compression rate. For archival or storage space-sensitive applications, the method relies on context mixing techniques that lead to further improvements of the compression rate. Implementations of smallWig can be executed in parallel on different sets of chromosomes using multiple processors, thereby enabling desirable scaling for future transcriptome Big Data platforms. The development of next-generation sequencing technologies has led to a dramatic decrease in the cost of DNA/RNA sequencing and expression profiling. RNA-seq has emerged as an important and inexpensive technology that provides information about whole transcriptomes of various species and organisms, as well as different organs and cellular communities. The vast volume of data generated by RNA-seq experiments has significantly increased data storage costs and communication bandwidth requirements. Current compression tools for RNA-seq data such as bigWig and cWig either use general-purpose compressors (gzip) or suboptimal compression schemes that leave significant room for improvement. To substantiate

  8. Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

    Directory of Open Access Journals (Sweden)

    Kiran Zahid

    2015-01-01

    Full Text Available Satellite RNAs (satRNAs are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS transgene fused with a Cucumber mosaic virus (CMV Y satellite RNA (Y-Sat sequence (35S-GUS:Sat was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

  9. Genome-wide Annotation, Identification, and Global Transcriptomic Analysis of Regulatory or Small RNA Gene Expression in Staphylococcus aureus.

    Science.gov (United States)

    Carroll, Ronan K; Weiss, Andy; Broach, William H; Wiemels, Richard E; Mogen, Austin B; Rice, Kelly C; Shaw, Lindsey N

    2016-02-09

    In Staphylococcus aureus, hundreds of small regulatory or small RNAs (sRNAs) have been identified, yet this class of molecule remains poorly understood and severely understudied. sRNA genes are typically absent from genome annotation files, and as a consequence, their existence is often overlooked, particularly in global transcriptomic studies. To facilitate improved detection and analysis of sRNAs in S. aureus, we generated updated GenBank files for three commonly used S. aureus strains (MRSA252, NCTC 8325, and USA300), in which we added annotations for >260 previously identified sRNAs. These files, the first to include genome-wide annotation of sRNAs in S. aureus, were then used as a foundation to identify novel sRNAs in the community-associated methicillin-resistant strain USA300. This analysis led to the discovery of 39 previously unidentified sRNAs. Investigating the genomic loci of the newly identified sRNAs revealed a surprising degree of inconsistency in genome annotation in S. aureus, which may be hindering the analysis and functional exploration of these elements. Finally, using our newly created annotation files as a reference, we perform a global analysis of sRNA gene expression in S. aureus and demonstrate that the newly identified tsr25 is the most highly upregulated sRNA in human serum. This study provides an invaluable resource to the S. aureus research community in the form of our newly generated annotation files, while at the same time presenting the first examination of differential sRNA expression in pathophysiologically relevant conditions. Despite a large number of studies identifying regulatory or small RNA (sRNA) genes in Staphylococcus aureus, their annotation is notably lacking in available genome files. In addition to this, there has been a considerable lack of cross-referencing in the wealth of studies identifying these elements, often leading to the same sRNA being identified multiple times and bearing multiple names. In this work

  10. MET-2-Dependent H3K9 Methylation Suppresses Transgenerational Small RNA Inheritance.

    Science.gov (United States)

    Lev, Itamar; Seroussi, Uri; Gingold, Hila; Bril, Roberta; Anava, Sarit; Rechavi, Oded

    2017-04-24

    In C. elegans, alterations to chromatin produce transgenerational effects, such as inherited increase in lifespan and gradual loss of fertility. Inheritance of histone modifications can be induced by double-stranded RNA-derived heritable small RNAs. Here, we show that the mortal germline phenotype, which is typical of met-2 mutants, defective in H3K9 methylation, depends on HRDE-1, an argonaute that carries small RNAs across generations, and is accompanied by accumulated transgenerational misexpression of heritable small RNAs. We discovered that MET-2 inhibits small RNA inheritance, and, as a consequence, induction of RNAi in met-2 mutants leads to permanent RNAi responses that do not terminate even after more than 30 generations. We found that potentiation of heritable RNAi in met-2 animals results from global hyperactivation of the small RNA inheritance machinery. Thus, changes in histone modifications can give rise to drastic transgenerational epigenetic effects, by controlling the overall potency of small RNA inheritance. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

    Science.gov (United States)

    Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

    2010-07-30

    Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.

  12. Thermodynamic control of small RNA-mediated gene silencing

    Directory of Open Access Journals (Sweden)

    Kumiko eUi-Tei

    2012-06-01

    Full Text Available Small interfering RNAs (siRNAs and microRNAs (miRNAs are crucial regulators of posttranscriptional gene silencing, which is referred to as RNA interference (RNAi or RNA silencing. In RNAi, siRNA loaded onto the RNA-induced silencing complex (RISC downregulates target gene expression by cleaving mRNA whose sequence is perfectly complementary to the siRNA guide strand. We previously showed that highly functional siRNAs possessed the following characteristics: A or U residues at nucleotide position 1 measured from the 5’ terminal, four to seven A/Us in positions 1–7, and G or C residues at position 19. This finding indicated that an RNA strand with a thermodynamically unstable 5’ terminal is easily retained in the RISC and functions as a guide strand. In addition, it is clear that unintended genes with complementarities only in the seed region (positions 2–8 are also downregulated by off-target effects. siRNA efficiency is mainly determined by the Watson-Crick base-pairing stability formed between the siRNA seed region and target mRNA. siRNAs with a low seed-target duplex melting temperature (Tm have little or no seed-dependent off-target activity. Thus, important parts of the RNA silencing machinery may be regulated by nucleotide base-pairing thermodynamic stability. A mechanistic understanding of thermodynamic control may enable an efficient target gene-specific RNAi for functional genomics and safe therapeutic applications.

  13. Responses of mRNA expression of PepT1 in small intestine to ...

    African Journals Online (AJOL)

    To study the effect of circulation small peptides concentration on mRNA expression in small intestine, graded amount of soybean small peptides (SSP) were infused into lactating goats through duodenal fistulas. Peptide-bound amino acid (PBAA) concentration in arterial plasma and the mRNA expression of PepT1 was ...

  14. Hybridization-based reconstruction of small non-coding RNA transcripts from deep sequencing data.

    Science.gov (United States)

    Ragan, Chikako; Mowry, Bryan J; Bauer, Denis C

    2012-09-01

    Recent advances in RNA sequencing technology (RNA-Seq) enables comprehensive profiling of RNAs by producing millions of short sequence reads from size-fractionated RNA libraries. Although conventional tools for detecting and distinguishing non-coding RNAs (ncRNAs) from reference-genome data can be applied to sequence data, ncRNA detection can be improved by harnessing the full information content provided by this new technology. Here we present NorahDesk, the first unbiased and universally applicable method for small ncRNAs detection from RNA-Seq data. NorahDesk utilizes the coverage-distribution of small RNA sequence data as well as thermodynamic assessments of secondary structure to reliably predict and annotate ncRNA classes. Using publicly available mouse sequence data from brain, skeletal muscle, testis and ovary, we evaluated our method with an emphasis on the performance for microRNAs (miRNAs) and piwi-interacting small RNA (piRNA). We compared our method with Dario and mirDeep2 and found that NorahDesk produces longer transcripts with higher read coverage. This feature makes it the first method particularly suitable for the prediction of both known and novel piRNAs.

  15. Small RNA expression and strain specificity in the rat

    Directory of Open Access Journals (Sweden)

    de Bruijn Ewart

    2010-04-01

    Full Text Available Abstract Background Digital gene expression (DGE profiling has become an established tool to study RNA expression. Here, we provide an in-depth analysis of small RNA DGE profiles from two different rat strains (BN-Lx and SHR from six different rat tissues (spleen, liver, brain, testis, heart, kidney. We describe the expression patterns of known and novel micro (miRNAs and piwi-interacting (piRNAs. Results We confirmed the expression of 588 known miRNAs (54 in antisense orientation and identified 56 miRNAs homologous to known human or mouse miRNAs, as well as 45 new rat miRNAs. Furthermore, we confirmed specific A to I editing in brain for mir-376a/b/c and identified mir-377 as a novel editing target. In accordance with earlier findings, we observed a highly tissue-specific expression pattern for all tissues analyzed. The brain was found to express the highest number of tissue-specific miRNAs, followed by testis. Notably, our experiments also revealed robust strain-specific differential miRNA expression in the liver that is caused by genetic variation between the strains. Finally, we identified two types of germline-specific piRNAs in testis, mapping either to transposons or in strand-specific clusters. Conclusions Taken together, the small RNA compendium described here advances the annotation of small RNAs in the rat genome. Strain and tissue-specific expression patterns furthermore provide a strong basis for studying the role of small RNAs in regulatory networks as well as biological process like physiology and neurobiology that are extensively studied in this model system.

  16. DSAP: deep-sequencing small RNA analysis pipeline.

    Science.gov (United States)

    Huang, Po-Jung; Liu, Yi-Chung; Lee, Chi-Ching; Lin, Wei-Chen; Gan, Richie Ruei-Chi; Lyu, Ping-Chiang; Tang, Petrus

    2010-07-01

    DSAP is an automated multiple-task web service designed to provide a total solution to analyzing deep-sequencing small RNA datasets generated by next-generation sequencing technology. DSAP uses a tab-delimited file as an input format, which holds the unique sequence reads (tags) and their corresponding number of copies generated by the Solexa sequencing platform. The input data will go through four analysis steps in DSAP: (i) cleanup: removal of adaptors and poly-A/T/C/G/N nucleotides; (ii) clustering: grouping of cleaned sequence tags into unique sequence clusters; (iii) non-coding RNA (ncRNA) matching: sequence homology mapping against a transcribed sequence library from the ncRNA database Rfam (http://rfam.sanger.ac.uk/); and (iv) known miRNA matching: detection of known miRNAs in miRBase (http://www.mirbase.org/) based on sequence homology. The expression levels corresponding to matched ncRNAs and miRNAs are summarized in multi-color clickable bar charts linked to external databases. DSAP is also capable of displaying miRNA expression levels from different jobs using a log(2)-scaled color matrix. Furthermore, a cross-species comparative function is also provided to show the distribution of identified miRNAs in different species as deposited in miRBase. DSAP is available at http://dsap.cgu.edu.tw.

  17. Novel dual small-molecule HIV inhibitors: scaffolds and discovery strategies.

    Science.gov (United States)

    Song, Anran; Yu, Haiqing; Wang, Changyuan; Zhu, Xingqi; Liu, Kexin; Ma, Xiaodong

    2015-01-01

    Searching for safe and effective treatments for HIV infection is still a great challenge worldwide in spite of the 27 marketed anti-HIV drugs and the powerful highly active antiretroviral therapy (HAART). As a promising prospect for generation of new HIV therapy drugs, multiple ligands (MDLs) were greatly focused on recently due to their lower toxicity, simplified dosing and patient adherence than single-target drugs. Till now, by disrupting two active sites or steps of HIV replications, a number of HIV dual inhibitors, such as CD4-gssucap120 inhibitors, CXCR4-gp20 inhibitors, RT-CXCR4 inhibitors, RT-protease inhibitors, RT-integrase inhibitors, and RTassociated functions inhibitors have been identified. Generally, these dual inhibitors were discovered mainly through screening approaches and design strategies. Of these compounds, the molecules bearing small skeletons exhibited strong anti-HIV activity and aroused great attention recently. Reviewing the progress of the dual small-molecule HIV inhibitors from the point of view of their scaffolds and discovery strategies will provide valuable information for producing more effective anti-HIV drugs. In this regard, novel dual small-molecule HIV inhibitors were illustrated, and their discovery paradigms as the major contents were also summarized in this manuscript.

  18. Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs.

    Directory of Open Access Journals (Sweden)

    Noah Fahlgren

    Full Text Available In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.

  19. Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs.

    Science.gov (United States)

    Fahlgren, Noah; Bollmann, Stephanie R; Kasschau, Kristin D; Cuperus, Josh T; Press, Caroline M; Sullivan, Christopher M; Chapman, Elisabeth J; Hoyer, J Steen; Gilbert, Kerrigan B; Grünwald, Niklaus J; Carrington, James C

    2013-01-01

    In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.

  20. Phytophthora Have Distinct Endogenous Small RNA Populations That Include Short Interfering and microRNAs

    Science.gov (United States)

    Fahlgren, Noah; Bollmann, Stephanie R.; Kasschau, Kristin D.; Cuperus, Josh T.; Press, Caroline M.; Sullivan, Christopher M.; Chapman, Elisabeth J.; Hoyer, J. Steen; Gilbert, Kerrigan B.; Grünwald, Niklaus J.; Carrington, James C.

    2013-01-01

    In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work. PMID:24204767

  1. Slicer-independent mechanism drives small-RNA strand separation during human RISC assembly.

    Science.gov (United States)

    Park, June Hyun; Shin, Chanseok

    2015-10-30

    Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1-4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that 'slicer-dependent unwinding' via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that 'slicer-independent unwinding' plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that 'slicer-dependency' for the unwinding was affected primarily by certain parameters such as temperature and Mg(2+). We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Small Molecule Drug Discovery at the Glucagon-Like Peptide-1 Receptor

    Directory of Open Access Journals (Sweden)

    Francis S. Willard

    2012-01-01

    Full Text Available The therapeutic success of peptide glucagon-like peptide-1 (GLP-1 receptor agonists for the treatment of type 2 diabetes mellitus has inspired discovery efforts aimed at developing orally available small molecule GLP-1 receptor agonists. Although the GLP-1 receptor is a member of the structurally complex class B1 family of GPCRs, in recent years, a diverse array of orthosteric and allosteric nonpeptide ligands has been reported. These compounds include antagonists, agonists, and positive allosteric modulators with intrinsic efficacy. In this paper, a comprehensive review of currently disclosed small molecule GLP-1 receptor ligands is presented. In addition, examples of “ligand bias” and “probe dependency” for the GLP-1 receptor are discussed; these emerging concepts may influence further optimization of known molecules or persuade designs of expanded screening strategies to identify novel chemical starting points for GLP-1 receptor drug discovery.

  3. The small RNA complement of adult Schistosoma haematobium.

    Directory of Open Access Journals (Sweden)

    Andreas J Stroehlein

    2018-05-01

    Full Text Available Blood flukes of the genus Schistosoma cause schistosomiasis-a neglected tropical disease (NTD that affects more than 200 million people worldwide. Studies of schistosome genomes have improved our understanding of the molecular biology of flatworms, but most of them have focused largely on protein-coding genes. Small non-coding RNAs (sncRNAs have been explored in selected schistosome species and are suggested to play essential roles in the post-transcriptional regulation of genes, and in modulating flatworm-host interactions. However, genome-wide small RNA data are currently lacking for key schistosomes including Schistosoma haematobium-the causative agent of urogenital schistosomiasis of humans.MicroRNAs (miRNAs and other sncRNAs of male and female adults of S. haematobium and small RNA transcription levels were explored by deep sequencing, genome mapping and detailed bioinformatic analyses.In total, 89 transcribed miRNAs were identified in S. haematobium-a similar complement to those reported for the congeners S. mansoni and S. japonicum. Of these miRNAs, 34 were novel, with no homologs in other schistosomes. Most miRNAs (n = 64 exhibited sex-biased transcription, suggestive of roles in sexual differentiation, pairing of adult worms and reproductive processes. Of the sncRNAs that were not miRNAs, some related to the spliceosome (n = 21, biogenesis of other RNAs (n = 3 or ribozyme functions (n = 16, whereas most others (n = 3798 were novel ('orphans' with unknown functions.This study provides the first genome-wide sncRNA resource for S. haematobium, extending earlier studies of schistosomes. The present work should facilitate the future curation and experimental validation of sncRNA functions in schistosomes to enhance our understanding of post-transcriptional gene regulation and of the roles that sncRNAs play in schistosome reproduction, development and parasite-host cross-talk.

  4. The small RNA complement of adult Schistosoma haematobium.

    Science.gov (United States)

    Stroehlein, Andreas J; Young, Neil D; Korhonen, Pasi K; Hall, Ross S; Jex, Aaron R; Webster, Bonnie L; Rollinson, David; Brindley, Paul J; Gasser, Robin B

    2018-05-01

    Blood flukes of the genus Schistosoma cause schistosomiasis-a neglected tropical disease (NTD) that affects more than 200 million people worldwide. Studies of schistosome genomes have improved our understanding of the molecular biology of flatworms, but most of them have focused largely on protein-coding genes. Small non-coding RNAs (sncRNAs) have been explored in selected schistosome species and are suggested to play essential roles in the post-transcriptional regulation of genes, and in modulating flatworm-host interactions. However, genome-wide small RNA data are currently lacking for key schistosomes including Schistosoma haematobium-the causative agent of urogenital schistosomiasis of humans. MicroRNAs (miRNAs) and other sncRNAs of male and female adults of S. haematobium and small RNA transcription levels were explored by deep sequencing, genome mapping and detailed bioinformatic analyses. In total, 89 transcribed miRNAs were identified in S. haematobium-a similar complement to those reported for the congeners S. mansoni and S. japonicum. Of these miRNAs, 34 were novel, with no homologs in other schistosomes. Most miRNAs (n = 64) exhibited sex-biased transcription, suggestive of roles in sexual differentiation, pairing of adult worms and reproductive processes. Of the sncRNAs that were not miRNAs, some related to the spliceosome (n = 21), biogenesis of other RNAs (n = 3) or ribozyme functions (n = 16), whereas most others (n = 3798) were novel ('orphans') with unknown functions. This study provides the first genome-wide sncRNA resource for S. haematobium, extending earlier studies of schistosomes. The present work should facilitate the future curation and experimental validation of sncRNA functions in schistosomes to enhance our understanding of post-transcriptional gene regulation and of the roles that sncRNAs play in schistosome reproduction, development and parasite-host cross-talk.

  5. RNA targeting by small molecules: Binding of protoberberine ...

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... Studies on RNA targeting by small molecules to specifically control certain cellular functions is an .... form secondary structures such as stem-loop, hairpin, etc. ..... paired third strand of the triplex without affecting the stability.

  6. From early lessons to new frontiers: the worm as a treasure trove of small RNA biology.

    Science.gov (United States)

    Youngman, Elaine M; Claycomb, Julie M

    2014-01-01

    In the past 20 years, the tiny soil nematode Caenorhabditis elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNA interference pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology.

  7. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    Science.gov (United States)

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. © 2015 Lama and Ryan; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  8. RNA isolation for transcriptomics of human and mouse small skin biopsies

    Directory of Open Access Journals (Sweden)

    Breit Timo M

    2011-10-01

    Full Text Available Abstract Background Isolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. As we lacked the dedicated equipment, i.e. homogenizer or bead-beater, needed for the available RNA from skin isolation methods, we adapted and tested our zebrafish single-embryo RNA-isolation protocol for RNA isolation from skin punch biopsies. Findings We tested our new RNA-isolation protocol in two experiments: a large-scale study with 97 human skin samples, and a small study with 16 mouse skin samples. Human skin was sampled with 4.0 mm biopsy punches and for the mouse skin different punch diameter sizes were tested; 1.0, 1.5, 2.0, and 2.5 mm. The average RNA yield in human samples was 1.5 μg with an average RNA quality RIN value of 8.1. For the mouse biopsies, the average RNA yield was 2.4 μg with an average RIN value of 7.5. For 96% of the human biopsies and 100% of the mouse biopsies we obtained enough high-quality RNA. The RNA samples were successfully tested in a transcriptomics analysis using the Affymetrix and Roche NimbleGen platforms. Conclusions Using our new RNA-isolation protocol, we were able to consistently isolate high-quality RNA, which is apt for further transcriptomics analysis. Furthermore, this method is already useable on biopsy material obtained with a punch diameter as small as 1.5 mm.

  9. Transposable-element associated small RNAs in Bombyx mori genome.

    Directory of Open Access Journals (Sweden)

    Yimei Cai

    Full Text Available Small RNAs are a group of regulatory RNA molecules that control gene expression at transcriptional or post-transcriptional levels among eukaryotes. The silkworm, Bombyx mori L., genome harbors abundant repetitive sequences derived from families of retrotransposons and transposons, which together constitute almost half of the genome space and provide ample resource for biogenesis of the three major small RNA families. We systematically discovered transposable-element (TE-associated small RNAs in B. mori genome based on a deep RNA-sequencing strategy and the effort yielded 182, 788 and 4,990 TE-associated small RNAs in the miRNA, siRNA and piRNA species, respectively. Our analysis suggested that the three small RNA species preferentially associate with different TEs to create sequence and functional diversity, and we also show evidence that a Bombyx non-LTR retrotransposon, bm1645, alone contributes to the generation of TE-associated small RNAs in a very significant way. The fact that bm1645-associated small RNAs partially overlap with each other implies a possibility that this element may be modulated by different mechanisms to generate different products with diverse functions. Taken together, these discoveries expand the small RNA pool in B. mori genome and lead to new knowledge on the diversity and functional significance of TE-associated small RNAs.

  10. From early lessons to new frontiers: The worm as a treasure trove of small RNA biology

    Directory of Open Access Journals (Sweden)

    Elaine M. Youngman

    2014-11-01

    Full Text Available In the past twenty years, the tiny soil nematode C. elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNAi pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology.

  11. The LncRNA Connectivity Map: Using LncRNA Signatures to Connect Small Molecules, LncRNAs, and Diseases.

    Science.gov (United States)

    Yang, Haixiu; Shang, Desi; Xu, Yanjun; Zhang, Chunlong; Feng, Li; Sun, Zeguo; Shi, Xinrui; Zhang, Yunpeng; Han, Junwei; Su, Fei; Li, Chunquan; Li, Xia

    2017-07-27

    Well characterized the connections among diseases, long non-coding RNAs (lncRNAs) and drugs are important for elucidating the key roles of lncRNAs in biological mechanisms in various biological states. In this study, we constructed a database called LNCmap (LncRNA Connectivity Map), available at http://www.bio-bigdata.com/LNCmap/ , to establish the correlations among diseases, physiological processes, and the action of small molecule therapeutics by attempting to describe all biological states in terms of lncRNA signatures. By reannotating the microarray data from the Connectivity Map database, the LNCmap obtained 237 lncRNA signatures of 5916 instances corresponding to 1262 small molecular drugs. We provided a user-friendly interface for the convenient browsing, retrieval and download of the database, including detailed information and the associations of drugs and corresponding affected lncRNAs. Additionally, we developed two enrichment analysis methods for users to identify candidate drugs for a particular disease by inputting the corresponding lncRNA expression profiles or an associated lncRNA list and then comparing them to the lncRNA signatures in our database. Overall, LNCmap could significantly improve our understanding of the biological roles of lncRNAs and provide a unique resource to reveal the connections among drugs, lncRNAs and diseases.

  12. Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ.

    Science.gov (United States)

    Mank, Nils N; Berghoff, Bork A; Hermanns, Yannick N; Klug, Gabriele

    2012-10-02

    The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

  13. Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa.

    Science.gov (United States)

    Morin, Ryan D; Aksay, Gozde; Dolgosheina, Elena; Ebhardt, H Alexander; Magrini, Vincent; Mardis, Elaine R; Sahinalp, S Cenk; Unrau, Peter J

    2008-04-01

    The diversity of microRNAs and small-interfering RNAs has been extensively explored within angiosperms by focusing on a few key organisms such as Oryza sativa and Arabidopsis thaliana. A deeper division of the plants is defined by the radiation of the angiosperms and gymnosperms, with the latter comprising the commercially important conifers. The conifers are expected to provide important information regarding the evolution of highly conserved small regulatory RNAs. Deep sequencing provides the means to characterize and quantitatively profile small RNAs in understudied organisms such as these. Pyrosequencing of small RNAs from O. sativa revealed, as expected, approximately 21- and approximately 24-nt RNAs. The former contained known microRNAs, and the latter largely comprised intergenic-derived sequences likely representing heterochromatin siRNAs. In contrast, sequences from Pinus contorta were dominated by 21-nt small RNAs. Using a novel sequence-based clustering algorithm, we identified sequences belonging to 18 highly conserved microRNA families in P. contorta as well as numerous clusters of conserved small RNAs of unknown function. Using multiple methods, including expressed sequence folding and machine learning algorithms, we found a further 53 candidate novel microRNA families, 51 appearing specific to the P. contorta library. In addition, alignment of small RNA sequences to the O. sativa genome revealed six perfectly conserved classes of small RNA that included chloroplast transcripts and specific types of genomic repeats. The conservation of microRNAs and other small RNAs between the conifers and the angiosperms indicates that important RNA silencing processes were highly developed in the earliest spermatophytes. Genomic mapping of all sequences to the O. sativa genome can be viewed at http://microrna.bcgsc.ca/cgi-bin/gbrowse/rice_build_3/.

  14. Small RNA sequencing reveals metastasis-related microRNAs in lung adenocarcinoma

    DEFF Research Database (Denmark)

    Daugaard, Iben; Venø, Morten T.; Yan, Yan

    2017-01-01

    The majority of lung cancer deaths are caused by metastatic disease. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and miRNA dysregulation can contribute to metastatic progression. Here, small RNA sequencing was used to profile the miRNA and piwi-interacting RNA (piRNA......) transcriptomes in relation to lung cancer metastasis. RNA-seq was performed using RNA extracted from formalin-fixed paraffin embedded (FFPE) lung adenocarcinomas (LAC) and brain metastases from 8 patients, and LACs from 8 patients without detectable metastatic disease. Impact on miRNA and piRNA transcriptomes...... was subtle with 9 miRNAs and 8 piRNAs demonstrating differential expression between metastasizing and non-metastasizing LACs. For piRNAs, decreased expression of piR-57125 was the most significantly associated with distant metastasis. Validation by RT-qPCR in a LAC cohort comprising 52 patients confirmed...

  15. Advances in targeted delivery of small interfering RNA using simple bioconjugates

    DEFF Research Database (Denmark)

    Nielsen, Christoffer; Kjems, Jørgen; Sorensen, Kristine Rothaus

    2014-01-01

    with a targeting moiety, in a simple bioconjugate construct. We discuss the use of different types of targeting moieties, as well as the different conjugation strategies employed for preparing these bioconjugate constructs that deliver the siRNA to target cells. We focus especially on the in-built or passive......Introduction: Development of drugs based on RNA interference by small interfering RNA (siRNA) has been progressing slowly due to a number of challenges associated with the in vivo behavior of siRNA. A central problem is controlling siRNA delivery to specific cell types. Here, we review existing...... literature on one type of strategy for solving the issue of cell-specific delivery of siRNA, namely delivering the siRNA as part of simple bioconjugate constructs. Areas covered: This review presents current experience from strategies aimed at targeting siRNA to specific cell types, by associating the siRNA...

  16. Translational regulation of gene expression by an anaerobically induced small non-coding RNA in Escherichia coli

    DEFF Research Database (Denmark)

    Boysen, Anders; Møller-Jensen, Jakob; Kallipolitis, Birgitte H.

    2010-01-01

    Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post...... that adaptation to anaerobic growth involves the action of a small regulatory RNA....... of at least one sRNA regulator. Here, we extend this view by the identification and characterization of a highly conserved, anaerobically induced small sRNA in E. coli, whose expression is strictly dependent on the anaerobic transcriptional fumarate and nitrate reductase regulator (FNR). The sRNA, named Fnr...

  17. Methods for small RNA preparation for digital gene expression profiling by next-generation sequencing

    NARCIS (Netherlands)

    Linsen, S.E.V.; Cuppen, E.

    2012-01-01

    Digital gene expression (DGE) profiling techniques are playing an eminent role in the detection, localization, and differential expression quantification of many small RNA species, including microRNAs (1-3). Procedures in small RNA library preparation techniques typically include adapter ligation by

  18. Approaches to Validate and Manipulate RNA Targets with Small Molecules in Cells.

    Science.gov (United States)

    Childs-Disney, Jessica L; Disney, Matthew D

    2016-01-01

    RNA has become an increasingly important target for therapeutic interventions and for chemical probes that dissect and manipulate its cellular function. Emerging targets include human RNAs that have been shown to directly cause cancer, metabolic disorders, and genetic disease. In this review, we describe various routes to obtain bioactive compounds that target RNA, with a particular emphasis on the development of small molecules. We use these cases to describe approaches that are being developed for target validation, which include target-directed cleavage, classic pull-down experiments, and covalent cross-linking. Thus, tools are available to design small molecules to target RNA and to identify the cellular RNAs that are their targets.

  19. Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles.

    Science.gov (United States)

    Wieben, E D; Nenninger, J M; Pederson, T

    1985-05-05

    Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.

  20. Final report for ER65039, The Role of Small RNA in Biomass Deposition

    Energy Technology Data Exchange (ETDEWEB)

    Hudson, Matthew E. [Univ. of Illinois, Urbana, IL (United States)

    2015-03-12

    Our objective in this project was to discover the role of sRNA in regulating both biomass biosynthesis and perenniality in the Andropogoneae feedstock grasses. Our central hypothesis was that there is a time-and space specific sRNA network playing a crucial role in regulating processes associated with cell wall biosynthesis, flowering time control, overwintering/juvenility, and nutrient sequestration in the feedstock grasses. To address this, we performed a large scale biological project consisting of the growth of material, generation of Illumina libraries, sequencing and analysis for small RNA, mRNA and Degradome / cmRNA. Our subsidiary objectives included analysis of the biology of small RNAs and the cell wall composition of Miscanthus. These objectives have all been completed, one publication is in print, one is submitted and several more are in progress.

  1. Radiolabeling small RNA with technetium-99m for visualizing cellular delivery and mouse biodistribution

    International Nuclear Information System (INIS)

    Liu Ning; Ding Hongliu; Vanderheyden, Jean-Luc; Zhu Zhihong; Zhang Yumin

    2007-01-01

    To develop a noninvasive direct method for the in vivo tracking of small interfering RNA (siRNA) used in RNA interference, two 18-nucleotide oligoribonucleotides were radiolabeled with technetium-99m ( 99m Tc-RNA). The ability of 99m Tc-RNA to track delivery was tested in cultured cells and living mice. The cellular delivery of 99m Tc-RNAs could be quantified by gamma counting and could be visualized by microautoradiography. Radiolabeled RNAs can be efficiently delivered into cells by reaching up to 3x10 5 molecules of small RNAs per cell. Moreover, RNAs were internalized with homogeneous distribution throughout the cytoplasm and nucleus. In tumor-bearing mice, whole-body images and biodistribution studies showed that 99m Tc-RNAs were delivered to almost all tissues after intravenous injection. The imaging of living animals allowed noninvasive and longitudinal monitoring of the in vivo delivery of these small RNAs. In conclusion, using 99m Tc radiolabeling, the delivery of small RNAs could be measured quantitatively in cultured cells and could be noninvasively visualized in living animals using a gamma camera. The results of this study could open up a new approach for measuring the in vivo delivery of small RNAs that might further facilitate the development of siRNAs as targeted therapies

  2. Using small RNA (sRNA) deep sequencing to understand global virus distribution in plants

    Science.gov (United States)

    Small RNAs (sRNAs), a class of regulatory RNAs, have been used to serve as the specificity determinants of suppressing gene expression in plants and animals. Next generation sequencing (NGS) uncovered the sRNA landscape in most organisms including their associated microbes. In the current study, w...

  3. Small interfering RNA delivery through positively charged polymer nanoparticles

    International Nuclear Information System (INIS)

    Dragoni, Luca; Cesana, Alberto; Moscatelli, Davide; Ferrari, Raffaele; Morbidelli, Massimo; Lupi, Monica; Falcetta, Francesca; Ubezio, Paolo; D’Incalci, Maurizio

    2016-01-01

    Small interfering RNA (siRNA) is receiving increasing attention with regard to the treatment of many genetic diseases, both acquired and hereditary, such as cancer and diabetes. Being a high molecular weight (MW) polyanion, siRNA is not able to cross a cell membrane, and in addition it is unstable in physiological conditions. Accordingly, a biocompatible nanocarrier able to deliver siRNA into cells is needed. In this work, we synthesized biocompatible positively charged nanoparticles (NPs) following a two-step process that involves ring opening polymerization (ROP) and emulsion free radical polymerization (EFRP). Firstly, we proved the possibility of fine tuning the NPs’ characteristics (e.g. size and surface charge) by changing the synthetic process parameters. Then the capability in loading and delivering undamaged siRNA into a cancer cell cytoplasm has been shown. This latter process occurs through the biodegradation of the polymer constituting the NPs, whose kinetics can be tuned by adjusting the polymer’s MW. Finally, the ability of NPs to carry siRNA inside the cells in order to inhibit their target gene has been demonstrated using green flourescent protein positive cells. (paper)

  4. Modeling the structure of RNA molecules with small-angle X-ray scattering data.

    Directory of Open Access Journals (Sweden)

    Michal Jan Gajda

    Full Text Available We propose a novel fragment assembly method for low-resolution modeling of RNA and show how it may be used along with small-angle X-ray solution scattering (SAXS data to model low-resolution structures of particles having as many as 12 independent secondary structure elements. We assessed this model-building procedure by using both artificial data on a previously proposed benchmark and publicly available data. With the artificial data, SAXS-guided models show better similarity to native structures than ROSETTA decoys. The publicly available data showed that SAXS-guided models can be used to reinterpret RNA structures previously deposited in the Protein Data Bank. Our approach allows for fast and efficient building of de novo models of RNA using approximate secondary structures that can be readily obtained from existing bioinformatic approaches. We also offer a rigorous assessment of the resolving power of SAXS in the case of small RNA structures, along with a small multimetric benchmark of the proposed method.

  5. Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages.

    Directory of Open Access Journals (Sweden)

    Peter Sarkies

    2015-02-01

    Full Text Available Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs. Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements.

  6. Diverse evolutionary trajectories for small RNA biogenesis genes in the oomycete genus Phytophthora

    Directory of Open Access Journals (Sweden)

    Stephanie eBollmann

    2016-03-01

    Full Text Available Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL, and RNA-dependent RNA polymerase (RDR through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P. sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed.

  7. Seryl-tRNA Synthetases in Translation and Beyond

    Directory of Open Access Journals (Sweden)

    Marko Močibob

    2016-06-01

    Full Text Available For a long time seryl-tRNA synthetases (SerRSs stood as an archetypal, canonical aminoacyl-tRNA synthetases (aaRS, exhibiting only basic tRNA aminoacylation activity and with no moonlighting functions beyond protein biosynthesis. The picture has changed substantially in recent years after the discovery that SerRSs play an important role in antibiotic production and resistance and act as a regulatory factor in vascular development, as well as after the discovery of mitochondrial morphogenesis factor homologous to SerRS in insects. In this review we summarize the recent research results from our laboratory, which advance the understanding of seryl-tRNA synthetases and further paint the dynamic picture of unexpected SerRS activities. SerRS from archaeon Methanothermobacter thermautotrophicus was shown to interact with the large ribosomal subunit and it was postulated to contribute to a more efficient translation by the"tRNA channeling" hypothesis. Discovery of the atypical SerRS in a small number of methanogenic archaea led to the discovery of a new family of enzymes in numerous bacteria - amino acid:[carrier protein] ligases (aa:CP ligases. These SerRS homologues resigned tRNA aminoacylation activity, and instead adopted carrier proteins as the acceptors of activated amino acids. The crystal structure of the aa:CP ligase complex with the carrier protein revealed that the interactions between two macromolecules are incomparable to tRNA binding by the aaRS and consequently represent a true evolutionary invention. Kinetic investigations of SerRSs and the accuracy of amino acid selection revealed that SerRSs possess pre-transfer proofreading activity, challenging the widely accepted presumption that hydrolytic proofreading activity must reside in an additional, separate editing domain, not present in SerRSs. Finally, the plant tRNA serylation system is discussed, which is particularly interesting due to the fact that protein biosynthesis takes place

  8. Mirnovo: genome-free prediction of microRNAs from small RNA sequencing data and single-cells using decision forests.

    Science.gov (United States)

    Vitsios, Dimitrios M; Kentepozidou, Elissavet; Quintais, Leonor; Benito-Gutiérrez, Elia; van Dongen, Stijn; Davis, Matthew P; Enright, Anton J

    2017-12-01

    The discovery of microRNAs (miRNAs) remains an important problem, particularly given the growth of high-throughput sequencing, cell sorting and single cell biology. While a large number of miRNAs have already been annotated, there may well be large numbers of miRNAs that are expressed in very particular cell types and remain elusive. Sequencing allows us to quickly and accurately identify the expression of known miRNAs from small RNA-Seq data. The biogenesis of miRNAs leads to very specific characteristics observed in their sequences. In brief, miRNAs usually have a well-defined 5' end and a more flexible 3' end with the possibility of 3' tailing events, such as uridylation. Previous approaches to the prediction of novel miRNAs usually involve the analysis of structural features of miRNA precursor hairpin sequences obtained from genome sequence. We surmised that it may be possible to identify miRNAs by using these biogenesis features observed directly from sequenced reads, solely or in addition to structural analysis from genome data. To this end, we have developed mirnovo, a machine learning based algorithm, which is able to identify known and novel miRNAs in animals and plants directly from small RNA-Seq data, with or without a reference genome. This method performs comparably to existing tools, however is simpler to use with reduced run time. Its performance and accuracy has been tested on multiple datasets, including species with poorly assembled genomes, RNaseIII (Drosha and/or Dicer) deficient samples and single cells (at both embryonic and adult stage). © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo

    NARCIS (Netherlands)

    Mook, Olaf R.; Baas, Frank; de Wissel, Marit B.; Fluiter, Kees

    2007-01-01

    RNA interference has become widely used as an experimental tool to study gene function. In addition, small interfering RNA (siRNA) may have great potential for the treatment of diseases. Recently, it was shown that siRNA can be used to mediate gene silencing in mouse models. Locally administered

  10. StarScan: a web server for scanning small RNA targets from degradome sequencing data.

    Science.gov (United States)

    Liu, Shun; Li, Jun-Hao; Wu, Jie; Zhou, Ke-Ren; Zhou, Hui; Yang, Jian-Hua; Qu, Liang-Hu

    2015-07-01

    Endogenous small non-coding RNAs (sRNAs), including microRNAs, PIWI-interacting RNAs and small interfering RNAs, play important gene regulatory roles in animals and plants by pairing to the protein-coding and non-coding transcripts. However, computationally assigning these various sRNAs to their regulatory target genes remains technically challenging. Recently, a high-throughput degradome sequencing method was applied to identify biologically relevant sRNA cleavage sites. In this study, an integrated web-based tool, StarScan (sRNA target Scan), was developed for scanning sRNA targets using degradome sequencing data from 20 species. Given a sRNA sequence from plants or animals, our web server performs an ultrafast and exhaustive search for potential sRNA-target interactions in annotated and unannotated genomic regions. The interactions between small RNAs and target transcripts were further evaluated using a novel tool, alignScore. A novel tool, degradomeBinomTest, was developed to quantify the abundance of degradome fragments located at the 9-11th nucleotide from the sRNA 5' end. This is the first web server for discovering potential sRNA-mediated RNA cleavage events in plants and animals, which affords mechanistic insights into the regulatory roles of sRNAs. The StarScan web server is available at http://mirlab.sysu.edu.cn/starscan/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Undesired small RNAs originate from an artificial microRNA precursor in transgenic petunia (Petunia hybrida.

    Directory of Open Access Journals (Sweden)

    Yulong Guo

    Full Text Available Although artificial microRNA (amiRNA technology has been used frequently in gene silencing in plants, little research has been devoted to investigating the accuracy of amiRNA precursor processing. In this work, amiRNAchs1 (amiRchs1, based on the Arabidopsis miR319a precursor, was expressed in order to suppress the expression of CHS genes in petunia. The transgenic plants showed the CHS gene-silencing phenotype. A modified 5' RACE technique was used to map small-RNA-directed cleavage sites and to detect processing intermediates of the amiRchs1 precursor. The results showed that the target CHS mRNAs were cut at the expected sites and that the amiRchs1 precursor was processed from loop to base. The accumulation of small RNAs in amiRchs1 transgenic petunia petals was analyzed using the deep-sequencing technique. The results showed that, alongside the accumulation of the desired artificial microRNAs, additional small RNAs that originated from other regions of the amiRNA precursor were also accumulated at high frequency. Some of these had previously been found to be accumulated at low frequency in the products of ath-miR319a precursor processing and some of them were accompanied by 3'-tailing variant. Potential targets of the undesired small RNAs were discovered in petunia and other Solanaceae plants. The findings draw attention to the potential occurrence of undesired target silencing induced by such additional small RNAs when amiRNA technology is used. No appreciable production of secondary small RNAs occurred, despite the fact that amiRchs1 was designed to have perfect complementarity to its CHS-J target. This confirmed that perfect pairing between an amiRNA and its targets is not the trigger for secondary small RNA production. In conjunction with the observation that amiRNAs with perfect complementarity to their target genes show high efficiency and specificity in gene silencing, this finding has an important bearing on future applications of ami

  12. Undesired small RNAs originate from an artificial microRNA precursor in transgenic petunia (Petunia hybrida).

    Science.gov (United States)

    Guo, Yulong; Han, Yao; Ma, Jing; Wang, Huiping; Sang, Xianchun; Li, Mingyang

    2014-01-01

    Although artificial microRNA (amiRNA) technology has been used frequently in gene silencing in plants, little research has been devoted to investigating the accuracy of amiRNA precursor processing. In this work, amiRNAchs1 (amiRchs1), based on the Arabidopsis miR319a precursor, was expressed in order to suppress the expression of CHS genes in petunia. The transgenic plants showed the CHS gene-silencing phenotype. A modified 5' RACE technique was used to map small-RNA-directed cleavage sites and to detect processing intermediates of the amiRchs1 precursor. The results showed that the target CHS mRNAs were cut at the expected sites and that the amiRchs1 precursor was processed from loop to base. The accumulation of small RNAs in amiRchs1 transgenic petunia petals was analyzed using the deep-sequencing technique. The results showed that, alongside the accumulation of the desired artificial microRNAs, additional small RNAs that originated from other regions of the amiRNA precursor were also accumulated at high frequency. Some of these had previously been found to be accumulated at low frequency in the products of ath-miR319a precursor processing and some of them were accompanied by 3'-tailing variant. Potential targets of the undesired small RNAs were discovered in petunia and other Solanaceae plants. The findings draw attention to the potential occurrence of undesired target silencing induced by such additional small RNAs when amiRNA technology is used. No appreciable production of secondary small RNAs occurred, despite the fact that amiRchs1 was designed to have perfect complementarity to its CHS-J target. This confirmed that perfect pairing between an amiRNA and its targets is not the trigger for secondary small RNA production. In conjunction with the observation that amiRNAs with perfect complementarity to their target genes show high efficiency and specificity in gene silencing, this finding has an important bearing on future applications of amiRNAs in gene

  13. Creation of transgenic rice plants producing small interfering RNA of Rice tungro spherical virus.

    Science.gov (United States)

    Le, Dung Tien; Chu, Ha Duc; Sasaya, Takahide

    2015-01-01

    Rice tungro spherical virus (RTSV), also known as Rice waika virus, does not cause visible symptoms in infected rice plants. However, the virus plays a critical role in spreading Rice tungro bacilliform virus (RTBV), which is the major cause of severe symptoms of rice tungro disease. Recent studies showed that RNA interference (RNAi) can be used to develop virus-resistance transgenic rice plants. In this report, we presented simple procedures and protocols needed for the creation of transgenic rice plants capable of producing small interfering RNA specific against RTSV sequences. Notably, our study showed that 60 out of 64 individual hygromycin-resistant lines (putative transgenic lines) obtained through transformation carried transgenes designed for producing hairpin double-stranded RNA. Northern blot analyses revealed the presence of small interfering RNA of 21- to 24-mer in 46 out of 56 confirmed transgenic lines. Taken together, our study indicated that transgenic rice plants carrying an inverted repeat of 500-bp fragments encoding various proteins of RTSV can produce small interfering RNA from the hairpin RNA transcribed from that transgene. In light of recent studies with other viruses, it is possible that some of these transgenic rice lines might be resistant to RTSV.

  14. Species-independent MicroRNA Gene Discovery

    KAUST Repository

    Kamanu, Timothy K.

    2012-01-01

    and other incurable diseases such as autism and Alzheimer’s. Functional miRNAs are excised from hairpin-like sequences that are known as miRNA genes. There are about 21,000 known miRNA genes, most of which have been determined using experimental methods. mi

  15. Identification of Bacterial Small RNAs by RNA Sequencing

    DEFF Research Database (Denmark)

    Gómez Lozano, María; Marvig, Rasmus Lykke; Molin, Søren

    2014-01-01

    sequencing (RNA-seq) is described that involves the preparation and analysis of three different sequencing libraries. As a signifi cant number of unique sRNAs are identifi ed in each library, the libraries can be used either alone or in combination to increase the number of sRNAs identifi ed. The approach......Small regulatory RNAs (sRNAs) in bacteria are known to modulate gene expression and control a variety of processes including metabolic reactions, stress responses, and pathogenesis in response to environmental signals. A method to identify bacterial sRNAs on a genome-wide scale based on RNA...... may be applied to identify sRNAs in any bacterium under different growth and stress conditions....

  16. Small RNA-Controlled Gene Regulatory Networks in Pseudomonas putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara

    evolved numerous mechanisms to controlgene expression in response to specific environmental signals. In addition to two-component systems, small regulatory RNAs (sRNAs) have emerged as major regulators of gene expression. The majority of sRNAs bind to mRNA and regulate their expression. They often have...... multiple targets and are incorporated into large regulatory networks and the RNA chaper one Hfq in many cases facilitates interactions between sRNAs and their targets. Some sRNAs also act by binding to protein targets and sequestering their function. In this PhD thesis we investigated the transcriptional....... Detailed insights into the mechanisms through which P. putida responds to different stress conditions and increased understanding of bacterial adaptation in natural and industrial settings were gained. Additionally, we identified genome-wide transcription start sites, andmany regulatory RNA elements...

  17. Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

    Science.gov (United States)

    Mars, Ruben A T; Nicolas, Pierre; Ciccolini, Mariano; Reilman, Ewoud; Reder, Alexander; Schaffer, Marc; Mäder, Ulrike; Völker, Uwe; van Dijl, Jan Maarten; Denham, Emma L

    2015-03-01

    Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions.

  18. Characterization of the Zika virus induced small RNA response in Aedes aegypti cells.

    Directory of Open Access Journals (Sweden)

    Margus Varjak

    2017-10-01

    Full Text Available RNA interference (RNAi controls arbovirus infections in mosquitoes. Two different RNAi pathways are involved in antiviral responses: the PIWI-interacting RNA (piRNA and exogenous short interfering RNA (exo-siRNA pathways, which are characterized by the production of virus-derived small RNAs of 25-29 and 21 nucleotides, respectively. The exo-siRNA pathway is considered to be the key mosquito antiviral response mechanism. In Aedes aegypti-derived cells, Zika virus (ZIKV-specific siRNAs were produced and loaded into the exo-siRNA pathway effector protein Argonaute 2 (Ago2; although the knockdown of Ago2 did not enhance virus replication. Enhanced ZIKV replication was observed in a Dcr2-knockout cell line suggesting that the exo-siRNA pathway is implicated in the antiviral response. Although ZIKV-specific piRNA-sized small RNAs were detected, these lacked the characteristic piRNA ping-pong signature motif and were bound to Ago3 but not Piwi5 or Piwi6. Silencing of PIWI proteins indicated that the knockdown of Ago3, Piwi5 or Piwi6 did not enhance ZIKV replication and only Piwi4 displayed antiviral activity. We also report that the expression of ZIKV capsid (C protein amplified the replication of a reporter alphavirus; although, unlike yellow fever virus C protein, it does not inhibit the exo-siRNA pathway. Our findings elucidate ZIKV-mosquito RNAi interactions that are important for understanding its spread.

  19. Synergistic Effect of Auto-Activation and Small RNA Regulation on Gene Expression

    Science.gov (United States)

    Xiong, Li-Ping; Ma, Yu-Qiang; Tang, Lei-Han

    2010-09-01

    Auto-activation and small ribonucleic acid (RNA)-mediated regulation are two important mechanisms in controlling gene expression. We study the synergistic effect of these two regulations on gene expression. It is found that under this combinatorial regulation, gene expression exhibits bistable behaviors at the transition regime, while each of these two regulations, if working solely, only leads to monostability. Within the stochastic framework, the base pairing strength between sRNA and mRNA plays an important role in controlling the transition time between on and off states. The noise strength of protein number in the off state approaches 1 and is smaller than that in the on state. The noise strength also depends on which parameters, the feedback strength or the synthesis rate of small RNA, are tuned in switching the gene expression on and off. Our findings may provide a new insight into gene-regulation mechanism and can be applied in synthetic biology.

  20. Synergistic Effect of Auto-Activation and Small RNA Regulation on Gene Expression

    International Nuclear Information System (INIS)

    Li-Ping, Xiong; Yu-Qiang, Ma; Lei-Han, Tang

    2010-01-01

    Auto-activation and small ribonucleic acid (RNA)-mediated regulation are two important mechanisms in controlling gene expression. We study the synergistic effect of these two regulations on gene expression. It is found that under this combinatorial regulation, gene expression exhibits bistable behaviors at the transition regime, while each of these two regulations, if working solely, only leads to monostability. Within the stochastic framework, the base pairing strength between sRNA and mRNA plays an important role in controlling the transition time between on and off states. The noise strength of protein number in the off state approaches 1 and is smaller than that in the on state. The noise strength also depends on which parameters, the feedback strength or the synthesis rate of small RNA, are tuned in switching the gene expression on and off. Our findings may provide a new insight into gene-regulation mechanism and can be applied in synthetic biology

  1. InFusion: Advancing Discovery of Fusion Genes and Chimeric Transcripts from Deep RNA-Sequencing Data.

    Directory of Open Access Journals (Sweden)

    Konstantin Okonechnikov

    Full Text Available Analysis of fusion transcripts has become increasingly important due to their link with cancer development. Since high-throughput sequencing approaches survey fusion events exhaustively, several computational methods for the detection of gene fusions from RNA-seq data have been developed. This kind of analysis, however, is complicated by native trans-splicing events, the splicing-induced complexity of the transcriptome and biases and artefacts introduced in experiments and data analysis. There are a number of tools available for the detection of fusions from RNA-seq data; however, certain differences in specificity and sensitivity between commonly used approaches have been found. The ability to detect gene fusions of different types, including isoform fusions and fusions involving non-coding regions, has not been thoroughly studied yet. Here, we propose a novel computational toolkit called InFusion for fusion gene detection from RNA-seq data. InFusion introduces several unique features, such as discovery of fusions involving intergenic regions, and detection of anti-sense transcription in chimeric RNAs based on strand-specificity. Our approach demonstrates superior detection accuracy on simulated data and several public RNA-seq datasets. This improved performance was also evident when evaluating data from RNA deep-sequencing of two well-established prostate cancer cell lines. InFusion identified 26 novel fusion events that were validated in vitro, including alternatively spliced gene fusion isoforms and chimeric transcripts that include intergenic regions. The toolkit is freely available to download from http:/bitbucket.org/kokonech/infusion.

  2. SCRAM: a pipeline for fast index-free small RNA read alignment and visualization.

    Science.gov (United States)

    Fletcher, Stephen J; Boden, Mikael; Mitter, Neena; Carroll, Bernard J

    2018-03-15

    Small RNAs play key roles in gene regulation, defense against viral pathogens and maintenance of genome stability, though many aspects of their biogenesis and function remain to be elucidated. SCRAM (Small Complementary RNA Mapper) is a novel, simple-to-use short read aligner and visualization suite that enhances exploration of small RNA datasets. The SCRAM pipeline is implemented in Go and Python, and is freely available under MIT license. Source code, multiplatform binaries and a Docker image can be accessed via https://sfletc.github.io/scram/. s.fletcher@uq.edu.au. Supplementary data are available at Bioinformatics online.

  3. psRNATarget: a plant small RNA target analysis server (2017 release).

    Science.gov (United States)

    Dai, Xinbin; Zhuang, Zhaohong; Zhao, Patrick Xuechun

    2018-04-30

    Plant regulatory small RNAs (sRNAs), which include most microRNAs (miRNAs) and a subset of small interfering RNAs (siRNAs), such as the phased siRNAs (phasiRNAs), play important roles in regulating gene expression. Although generated from genetically distinct biogenesis pathways, these regulatory sRNAs share the same mechanisms for post-translational gene silencing and translational inhibition. psRNATarget was developed to identify plant sRNA targets by (i) analyzing complementary matching between the sRNA sequence and target mRNA sequence using a predefined scoring schema and (ii) by evaluating target site accessibility. This update enhances its analytical performance by developing a new scoring schema that is capable of discovering miRNA-mRNA interactions at higher 'recall rates' without significantly increasing total prediction output. The scoring procedure is customizable for the users to search both canonical and non-canonical targets. This update also enables transmitting and analyzing 'big' data empowered by (a) the implementation of multi-threading chunked file uploading, which can be paused and resumed, using HTML5 APIs and (b) the allocation of significantly more computing nodes to its back-end Linux cluster. The updated psRNATarget server has clear, compelling and user-friendly interfaces that enhance user experiences and present data clearly and concisely. The psRNATarget is freely available at http://plantgrn.noble.org/psRNATarget/.

  4. Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling.

    Science.gov (United States)

    Asha, Srinivasan; Sreekumar, Sweda; Soniya, E V

    2016-01-01

    Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

  5. Preparation of highly multiplexed small RNA sequencing libraries.

    Science.gov (United States)

    Persson, Helena; Søkilde, Rolf; Pirona, Anna Chiara; Rovira, Carlos

    2017-08-01

    MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.

  6. Identification of the miRNA-mRNA regulatory network of small cell osteosarcoma based on RNA-seq.

    Science.gov (United States)

    Xie, Lin; Liao, Yedan; Shen, Lida; Hu, Fengdi; Yu, Sunlin; Zhou, Yonghong; Zhang, Ya; Yang, Yihao; Li, Dongqi; Ren, Minyan; Yuan, Zhongqin; Yang, Zuozhang

    2017-06-27

    Small cell osteosarcoma (SCO) is a rare subtype of osteosarcoma characterized by highly aggressive progression and a poor prognosis. The miRNA and mRNA expression profiles of peripheral blood mononuclear cells (PBMCs) were obtained in 3 patients with SCO and 10 healthy individuals using high-throughput RNA-sequencing. We identified 37 dysregulated miRNAs and 1636 dysregulated mRNAs in patients with SCO compared to the healthy controls. Specifically, the 37 dysregulated miRNAs consisted of 27 up-regulated miRNAs and 10 down-regulated miRNAs; the 1636 dysregulated mRNAs consisted of 555 up-regulated mRNAs and 1081 down-regulated mRNAs. The target-genes of miRNAs were predicted, and 1334 negative correlations between miRNAs and mRNAs were used to construct an miRNA-mRNA regulatory network. Dysregulated genes were significantly enriched in pathways related to cancer, mTOR signaling and cell cycle signaling. Specifically, hsa-miR-26b-5p, hsa-miR-221-3p and hsa-miR-125b-2-3p were significantly dysregulated miRNAs and exhibited a high degree of connectivity with target genes. Overall, the expression of dysregulated genes in tumor tissues and peripheral blood samples of patients with SCO measured by quantitative real-time polymerase chain reaction corroborated with our bioinformatics analyses based on the expression profiles of PBMCs from patients with SCO. Thus, hsa-miR-26b-5p, hsa-miR-221-3p and hsa-miR-125b-2-3p may be involved in SCO tumorigenesis.

  7. Exploiting Pre-rRNA Processing in Diamond Blackfan Anemia Gene Discovery and Diagnosis

    Science.gov (United States)

    Farrar, Jason E.; Quarello, Paola; Fisher, Ross; O’Brien, Kelly A.; Aspesi, Anna; Parrella, Sara; Henson, Adrianna L.; Seidel, Nancy E.; Atsidaftos, Eva; Prakash, Supraja; Bari, Shahla; Garelli, Emanuela; Arceci, Robert J.; Dianzani, Irma; Ramenghi, Ugo; Vlachos, Adrianna; Lipton, Jeffrey M.; Bodine, David M.; Ellis, Steven R.

    2014-01-01

    Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single-copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre-rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre-rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre-60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene. PMID:25042156

  8. Gene expression and stress response mediated by the epigenetic regulation of a transposable element small RNA.

    Directory of Open Access Journals (Sweden)

    Andrea D McCue

    2012-02-01

    Full Text Available The epigenetic activity of transposable elements (TEs can influence the regulation of genes; though, this regulation is confined to the genes, promoters, and enhancers that neighbor the TE. This local cis regulation of genes therefore limits the influence of the TE's epigenetic regulation on the genome. TE activity is suppressed by small RNAs, which also inhibit viruses and regulate the expression of genes. The production of TE heterochromatin-associated endogenous small interfering RNAs (siRNAs in the reference plant Arabidopsis thaliana is mechanistically distinct from gene-regulating small RNAs, such as microRNAs or trans-acting siRNAs (tasiRNAs. Previous research identified a TE small RNA that potentially regulates the UBP1b mRNA, which encodes an RNA-binding protein involved in stress granule formation. We demonstrate that this siRNA, siRNA854, is under the same trans-generational epigenetic control as the Athila family LTR retrotransposons from which it is produced. The epigenetic activation of Athila elements results in a shift in small RNA processing pathways, and new 21-22 nucleotide versions of Athila siRNAs are produced by protein components normally not responsible for processing TE siRNAs. This processing results in siRNA854's incorporation into ARGONAUTE1 protein complexes in a similar fashion to gene-regulating tasiRNAs. We have used reporter transgenes to demonstrate that the UPB1b 3' untranslated region directly responds to the epigenetic status of Athila TEs and the accumulation of siRNA854. The regulation of the UPB1b 3' untranslated region occurs both on the post-transcriptional and translational levels when Athila TEs are epigenetically activated, and this regulation results in the phenocopy of the ubp1b mutant stress-sensitive phenotype. This demonstrates that a TE's epigenetic activity can modulate the host organism's stress response. In addition, the ability of this TE siRNA to regulate a gene's expression in trans blurs

  9. The early history of tRNA recognition by aminoacyl-tRNA synthetases

    Indian Academy of Sciences (India)

    Madhu

    2006-10-04

    Oct 4, 2006 ... Discovery of aminoacyl-tRNA synthetases and importance ... The pioneering work of Fritz Lipmann on the high-energy ... the peculiar structural and functional relationships tRNAs ... a bulk of only 20 families of tRNA molecules in contrast ...... balance of tRNA and aminoacyl-tRNA synthetase; Science 242.

  10. A survey of small RNA population during FR-induced apical hook opening

    Directory of Open Access Journals (Sweden)

    Ying eLi

    2014-04-01

    Full Text Available Photomorphogenesis is a mechanism employed by plants to regulate their architecture and developmental program in response to light conditions. As they emerge into light for the first time, dark-grown seedlings employ a rapid and finely-controlled photomorphogenic signaling network. Small RNAs have increasingly been revealed to play an important role in regulating multiple aspects of plant development, by modulating the stability of mRNAs. The rapid alteration of the mRNA transcriptome is a known hallmark of the de-etiolation response, thus we investigated the small RNA transcriptome during this process in specific seedling tissues. Here we describe a survey of the small RNA expression profile in four tissues of etiolated soybean seedlings, the cotyledons, hypocotyl and the convex and concave sides of the apical hook. We also investigate how this profile responds to a one-hour far-red light treatment. Our data suggests that miRNAs show a different global profile between these tissues and treatments, suggesting a possible role for tissue- and treatment-specific expression in the differential morphology of the seedling on de-etiolation. Further evidence for the role of miRNA in light-regulated development is given by the de-etiolation responses of a hypomorphic ago1 mutant, which displays reduced and delayed photomorphogenic responses in apical hook and cotyledon angle to far-red light.

  11. Optimization of miRNA-seq data preprocessing.

    Science.gov (United States)

    Tam, Shirley; Tsao, Ming-Sound; McPherson, John D

    2015-11-01

    The past two decades of microRNA (miRNA) research has solidified the role of these small non-coding RNAs as key regulators of many biological processes and promising biomarkers for disease. The concurrent development in high-throughput profiling technology has further advanced our understanding of the impact of their dysregulation on a global scale. Currently, next-generation sequencing is the platform of choice for the discovery and quantification of miRNAs. Despite this, there is no clear consensus on how the data should be preprocessed before conducting downstream analyses. Often overlooked, data preprocessing is an essential step in data analysis: the presence of unreliable features and noise can affect the conclusions drawn from downstream analyses. Using a spike-in dilution study, we evaluated the effects of several general-purpose aligners (BWA, Bowtie, Bowtie 2 and Novoalign), and normalization methods (counts-per-million, total count scaling, upper quartile scaling, Trimmed Mean of M, DESeq, linear regression, cyclic loess and quantile) with respect to the final miRNA count data distribution, variance, bias and accuracy of differential expression analysis. We make practical recommendations on the optimal preprocessing methods for the extraction and interpretation of miRNA count data from small RNA-sequencing experiments. © The Author 2015. Published by Oxford University Press.

  12. Small angle scattering study of the structure and organization of RNA and protein in Brome Mosaic Virus (BMV)

    Science.gov (United States)

    Das, Narayan C.; Warren, Garfield T.; Cheng, Si; Kao, C. Cheng; Ni, Peng; Dragnea, Bogdan; Sokol, Paul E.

    2012-02-01

    Brome mosaic virus (BMV) is a small icosahedral of the alpha virus-like superfamily of RNA with a segmented positive-strand RNA genome and a mean diameter ˜ 268å that offers high levels of RNA synthesis and virus production in plants. BMV also tightly regulates the packaging of its four RNAs (RNA1 through RNA4) into three separate particles; RNA1 and RNA2 are encapsidated separately while one copy each of RNA3 and RNA4 are normally packaged together. Small angle neutron scattering (SANS) and small angle X-ray scattering (SAXS) were applied to study the size, shape and protein-RNA organization of BMV. D2O/H2O mixture was used to enhance contrast in SANS measurement. The radial distribution of BMV from the Fourier transform of scattering spectrum gives a clear indication of RNA packing, and distribution and their structure in the BMV. The result reveals that the virus is about 266 å in diameter and is composed of RNA inside the virion coated with a protein shell.

  13. Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates.

    Science.gov (United States)

    Flores-Jasso, C Fabián; Salomon, William E; Zamore, Phillip D

    2013-02-01

    Small interfering RNAs (siRNAs) direct Argonaute proteins, the core components of the RNA-induced silencing complex (RISC), to cleave complementary target RNAs. Here, we describe a method to purify active RISC containing a single, unique small RNA guide sequence. We begin by capturing RISC using a complementary 2'-O-methyl oligonucleotide tethered to beads. Unlike other methods that capture RISC but do not allow its recovery, our strategy purifies active, soluble RISC in good yield. The method takes advantage of the finding that RISC partially paired to a target through its siRNA guide dissociates more than 300 times faster than a fully paired siRNA in RISC. We use this strategy to purify fly Ago1- and Ago2-RISC, as well as mouse AGO2-RISC. The method can discriminate among RISCs programmed with different guide strands, making it possible to deplete and recover specific RISC populations. Endogenous microRNA:Argonaute complexes can also be purified from cell lysates. Our method scales readily and takes less than a day to complete.

  14. NATpipe: an integrative pipeline for systematical discovery of natural antisense transcripts (NATs) and phase-distributed nat-siRNAs from de novo assembled transcriptomes

    Science.gov (United States)

    Yu, Dongliang; Meng, Yijun; Zuo, Ziwei; Xue, Jie; Wang, Huizhong

    2016-01-01

    Nat-siRNAs (small interfering RNAs originated from natural antisense transcripts) are a class of functional small RNA (sRNA) species discovered in both plants and animals. These siRNAs are highly enriched within the annealed regions of the NAT (natural antisense transcript) pairs. To date, great research efforts have been taken for systematical identification of the NATs in various organisms. However, developing a freely available and easy-to-use program for NAT prediction is strongly demanded by researchers. Here, we proposed an integrative pipeline named NATpipe for systematical discovery of NATs from de novo assembled transcriptomes. By utilizing sRNA sequencing data, the pipeline also allowed users to search for phase-distributed nat-siRNAs within the perfectly annealed regions of the NAT pairs. Additionally, more reliable nat-siRNA loci could be identified based on degradome sequencing data. A case study on the non-model plant Dendrobium officinale was performed to illustrate the utility of NATpipe. Finally, we hope that NATpipe would be a useful tool for NAT prediction, nat-siRNA discovery, and related functional studies. NATpipe is available at www.bioinfolab.cn/NATpipe/NATpipe.zip. PMID:26858106

  15. Small RNA-directed epigenetic natural variation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Jixian Zhai

    2008-04-01

    Full Text Available Progress in epigenetics has revealed mechanisms that can heritably regulate gene function independent of genetic alterations. Nevertheless, little is known about the role of epigenetics in evolution. This is due in part to scant data on epigenetic variation among natural populations. In plants, small interfering RNA (siRNA is involved in both the initiation and maintenance of gene silencing by directing DNA methylation and/or histone methylation. Here, we report that, in the model plant Arabidopsis thaliana, a cluster of approximately 24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC, a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities. We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression. DNA methylation of the Ler allele MPF is dependent on genes in known silencing pathways, and such methylation is transmissible to Col by genetic crosses, although with varying degrees of penetrance. A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of approximately 24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences. Methylation analysis revealed that 88% of the examined loci (37 out of 42 were specifically methylated in Ler but not Col, suggesting that small RNA can direct epigenetic differences between two closely related Arabidopsis ecotypes.

  16. Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls.

    Science.gov (United States)

    Thomas, Andreas; Walpurgis, Katja; Delahaut, Philippe; Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

    2013-01-01

    Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, 'experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls. Copyright © 2013 John Wiley & Sons, Ltd.

  17. Deep-sequencing protocols influence the results obtained in small-RNA sequencing.

    Directory of Open Access Journals (Sweden)

    Joern Toedling

    Full Text Available Second-generation sequencing is a powerful method for identifying and quantifying small-RNA components of cells. However, little attention has been paid to the effects of the choice of sequencing platform and library preparation protocol on the results obtained. We present a thorough comparison of small-RNA sequencing libraries generated from the same embryonic stem cell lines, using different sequencing platforms, which represent the three major second-generation sequencing technologies, and protocols. We have analysed and compared the expression of microRNAs, as well as populations of small RNAs derived from repetitive elements. Despite the fact that different libraries display a good correlation between sequencing platforms, qualitative and quantitative variations in the results were found, depending on the protocol used. Thus, when comparing libraries from different biological samples, it is strongly recommended to use the same sequencing platform and protocol in order to ensure the biological relevance of the comparisons.

  18. A Capture-SELEX Strategy for Multiplexed Selection of RNA Aptamers Against Small Molecules

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm; Doessing, Holger B.; Long, Katherine S.

    2018-01-01

    -SELEX, a selection strategy that uses an RNA library to yield ligand-responsive RNA aptamers targeting small organic molecules in solution. To demonstrate the power of this method we selected several aptamers with specificity towards either the natural sweetener rebaudioside A or the food-coloring agent carminic...

  19. Oasis: online analysis of small RNA deep sequencing data.

    Science.gov (United States)

    Capece, Vincenzo; Garcia Vizcaino, Julio C; Vidal, Ramon; Rahman, Raza-Ur; Pena Centeno, Tonatiuh; Shomroni, Orr; Suberviola, Irantzu; Fischer, Andre; Bonn, Stefan

    2015-07-01

    Oasis is a web application that allows for the fast and flexible online analysis of small-RNA-seq (sRNA-seq) data. It was designed for the end user in the lab, providing an easy-to-use web frontend including video tutorials, demo data and best practice step-by-step guidelines on how to analyze sRNA-seq data. Oasis' exclusive selling points are a differential expression module that allows for the multivariate analysis of samples, a classification module for robust biomarker detection and an advanced programming interface that supports the batch submission of jobs. Both modules include the analysis of novel miRNAs, miRNA targets and functional analyses including GO and pathway enrichment. Oasis generates downloadable interactive web reports for easy visualization, exploration and analysis of data on a local system. Finally, Oasis' modular workflow enables for the rapid (re-) analysis of data. Oasis is implemented in Python, R, Java, PHP, C++ and JavaScript. It is freely available at http://oasis.dzne.de. stefan.bonn@dzne.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

  20. Small RNA Deep Sequencing and the Effects of microRNA408 on Root Gravitropic Bending in Arabidopsis

    Science.gov (United States)

    Li, Huasheng; Lu, Jinying; Sun, Qiao; Chen, Yu; He, Dacheng; Liu, Min

    2015-11-01

    MicroRNA (miRNA) is a non-coding small RNA composed of 20 to 24 nucleotides that influences plant root development. This study analyzed the miRNA expression in Arabidopsis root tip cells using Illumina sequencing and real-time PCR before (sample 0) and 15 min after (sample 15) a 3-D clinostat rotational treatment was administered. After stimulation was performed, the expression levels of seven miRNA genes, including Arabidopsis miR160, miR161, miR394, miR402, miR403, miR408, and miR823, were significantly upregulated. Illumina sequencing results also revealed two novel miRNAsthat have not been previously reported, The target genes of these miRNAs included pentatricopeptide repeat-containing protein and diadenosine tetraphosphate hydrolase. An overexpression vector of Arabidopsis miR408 was constructed and transferred to Arabidopsis plant. The roots of plants over expressing miR408 exhibited a slower reorientation upon gravistimulation in comparison with those of wild-type. This result indicate that miR408 could play a role in root gravitropic response.

  1. High-throughput sequencing of RNA silencing-associated small RNAs in olive (Olea europaea L..

    Directory of Open Access Journals (Sweden)

    Livia Donaire

    Full Text Available Small RNAs (sRNAs of 20 to 25 nucleotides (nt in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.. sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive.

  2. High throughput sequencing of small RNA component of leaves and inflorescence revealed conserved and novel miRNAs as well as phasiRNA loci in chickpea.

    Science.gov (United States)

    Srivastava, Sangeeta; Zheng, Yun; Kudapa, Himabindu; Jagadeeswaran, Guru; Hivrale, Vandana; Varshney, Rajeev K; Sunkar, Ramanjulu

    2015-06-01

    Among legumes, chickpea (Cicer arietinum L.) is the second most important crop after soybean. MicroRNAs (miRNAs) play important roles by regulating target gene expression important for plant development and tolerance to stress conditions. Additionally, recently discovered phased siRNAs (phasiRNAs), a new class of small RNAs, are abundantly produced in legumes. Nevertheless, little is known about these regulatory molecules in chickpea. The small RNA population was sequenced from leaves and flowers of chickpea to identify conserved and novel miRNAs as well as phasiRNAs/phasiRNA loci. Bioinformatics analysis revealed 157 miRNA loci for the 96 highly conserved and known miRNA homologs belonging to 38 miRNA families in chickpea. Furthermore, 20 novel miRNAs belonging to 17 miRNA families were identified. Sequence analysis revealed approximately 60 phasiRNA loci. Potential target genes likely to be regulated by these miRNAs were predicted and some were confirmed by modified 5' RACE assay. Predicted targets are mostly transcription factors that might be important for developmental processes, and others include superoxide dismutases, plantacyanin, laccases and F-box proteins that could participate in stress responses and protein degradation. Overall, this study provides an inventory of miRNA-target gene interactions for chickpea, useful for the comparative analysis of small RNAs among legumes. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  3. Application of small RNA technology for improved control of parasitic helminths.

    Science.gov (United States)

    Britton, Collette; Winter, Alan D; Marks, Neil D; Gu, Henry; McNeilly, Tom N; Gillan, Victoria; Devaney, Eileen

    2015-08-15

    Over the last decade microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important regulators of post-transcriptional gene expression. miRNAs are short, non-coding RNAs that regulate a variety of processes including cancer, organ development and immune function. This class of small RNAs bind with partial complementarity to their target mRNA sequences, most often in the 3'UTR, to negatively regulate gene expression. In parasitic helminths, miRNAs are being increasingly studied for their potential roles in development and host-parasite interactions. The availability of genome data, combined with small RNA sequencing, has paved the way to profile miRNAs expressed at particular developmental stages for many parasitic helminths. While some miRNAs are conserved across species, others appear to be unique to specific parasites, suggesting important roles in adaptation and survival in the host environment. Some miRNAs are released from parasites, in exosomes or in protein complexes, and the potential effects of these on host immune function are being increasingly studied. In addition, release of miRNAs from schistosome and filarial parasites into host plasma can be exploited for the development of specific and sensitive diagnostic biomarkers of infection. Interfering with miRNA function, as well as silencing key components of the pathways they regulate, will progress our understanding of parasite development and provide a novel approach to therapeutic control. RNA interference (RNAi) by siRNAs has proven to be inconsistent in parasitic nematodes. However, the recent successes reported for schistosome and liver fluke RNAi, encourage further efforts to enhance delivery of RNA and improve in vitro culture systems and assays to monitor phenotypic effects in nematodes. These improvements are important for the establishment of reliable functional genomic platforms for novel drug and vaccine development. In this review we focus on the important roles of mi

  4. P2X7 mRNA expression in non-small cell lung cancer: MicroRNA regulation and prognostic value

    OpenAIRE

    BOLDRINI, LAURA; GIORDANO, MIRELLA; ALÌ, GRETA; MELFI, FRANCA; ROMANO, GAETANO; LUCCHI, MARCO; FONTANINI, GABRIELLA

    2014-01-01

    The human P2X7 receptor is significant and exhibits several functions in neoplasia. At present, little is known with regard to its regulation. P2X7 expression may be regulated post-transcriptionally and putative microRNA (miRNA) binding sites are considered to be involved. The aim of this study was to determine whether miRNAs (miR-21, let-7 g and miR-205) regulate P2X7 mRNA stability. In addition, the impact of P2X7 expression in patients with non-small cell lung cancer (NSCLC) was investigat...

  5. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown

    Science.gov (United States)

    Moore, Chris B.; Guthrie, Elizabeth H.; Huang, Max Tze-Han; Taxman, Debra J.

    2013-01-01

    Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. PMID:20387148

  6. MicroRNA discovery and analysis of pinewood nematode Bursaphelenchus xylophilus by deep sequencing.

    Directory of Open Access Journals (Sweden)

    Qi-Xing Huang

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are considered to be very important in regulating the growth, development, behavior and stress response in animals and plants in post-transcriptional gene regulation. Pinewood nematode, Bursaphelenchus xylophilus, is an important invasive plant parasitic nematode in Asia. To have a comprehensive knowledge about miRNAs of the nematode is necessary for further in-depth study on roles of miRNAs in the ecological adaptation of the invasive species. METHODS AND FINDINGS: Five small RNA libraries were constructed and sequenced by Illumina/Solexa deep-sequencing technology. A total of 810 miRNA candidates (49 conserved and 761 novel were predicted by a computational pipeline, of which 57 miRNAs (20 conserved and 37 novel encoded by 53 miRNA precursors were identified by experimental methods. Ten novel miRNAs were considered to be species-specific miRNAs of B. xylophilus. Comparison of expression profiles of miRNAs in the five small RNA libraries showed that many miRNAs exhibited obviously different expression levels in the third-stage dispersal juvenile and at a cold-stressed status. Most of the miRNAs exhibited obviously down-regulated expression in the dispersal stage. But differences among the three geographic libraries were not prominent. A total of 979 genes were predicted to be targets of these authentic miRNAs. Among them, seven heat shock protein genes were targeted by 14 miRNAs, and six FMRFamide-like neuropeptides genes were targeted by 17 miRNAs. A real-time quantitative polymerase chain reaction was used to quantify the mRNA expression levels of target genes. CONCLUSIONS: Basing on the fact that a negative correlation existed between the expression profiles of miRNAs and the mRNA expression profiles of their target genes (hsp, flp by comparing those of the nematodes at a cold stressed status and a normal status, we suggested that miRNAs might participate in ecological adaptation and behavior regulation of the

  7. History, Discovery, and Classification of lncRNAs.

    Science.gov (United States)

    Jarroux, Julien; Morillon, Antonin; Pinskaya, Marina

    2017-01-01

    The RNA World Hypothesis suggests that prebiotic life revolved around RNA instead of DNA and proteins. Although modern cells have changed significantly in 4 billion years, RNA has maintained its central role in cell biology. Since the discovery of DNA at the end of the nineteenth century, RNA has been extensively studied. Many discoveries such as housekeeping RNAs (rRNA, tRNA, etc.) supported the messenger RNA model that is the pillar of the central dogma of molecular biology, which was first devised in the late 1950s. Thirty years later, the first regulatory non-coding RNAs (ncRNAs) were initially identified in bacteria and then in most eukaryotic organisms. A few long ncRNAs (lncRNAs) such as H19 and Xist were characterized in the pre-genomic era but remained exceptions until the early 2000s. Indeed, when the sequence of the human genome was published in 2001, studies showed that only about 1.2% encodes proteins, the rest being deemed "non-coding." It was later shown that the genome is pervasively transcribed into many ncRNAs, but their functionality remained controversial. Since then, regulatory lncRNAs have been characterized in many species and were shown to be involved in processes such as development and pathologies, revealing a new layer of regulation in eukaryotic cells. This newly found focus on lncRNAs, together with the advent of high-throughput sequencing, was accompanied by the rapid discovery of many novel transcripts which were further characterized and classified according to specific transcript traits.In this review, we will discuss the many discoveries that led to the study of lncRNAs, from Friedrich Miescher's "nuclein" in 1869 to the elucidation of the human genome and transcriptome in the early 2000s. We will then focus on the biological relevance during lncRNA evolution and describe their basic features as genes and transcripts. Finally, we will present a non-exhaustive catalogue of lncRNA classes, thus illustrating the vast complexity of

  8. Strategies for Discovery of Small Molecule Radiation Protectors and Radiation Mitigators

    Directory of Open Access Journals (Sweden)

    Joel S Greenberger

    2012-01-01

    Full Text Available Mitochondrial targeted radiation damage protectors (delivered prior to irradiation and mitigators (delivered after irradiation, but before the appearance of symptoms associated with radiation syndrome have been a recent focus in drug discovery for 1 normal tissue radiation protection during fractionated radiotherapy, and 2 radiation terrorism counter measures. Several categories of such molecules have been discovered: nitroxide-linked hybrid molecules, including GS-nitroxide, GS-nitric oxide synthase inhibitors, p53/mdm2/mdm4 inhibitors, and pharmaceutical agents including inhibitors of the phosphoinositide-3-kinase pathway and the anti-seizure medicine, carbamazepine. Evaluation of potential new irradiation dose modifying molecules to protect normal tissue includes: clonagenic radiation survival curves; assays for apoptosis and DNA repair, and irradiation-induced depletion of antioxidant stores. Studies of organ specific radioprotection and in total body irradiation-induced hematopoietic syndrome in the mouse model for protection/mitigation facilitate rational means by which to move candidate small molecule drugs along the drug discovery pipeline into clinical development.

  9. Strategies for Discovery of Small Molecule Radiation Protectors and Radiation Mitigators

    Energy Technology Data Exchange (ETDEWEB)

    Greenberger, Joel S.; Clump, David [Radiation Oncology Department, University of Pittsburgh Cancer Institute, Pittsburgh, PA (United States); Kagan, Valerian [Environmental and Occupational Health Department, University of Pittsburgh, Pittsburgh, PA (United States); Bayir, Hülya [Critical Care Medicine Department, University of Pittsburgh Medical Center, Pittsburgh, PA (United States); Lazo, John S. [Pharmacology Department, University of Virginia, Charlottesville, VA (United States); Wipf, Peter [Department of Chemistry, Accelerated Chemical Discovery Center, University of Pittsburgh, Pittsburgh, PA (United States); Li, Song; Gao, Xiang [Pharmaceutical Science Department, University of Pittsburgh, Pittsburgh, PA (United States); Epperly, Michael W., E-mail: greenbergerjs@upmc.edu [Radiation Oncology Department, University of Pittsburgh Cancer Institute, Pittsburgh, PA (United States)

    2012-01-13

    Mitochondrial targeted radiation damage protectors (delivered prior to irradiation) and mitigators (delivered after irradiation, but before the appearance of symptoms associated with radiation syndrome) have been a recent focus in drug discovery for (1) normal tissue radiation protection during fractionated radiotherapy, and (2) radiation terrorism counter measures. Several categories of such molecules have been discovered: nitroxide-linked hybrid molecules, including GS-nitroxide, GS-nitric oxide synthase inhibitors, p53/mdm2/mdm4 inhibitors, and pharmaceutical agents including inhibitors of the phosphoinositide-3-kinase pathway and the anti-seizure medicine, carbamazepine. Evaluation of potential new radiation dose modifying molecules to protect normal tissue includes: clonogenic radiation survival curves, assays for apoptosis and DNA repair, and irradiation-induced depletion of antioxidant stores. Studies of organ specific radioprotection and in total body irradiation-induced hematopoietic syndrome in the mouse model for protection/mitigation facilitate rational means by which to move candidate small molecule drugs along the drug discovery pipeline into clinical development.

  10. Mycoplasma non-coding RNA: identification of small RNAs and targets

    Directory of Open Access Journals (Sweden)

    Franciele Maboni Siqueira

    2016-10-01

    Full Text Available Abstract Background Bacterial non-coding RNAs act by base-pairing as regulatory elements in crucial biological processes. We performed the identification of trans-encoded small RNAs (sRNA from the genomes of Mycoplama hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis, which are Mycoplasma species that have been identified in the porcine respiratory system. Results A total of 47, 15 and 11 putative sRNAs were predicted in M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively. A comparative genomic analysis revealed the presence of species or lineage specific sRNA candidates. Furthermore, the expression profile of some M. hyopneumoniae sRNAs was determined by a reverse transcription amplification approach, in three different culture conditions. All tested sRNAs were transcribed in at least one condition. A detailed investigation revealed a differential expression profile for two M. hyopneumoniae sRNAs in response to oxidative and heat shock stress conditions, suggesting that their expression is influenced by environmental signals. Moreover, we analyzed sRNA-mRNA hybrids and accessed putative target genes for the novel sRNA candidates. The majority of the sRNAs showed interaction with multiple target genes, some of which could be linked to pathogenesis and cell homeostasis activity. Conclusion This study contributes to our knowledge of Mycoplasma sRNAs and their response to environmental changes. Furthermore, the mRNA target prediction provides a perspective for the characterization and comprehension of the function of the sRNA regulatory mechanisms.

  11. Biomarker MicroRNAs for Diagnosis of Oral Squamous Cell Carcinoma Identified Based on Gene Expression Data and MicroRNA-mRNA Network Analysis

    Science.gov (United States)

    Zhang, Hui; Li, Tangxin; Zheng, Linqing

    2017-01-01

    Oral squamous cell carcinoma is one of the most malignant tumors with high mortality rate worldwide. Biomarker discovery is critical for early diagnosis and precision treatment of this disease. MicroRNAs are small noncoding RNA molecules which often regulate essential biological processes and are good candidates for biomarkers. By integrative analysis of both the cancer-associated gene expression data and microRNA-mRNA network, miR-148b-3p, miR-629-3p, miR-27a-3p, and miR-142-3p were screened as novel diagnostic biomarkers for oral squamous cell carcinoma based on their unique regulatory abilities in the network structure of the conditional microRNA-mRNA network and their important functions. These findings were confirmed by literature verification and functional enrichment analysis. Future experimental validation is expected for the further investigation of their molecular mechanisms. PMID:29098014

  12. MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer

    OpenAIRE

    Minxia Liu; Kecheng Zhou; Yi Cao

    2016-01-01

    MicroRNAs (miRNAs) have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC) and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D) and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfectio...

  13. Diversity of antisense and other non-coding RNAs in Archaea revealed by comparative small RNA sequencing in four Pyrobaculum species

    Directory of Open Access Journals (Sweden)

    David L Bernick

    2012-07-01

    Full Text Available A great diversity of small, non-coding RNA molecules with roles in gene regulation and RNA processing have been intensely studied in eukaryotic and bacterial model organisms, yet our knowledge of possible parallel roles for small RNAs in archaea is limited. We employed RNA-seq to identify novel small RNA across multiple species of the hyperthermophilic genus Pyrobaculum, known for unusual RNA gene characteristics. By comparing transcriptional data collected in parallel among four species, we were able to identify conserved RNA genes fitting into known and novel families. Among our findings, we highlight three novel cis-antisense small RNAs encoded opposite to key regulatory (ferric uptake regulator, metabolic (triose-phosphate isomerase, and core transcriptional apparatus genes (transcription factor B. We also found a large increase in the number of conserved C/D box small RNA genes over what had been previously recognized; many of these genes are encoded antisense to protein coding genes. The conserved opposition to orthologous genes across the Pyrobaculum genus suggests similarities to other cis-antisense regulatory systems. Furthermore, the genus-specific nature of these small RNAs indicates they are relatively recent, stable adaptations.

  14. In vivo efficacy and off-target effects of locked nucleic acid (LNA) and unlocked nucleic acid (UNA) modified siRNA and small internally segmented interfering RNA (sisiRNA) in mice bearing human tumor xenografts

    NARCIS (Netherlands)

    Mook, O. R. F.; Vreijling, Jeroen; Wengel, Suzy L.; Wengel, Jesper; Zhou, Chuanzheng; Chattopadhyaya, Jyoti; Baas, Frank; Fluiter, Kees

    2010-01-01

    The clinical use of small interfering RNA (siRNA) is hampered by poor uptake by tissues and instability in circulation. In addition, off-target effects pose a significant additional problem for therapeutic use of siRNA. Chemical modifications of siRNA have been reported to increase stability and

  15. A systems biology approach for miRNA-mRNA expression patterns analysis in non-small cell lung cancer.

    Science.gov (United States)

    Najafi, Ali; Tavallaei, Mahmood; Hosseini, Sayed Mostafa

    2016-01-01

    Non-small cell lung cancers (NSCLCs) is a prevalent and heterogeneous subtype of lung cancer accounting for 85 percent of patients. MicroRNAs (miRNAs), a class of small endogenous non-coding RNAs, incorporate into regulation of gene expression post-transcriptionally. Therefore, deregulation of miRNAs' expression has provided further layers of complexity to the molecular etiology and pathogenesis of different diseases and malignancies. Although, until now considerable number of studies has been carried out to illuminate this complexity in NSCLC, they have remained less effective in their goal due to lack of a holistic and integrative systems biology approach which considers all natural elaborations of miRNAs' function. It is able to reliably nominate most affected signaling pathways and therapeutic target genes by deregulated miRNAs during a particular pathological condition. Herein, we utilized a holistic systems biology approach, based on appropriate re-analyses of microarray datasets followed by reliable data filtering, to analyze integrative and combinatorial deregulated miRNA-mRNA interaction network in NSCLC, aiming to ascertain miRNA-dysregulated signaling pathway and potential therapeutic miRNAs and mRNAs which represent a lion' share during various aspects of NSCLC's pathogenesis. Our systems biology approach introduced and nominated 1) important deregulated miRNAs in NSCLCs compared with normal tissue 2) significant and confident deregulated mRNAs which were anti-correlatively targeted by deregulated miRNA in NSCLCs and 3) dysregulated signaling pathways in association with deregulated miRNA-mRNAs interactions in NSCLCs. These results introduce possible mechanism of function of deregulated miRNAs and mRNAs in NSCLC that could be used as potential therapeutic targets.

  16. Post-transcriptional bursting in genes regulated by small RNA molecules

    Science.gov (United States)

    Rodrigo, Guillermo

    2018-03-01

    Gene expression programs in living cells are highly dynamic due to spatiotemporal molecular signaling and inherent biochemical stochasticity. Here we study a mechanism based on molecule-to-molecule variability at the RNA level for the generation of bursts of protein production, which can lead to heterogeneity in a cell population. We develop a mathematical framework to show numerically and analytically that genes regulated post transcriptionally by small RNA molecules can exhibit such bursts due to different states of translation activity (on or off), mostly revealed in a regime of few molecules. We exploit this framework to compare transcriptional and post-transcriptional bursting and also to illustrate how to tune the resulting protein distribution with additional post-transcriptional regulations. Moreover, because RNA-RNA interactions are predictable with an energy model, we define the kinetic constants of on-off switching as functions of the two characteristic free-energy differences of the system, activation and formation, with a nonequilibrium scheme. Overall, post-transcriptional bursting represents a distinctive principle linking gene regulation to gene expression noise, which highlights the importance of the RNA layer beyond the simple information transfer paradigm and significantly contributes to the understanding of the intracellular processes from a first-principles perspective.

  17. sRNAtoolboxVM: Small RNA Analysis in a Virtual Machine.

    Science.gov (United States)

    Gómez-Martín, Cristina; Lebrón, Ricardo; Rueda, Antonio; Oliver, José L; Hackenberg, Michael

    2017-01-01

    High-throughput sequencing (HTS) data for small RNAs (noncoding RNA molecules that are 20-250 nucleotides in length) can now be routinely generated by minimally equipped wet laboratories; however, the bottleneck in HTS-based research has shifted now to the analysis of such huge amount of data. One of the reasons is that many analysis types require a Linux environment but computers, system administrators, and bioinformaticians suppose additional costs that often cannot be afforded by small to mid-sized groups or laboratories. Web servers are an alternative that can be used if the data is not subjected to privacy issues (what very often is an important issue with medical data). However, in any case they are less flexible than stand-alone programs limiting the number of workflows and analysis types that can be carried out.We show in this protocol how virtual machines can be used to overcome those problems and limitations. sRNAtoolboxVM is a virtual machine that can be executed on all common operating systems through virtualization programs like VirtualBox or VMware, providing the user with a high number of preinstalled programs like sRNAbench for small RNA analysis without the need to maintain additional servers and/or operating systems.

  18. Immunotherapy of hepatocellular carcinoma with small double-stranded RNA

    International Nuclear Information System (INIS)

    Kabilova, Tatyana O; Chernolovskaya, Elena L; Kovtonyuk, Larisa V; Zonov, Evgeniy V; Ryabchikova, Elena I; Popova, Nelly A; Nikolin, Valeriy P; Kaledin, Vasiliy I; Zenkova, Marina A; Vlassov, Valentin V

    2014-01-01

    Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, immunotherapy is considered a promising therapeutic approach. Small interfering RNAs (siRNAs), depending on their structure and sequence, can trigger the innate immune system, which can potentially enhance the adaptive anticancer immune response in the tumor-bearing subjects. Immunostimulatory properties of nucleic acids can be applied to develop adjuvants for HCC treatment. The transplantable HCC G-29 tumor in male CBA/LacSto (CBA) mice was used to study the effects of immunostimulatory RNA on tumor growth. Tumor size, metastases area in different organs of mice and mouse survival rate were analyzed. Furthermore the mouse serum IFN-α levels were measured using ELISA. In the present study, we found that a 19-bp RNA duplex (ImmunoStimulattory RNA or isRNA) with 3-nt overhangs at the 3′-ends of specific sequence displays immunostimulatory, antitumor, and antimetastatic activities in mice bearing HCC G-29. Our results demonstrate that isRNA strongly increases the level of interferon-α (IFN-α) by up to 25-fold relative to the level in mice injected with Lipofectamine alone (Mock), and to a lesser extent increases the level of proinflammatory cytokine interleukin-6 (IL-6) (by up to 5.5-fold relative to the Mock level), in mice blood serum. We showed that isRNA reliably (P < 0.05) inhibits primary tumor growth in mice compared to the mock group. Furthermore, injections of isRNA significantly enhanced necrotic processes in the center of the primary tumor, and decreased by twofold the width of the undifferentiated peripheral zone and the number of mitotic cells in this zone. The results showed that isRNA efficiently reduces the area of metastases in the liver, kidneys, and heart of CBA/LacSto mice with HCC. The obtained results clearly demonstrate immunostimulatory and antimetastatic properties of the isRNAs in

  19. Small RNA pathways and diversity in model legumes: lessons from genomics.

    Directory of Open Access Journals (Sweden)

    Pilar eBustos-Sanmamed

    2013-07-01

    Full Text Available Small non coding RNAs (smRNA participate in the regulation of development, cell differentiation, adaptation to environmental constraints and defense responses in plants. They negatively regulate gene expression by degrading specific mRNA targets, repressing their translation or modifying chromatin conformation through homologous interaction with target loci. MicroRNAs (miRNA and short-interfering RNAs (siRNA are generated from long double stranded RNA (dsRNA that are cleaved into 20- to 24-nucleotide dsRNAs by RNase III proteins called DICERs (DCL. One strand of the duplex is then loaded onto effective complexes containing different ARGONAUTE (AGO proteins. In this review, we explored smRNA diversity in model legumes and compiled available data from miRBAse, the miRNA database, and from 22 reports of smRNA deep sequencing or miRNA identification genome-wide in Medicago truncatula, Glycine max and Lotus japonicus. In addition to conserved miRNAs present in other plant species, 229, 179 and 35 novel miRNA families were identified respectively in these 3 legumes, among which several seems legume-specific. New potential functions of several miRNAs in the legume-specific nodulation process are discussed. Furthermore, a new category of siRNA, the phased siRNAs, which seems to mainly regulate disease-resistance genes, was recently discovered in legumes. Despite that the genome sequence of model legumes are not yet fully completed, further analysis was performed by database mining of gene families and protein characteristics of DCLs and AGOs in these genomes. Although most components of the smRNA pathways are conserved, identifiable homologs of key smRNA players from non-legumes could not yet be detected in M. truncatula available genomic and expressed sequence databases. In addition, an important gene diversification was observed in the three legumes. Functional significance of these variant isoforms may reflect peculiarities of smRNA biogenesis in

  20. Antibiotic discovery throughout the Small World Initiative: A molecular strategy to identify biosynthetic gene clusters involved in antagonistic activity.

    Science.gov (United States)

    Davis, Elizabeth; Sloan, Tyler; Aurelius, Krista; Barbour, Angela; Bodey, Elijah; Clark, Brigette; Dennis, Celeste; Drown, Rachel; Fleming, Megan; Humbert, Allison; Glasgo, Elizabeth; Kerns, Trent; Lingro, Kelly; McMillin, MacKenzie; Meyer, Aaron; Pope, Breanna; Stalevicz, April; Steffen, Brittney; Steindl, Austin; Williams, Carolyn; Wimberley, Carmen; Zenas, Robert; Butela, Kristen; Wildschutte, Hans

    2017-06-01

    The emergence of bacterial pathogens resistant to all known antibiotics is a global health crisis. Adding to this problem is that major pharmaceutical companies have shifted away from antibiotic discovery due to low profitability. As a result, the pipeline of new antibiotics is essentially dry and many bacteria now resist the effects of most commonly used drugs. To address this global health concern, citizen science through the Small World Initiative (SWI) was formed in 2012. As part of SWI, students isolate bacteria from their local environments, characterize the strains, and assay for antibiotic production. During the 2015 fall semester at Bowling Green State University, students isolated 77 soil-derived bacteria and genetically characterized strains using the 16S rRNA gene, identified strains exhibiting antagonistic activity, and performed an expanded SWI workflow using transposon mutagenesis to identify a biosynthetic gene cluster involved in toxigenic compound production. We identified one mutant with loss of antagonistic activity and through subsequent whole-genome sequencing and linker-mediated PCR identified a 24.9 kb biosynthetic gene locus likely involved in inhibitory activity in that mutant. Further assessment against human pathogens demonstrated the inhibition of Bacillus cereus, Listeria monocytogenes, and methicillin-resistant Staphylococcus aureus in the presence of this compound, thus supporting our molecular strategy as an effective research pipeline for SWI antibiotic discovery and genetic characterization. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  1. Diagnostic and prognostic signatures from the small non-coding RNA transcriptome in prostate cancer

    DEFF Research Database (Denmark)

    Martens-Uzunova, E S; Jalava, S E; Dits, N F

    2011-01-01

    Prostate cancer (PCa) is the most frequent male malignancy and the second most common cause of cancer-related death in Western countries. Current clinical and pathological methods are limited in the prediction of postoperative outcome. It is becoming increasingly evident that small non-coding RNA...... signatures of 102 fresh-frozen patient samples during PCa progression by miRNA microarrays. Both platforms were cross-validated by quantitative reverse transcriptase-PCR. Besides the altered expression of several miRNAs, our deep sequencing analyses revealed strong differential expression of small nucleolar...... RNAs (snoRNAs) and transfer RNAs (tRNAs). From microarray analysis, we derived a miRNA diagnostic classifier that accurately distinguishes normal from cancer samples. Furthermore, we were able to construct a PCa prognostic predictor that independently forecasts postoperative outcome. Importantly...

  2. A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples

    Science.gov (United States)

    de Vries, Michel; Deijs, Martin; Canuti, Marta; van Schaik, Barbera D. C.; Faria, Nuno R.; van de Garde, Martijn D. B.; Jachimowski, Loes C. M.; Jebbink, Maarten F.; Jakobs, Marja; Luyf, Angela C. M.; Coenjaerts, Frank E. J.; Claas, Eric C. J.; Molenkamp, Richard; Koekkoek, Sylvie M.; Lammens, Christine; Leus, Frank; Goossens, Herman; Ieven, Margareta; Baas, Frank; van der Hoek, Lia

    2011-01-01

    In 5–40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3′-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3–7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material. PMID:21283679

  3. A sensitive assay for virus discovery in respiratory clinical samples.

    Directory of Open Access Journals (Sweden)

    Michel de Vries

    Full Text Available In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3'-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454. Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18. The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6. Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.

  4. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    DEFF Research Database (Denmark)

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, S. G.

    2011-01-01

    significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular......Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24...... and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated...

  5. Identification of rat lung-specific microRNAs by microRNA microarray: valuable discoveries for the facilitation of lung research

    OpenAIRE

    Chintagari Narendranath; Chen Zhongming; Gou Deming; Weng Tingting; Wang Yang; Liu Lin

    2007-01-01

    Abstract Background An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. Results In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to d...

  6. Production of virus-derived ping-pong-dependent piRNA-like small RNAs in the mosquito soma.

    Directory of Open Access Journals (Sweden)

    Elaine M Morazzani

    2012-01-01

    Full Text Available The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs. However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma. We also present evidence that suggests virus-derived piRNA-like small RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication.

  7. Reexamining the P-Element Invasion of Drosophila melanogaster Through the Lens of piRNA Silencing

    Science.gov (United States)

    Kelleher, Erin S.

    2016-01-01

    Transposable elements (TEs) are both important drivers of genome evolution and genetic parasites with potentially dramatic consequences for host fitness. The recent explosion of research on regulatory RNAs reveals that small RNA-mediated silencing is a conserved genetic mechanism through which hosts repress TE activity. The invasion of the Drosophila melanogaster genome by P elements, which happened on a historical timescale, represents an incomparable opportunity to understand how small RNA-mediated silencing of TEs evolves. Repression of P-element transposition emerged almost concurrently with its invasion. Recent studies suggest that this repression is implemented in part, and perhaps predominantly, by the Piwi-interacting RNA (piRNA) pathway, a small RNA-mediated silencing pathway that regulates TE activity in many metazoan germlines. In this review, I consider the P-element invasion from both a molecular and evolutionary genetic perspective, reconciling classic studies of P-element regulation with the new mechanistic framework provided by the piRNA pathway. I further explore the utility of the P-element invasion as an exemplar of the evolution of piRNA-mediated silencing. In light of the highly-conserved role for piRNAs in regulating TEs, discoveries from this system have taxonomically broad implications for the evolution of repression. PMID:27516614

  8. Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella.

    Science.gov (United States)

    Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

    2015-08-25

    Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

  9. Small RNA sequencing reveals a comprehensive miRNA signature of BRCA1-associated high-grade serous ovarian cancer

    NARCIS (Netherlands)

    Brouwer, Jan; Kluiver, Joost; de Almeida, Rodrigo C.; Modderman, Rutger; Terpstra, Martijn; Kok, Klaas; Withoff, Sebo; Hollema, Harry; Reitsma, Welmoed; de Bock, Geertruida H.; Mourits, Marian J. E.; van den Berg, Anke

    2016-01-01

    AimsBRCA1 mutation carriers are at increased risk of developing high-grade serous ovarian cancer (HGSOC), a malignancy that originates from fallopian tube epithelium. We aimed to identify differentially expressed known and novel miRNAs in BRCA1-associated HGSOC. Methods Small RNA sequencing was

  10. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

    Science.gov (United States)

    Valli, Adrian; Busnadiego, Idoia; Maliogka, Varvara; Ferrero, Diego; Castón, José R; Rodríguez, José Francisco; García, Juan Antonio

    2012-01-01

    RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  11. Small RNA-Mediated Epigenetic Myostatin Silencing

    Directory of Open Access Journals (Sweden)

    Thomas C Roberts

    2012-01-01

    Full Text Available Myostatin (Mstn is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

  12. tRNA--the golden standard in molecular biology.

    Science.gov (United States)

    Barciszewska, Mirosława Z; Perrigue, Patrick M; Barciszewski, Jan

    2016-01-01

    Transfer RNAs (tRNAs) represent a major class of RNA molecules. Their primary function is to help decode a messenger RNA (mRNA) sequence in order to synthesize protein and thus ensures the precise translation of genetic information that is imprinted in DNA. The discovery of tRNA in the late 1950's provided critical insight into a genetic machinery when little was known about the central dogma of molecular biology. In 1965, Robert Holley determined the first nucleotide sequence of alanine transfer RNA (tRNA(Ala)) which earned him the 1968 Nobel Prize in Physiology or Medicine. Today, tRNA is one of the best described and characterized biological molecules. Here we review some of the key historical events in tRNA research which led to breakthrough discoveries and new developments in molecular biology.

  13. Discovery of new planetary nebulae in the Small Magellanic Cloud

    International Nuclear Information System (INIS)

    Drašković, D.; Reid, W. A.; Parker, Q. A.; Stupar, M.

    2016-01-01

    We present six new planetary nebulae (PNe) discovered in the Small Magellanic Cloud (SMC) from deep UK Schmidt telescope (UKST) narrow band Ha and broad-band short- red “SR” continuum images and confirmed spectroscopically. These 6 preliminary discoveries provide a 6% increase to the previously known SMC PN population of ∼⃒100. Once spectroscopic follow-up of all our newly identified candidates is complete, we expect to increase the total number of known SMC PNe by up to 50%. This will permit a significant improvement to determination of the SMC PN luminosity function (PNLF) and enable further insights into the chemical evolution and kinematics of the SMC PN population. (paper)

  14. Cellular Response to Ionizing Radiation: A MicroRNA Story

    Science.gov (United States)

    Halimi, Mohammad; Asghari, S. Mohsen; Sariri, Reyhaneh; Moslemi, Dariush; Parsian, Hadi

    2012-01-01

    MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They play a crucial role in diverse cellular pathways. Ionizing radiation (IR) is one of the most important treatment protocols for patients that suffer from cancer and affects directly or indirectly cellular integration. Recently it has been discovered that microRNA-mediated gene regulation interferes with radio-related pathways in ionizing radiation. Here, we review the recent discoveries about miRNAs in cellular response to IR. Thoroughly understanding the mechanism of miRNAs in radiation response, it will be possible to design new strategies for improving radiotherapy efficiency and ultimately cancer treatment. PMID:24551775

  15. Engineering a Functional Small RNA Negative Autoregulation Network with Model-Guided Design.

    Science.gov (United States)

    Hu, Chelsea Y; Takahashi, Melissa K; Zhang, Yan; Lucks, Julius B

    2018-05-22

    RNA regulators are powerful components of the synthetic biology toolbox. Here, we expand the repertoire of synthetic gene networks built from these regulators by constructing a transcriptional negative autoregulation (NAR) network out of small RNAs (sRNAs). NAR network motifs are core motifs of natural genetic networks, and are known for reducing network response time and steady state signal. Here we use cell-free transcription-translation (TX-TL) reactions and a computational model to design and prototype sRNA NAR constructs. Using parameter sensitivity analysis, we design a simple set of experiments that allow us to accurately predict NAR function in TX-TL. We transfer successful network designs into Escherichia coli and show that our sRNA transcriptional network reduces both network response time and steady-state gene expression. This work broadens our ability to construct increasingly sophisticated RNA genetic networks with predictable function.

  16. RNA structure alignment by a unit-vector approach.

    Science.gov (United States)

    Capriotti, Emidio; Marti-Renom, Marc A

    2008-08-15

    The recent discovery of tiny RNA molecules such as microRNAs and small interfering RNA are transforming the view of RNA as a simple information transfer molecule. Similar to proteins, the native three-dimensional structure of RNA determines its biological activity. Therefore, classifying the current structural space is paramount for functionally annotating RNA molecules. The increasing numbers of RNA structures deposited in the PDB requires more accurate, automatic and benchmarked methods for RNA structure comparison. In this article, we introduce a new algorithm for RNA structure alignment based on a unit-vector approach. The algorithm has been implemented in the SARA program, which results in RNA structure pairwise alignments and their statistical significance. The SARA program has been implemented to be of general applicability even when no secondary structure can be calculated from the RNA structures. A benchmark against the ARTS program using a set of 1275 non-redundant pairwise structure alignments results in inverted approximately 6% extra alignments with at least 50% structurally superposed nucleotides and base pairs. A first attempt to perform RNA automatic functional annotation based on structure alignments indicates that SARA can correctly assign the deepest SCOR classification to >60% of the query structures. The SARA program is freely available through a World Wide Web server http://sgu.bioinfo.cipf.es/services/SARA/. Supplementary data are available at Bioinformatics online.

  17. RNA-Seq analysis uncovers non-coding small RNA system of Mycobacterium neoaurum in the metabolism of sterols to accumulate steroid intermediates.

    Science.gov (United States)

    Liu, Min; Zhu, Zhan-Tao; Tao, Xin-Yi; Wang, Feng-Qing; Wei, Dong-Zhi

    2016-04-25

    Understanding the metabolic mechanism of sterols to produce valuable steroid intermediates in mycobacterium by a noncoding small RNA (sRNA) view is still limited. In the work, RNA-seq was implemented to investigate the noncoding transcriptome of Mycobacterium neoaurum (Mn) in the transformation process of sterols to valuable steroid intermediates, including 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA). A total of 263 sRNA candidates were predicted from the intergenic regions in Mn. Differential expression of sRNA candidates was explored in the wide type Mn with vs without sterol addition, and the steroid intermediate producing Mn strains vs wide type Mn with sterol addition, respectively. Generally, sRNA candidates were differentially expressed in various strains, but there were still some shared candidates with outstandingly upregulated or downregulated expression in these steroid producing strains. Accordingly, four regulatory networks were constructed to reveal the direct and/or indirect interactions between sRNA candidates and their target genes in four groups, including wide type Mn with vs without sterol addition, 9OHAD, ADD, and BNA producing strains vs wide type Mn with sterol addition, respectively. Based on these constructed networks, several highly focused sRNA candidates were discovered to be prevalent in the networks, which showed comprehensive regulatory roles in various cellular processes, including lipid transport and metabolism, amino acid transport and metabolism, signal transduction, cell envelope biosynthesis and ATP synthesis. To explore the functional role of sRNA candidates in Mn cells, we manipulated the overexpression of candidates 131 and 138 in strain Mn-9OHAD, which led to enhanced production of 9OHAD from 1.5- to 2.3-fold during 6 d' fermentation and a slight effect on growth rate. This study revealed the complex and important regulatory

  18. Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells.

    Directory of Open Access Journals (Sweden)

    Jaclyn C Scott

    2010-10-01

    Full Text Available The exogenous RNA interference (RNAi pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (siRNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2 cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

  19. Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection.

    Science.gov (United States)

    Qiao, Yongli; Shi, Jinxia; Zhai, Yi; Hou, Yingnan; Ma, Wenbo

    2015-05-05

    A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate-glutamate-alanine-histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection.

  20. Genome-Wide Analysis of miRNA targets in Brachypodium and Biomass Energy Crops

    Energy Technology Data Exchange (ETDEWEB)

    Green, Pamela J. [Univ. of Delaware, Newark, DE (United States)

    2015-08-11

    MicroRNAs (miRNAs) contribute to the control of numerous biological processes through the regulation of specific target mRNAs. Although the identities of these targets are essential to elucidate miRNA function, the targets are much more difficult to identify than the small RNAs themselves. Before this work, we pioneered the genome-wide identification of the targets of Arabidopsis miRNAs using an approach called PARE (German et al., Nature Biotech. 2008; Nature Protocols, 2009). Under this project, we applied PARE to Brachypodium distachyon (Brachypodium), a model plant in the Poaceae family, which includes the major food grain and bioenergy crops. Through in-depth global analysis and examination of specific examples, this research greatly expanded our knowledge of miRNAs and target RNAs of Brachypodium. New regulation in response to environmental stress or tissue type was found, and many new miRNAs were discovered. More than 260 targets of new and known miRNAs with PARE sequences at the precise sites of miRNA-guided cleavage were identified and characterized. Combining PARE data with the small RNA data also identified the miRNAs responsible for initiating approximately 500 phased loci, including one of the novel miRNAs. PARE analysis also revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. The project included generation of small RNA and PARE resources for bioenergy crops, to facilitate ongoing discovery of conserved miRNA-target RNA regulation. By associating specific miRNA-target RNA pairs with known physiological functions, the research provides insights about gene regulation in different tissues and in response to environmental stress. This, and release of new PARE and small RNA data sets should contribute basic knowledge to enhance breeding and may suggest new strategies for improvement of biomass energy crops.

  1. Small Molecule, Big Prospects: MicroRNA in Pregnancy and Its Complications

    Directory of Open Access Journals (Sweden)

    Meng Cai

    2017-01-01

    Full Text Available MicroRNAs are small, noncoding RNA molecules that regulate target gene expression in the posttranscriptional level. Unlike siRNA, microRNAs are “fine-tuners” rather than “switches” in the regulation of gene expression; thus they play key roles in maintaining tissue homeostasis. The aberrant microRNA expression is implicated in the disease process. To date, numerous studies have demonstrated the regulatory roles of microRNAs in various pathophysiological conditions. In contrast, the study of microRNA in pregnancy and its associated complications, such as preeclampsia (PE, fetal growth restriction (FGR, and preterm labor, is a young field. Over the last decade, the knowledge of pregnancy-related microRNAs has increased and the molecular mechanisms by which microRNAs regulate pregnancy or its associated complications are emerging. In this review, we focus on the recent advances in the research of pregnancy-related microRNAs, especially their function in pregnancy-associated complications and the potential clinical applications. Here microRNAs that associate with pregnancy are classified as placenta-specific, placenta-associated, placenta-derived circulating, and uterine microRNA according to their localization and origin. MicroRNAs offer a great potential for developing diagnostic and therapeutic targets in pregnancy-related disorders.

  2. Identifying small RNAs derived from maternal- and somatic-type rRNAs in zebrafish development.

    Science.gov (United States)

    Locati, Mauro D; Pagano, Johanna F B; Abdullah, Farah; Ensink, Wim A; van Olst, Marina; van Leeuwen, Selina; Nehrdich, Ulrike; Spaink, Herman P; Rauwerda, Han; Jonker, Martijs J; Dekker, Rob J; Breit, Timo M

    2018-02-09

    rRNAs are non-coding RNAs present in all prokaryotes and eukaryotes. In eukaryotes there are four rRNAs: 18S, 5.8S, 28S, originating from a common precursor (45S), and 5S. We have recently discovered the existence of two distinct developmental types of rRNA: a maternal-type, present in eggs and a somatic-type, expressed in adult tissues. Lately, next-generation sequencing has allowed the discovery of new small-RNAs deriving from longer non-coding RNAs, including small-RNAs from rRNAs (srRNAs). Here, we systemically investigated srRNAs of maternal- or somatic-type 18S, 5.8S, 28S, with small-RNAseq from many zebrafish developmental stages. We identified new srRNAs for each rRNA. For 5.8S, we found srRNA consisting of the 5' or 3' halves, with only the latter having different sequence for the maternal- and somatic-types. For 18S, we discovered 21 nt srRNA from the 5' end of the 18S rRNA with a striking resemblance to microRNAs; as it is likely processed from a stem-loop precursor and present in human and mouse Argonaute-complexed small-RNA. For 28S, an abundant 80 nt srRNA from the 3' end of the 28S rRNA was found. The expression levels during embryogenesis of these srRNA indicate they are not generated from rRNA degradation and might have a role in the zebrafish development.

  3. [Construction of lentiviral mediated CyPA siRNA and its functions in non-small cell lung cancer].

    Science.gov (United States)

    FENG, Yan-ming; WU, Yi-ming; TU, Xin-ming; XU, Zheng-shun; WU, Wei-dong

    2010-02-01

    To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer. First, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM. It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (Ppathways may lead to new targeted therapies for non-small cell lung cancer.

  4. siRNA Treatment: “A Sword-in-the-Stone” for Acute Brain Injuries

    Directory of Open Access Journals (Sweden)

    Jerome Badaut

    2013-09-01

    Full Text Available Ever since the discovery of small interfering ribonucleic acid (siRNA a little over a decade ago, it has been highly sought after for its potential as a therapeutic agent for many diseases. In this review, we discuss the promising possibility of siRNA to be used as a drug to treat acute brain injuries such as stroke and traumatic brain injury. First, we will give a brief and basic overview of the principle of RNA interference as an effective mechanism to decrease specific protein expression. Then, we will review recent in vivo studies describing siRNA research experiments/treatment options for acute brain diseases. Lastly, we will discuss the future of siRNA as a clinical therapeutic strategy against brain diseases and injuries, while addressing the current obstacles to effective brain delivery.

  5. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

    Directory of Open Access Journals (Sweden)

    Adrian Valli

    Full Text Available RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  6. The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

    Science.gov (United States)

    Pantano, Lorena; Jodar, Meritxell; Bak, Mads; Ballescà, Josep Lluís; Tommerup, Niels; Oliva, Rafael; Vavouri, Tanya

    2015-01-01

    At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline. PMID:25904136

  7. Biochemistry and Function of the RNA Exosomes

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon; Chlebowski, Aleksander; Dziembowski, Andrzej

    2012-01-01

    Discovery of the evolutionary conserved RNA exosome was a milestone in RNA biology. First identified as an activity essential for the processing of ribosomal RNA, the exosome has since proved to be central for RNA processing and degradation in both the nucleus and the cytoplasm of eukaryotic cell...

  8. MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Minxia Liu

    2016-09-01

    Full Text Available MicroRNAs (miRNAs have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC.

  9. MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Liu, Minxia; Zhou, Kecheng; Cao, Yi

    2016-09-26

    MicroRNAs (miRNAs) have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC) and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D) and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC.

  10. Targeted Sterically Stabilized Phospholipid siRNA Nanomedicine for Hepatic and Renal Fibrosis

    Directory of Open Access Journals (Sweden)

    Fatima Khaja

    2016-01-01

    Full Text Available Since its discovery, small interfering RNA (siRNA has been considered a potent tool for modulating gene expression. It has the ability to specifically target proteins via selective degradation of messenger RNA (mRNA not easily accessed by conventional drugs. Hence, RNA interference (RNAi therapeutics have great potential in the treatment of many diseases caused by faulty protein expression such as fibrosis and cancer. However, for clinical application siRNA faces a number of obstacles, such as poor in vivo stability, and off-target effects. Here we developed a unique targeted nanomedicine to tackle current siRNA delivery issues by formulating a biocompatible, biodegradable and relatively inexpensive nanocarrier of sterically stabilized phospholipid nanoparticles (SSLNPs. This nanocarrier is capable of incorporating siRNA in its core through self-association with a novel cationic lipid composed of naturally occuring phospholipids and amino acids. This overall assembly protects and delivers sufficient amounts of siRNA to knockdown over-expressed protein in target cells. The siRNA used in this study, targets connective tissue growth factor (CTGF, an important regulator of fibrosis in both hepatic and renal cells. Furthermore, asialoglycoprotein receptors are targeted by attaching the galactosamine ligand to the nanocarries which enhances the uptake of nanoparticles by hepatocytes and renal tubular epithelial cells, the major producers of CTGF in fibrosis. On animals this innovative nanoconstruct, small interfering RNA in sterically stabilized phospholipid nanoparticles (siRNA-SSLNP, showed favorable pharmacokinetic properties and accumulated mostly in hepatic and renal tissues making siRNA-SSLNP a suitable system for targeting liver and kidney fibrotic diseases.

  11. A conserved small RNA promotes silencing of the outer membrane protein YbfM

    DEFF Research Database (Denmark)

    Rasmussen, Anders Aamann; Johansen, Jesper; Nielsen, Jesper S

    2009-01-01

    important physiological role of regulatory RNA molecules in Gram-negative bacteria is to modulate the cell surface and/or to prevent accumulation of OMPs in the envelope. Here, we extend the OMP-sRNA network by showing that the expression of the outer membrane protein YbfM is silenced by a conserved sRNA......In the past few years an increasing number of small non-coding RNAs (sRNAs) in enterobacteria have been found to negatively regulate the expression of outer membrane proteins (OMPs) at the post-transcriptional level. These RNAs act under various growth and stress conditions, suggesting that one......, designated MicM (also known as RybC/SroB). The regulation is strictly dependent on the RNA chaperone Hfq, and mutational analysis indicates that MicM sequesters the ribosome binding site of ybfM mRNA by an antisense mechanism. Furthermore, we provide evidence that Hfq strongly enhances the on-rate of duplex...

  12. Understanding alcoholism through microRNA signatures in brains of human alcoholics

    Directory of Open Access Journals (Sweden)

    R. Dayne eMayfield

    2012-04-01

    Full Text Available Advances in the fields of genomics and genetics in the last decade have identified a large number of genes that can potentially influence alcohol-drinking behavior in humans as well as animal models. Consequently, the task of identifying efficient molecular targets that could be used to develop effective therapeutics against the disease has become increasingly daunting. One of the reasons for this is the fact that each of the many alcohol-responsive genes only contributes a small effect to the overall mechanism and disease phenotype, as is characteristic of complex traits. Current research trends are hence shifting towards the analysis of gene networks rather than emphasizing individual genes. The discovery of microRNAs and their mechanisms of action on regulation of transcript level and protein translation have made evident the utility of these small non-coding RNA molecules that act as central coordinators of multiple cross-communicating cellular pathways. Cells exploit the fact that a single microRNA can target hundreds of mRNA transcripts and that a single mRNA transcript can be simultaneously targeted by distinct microRNAs, to ensure fine-tuned and/or redundant control over a large number of cellular functions. By the same token, we can use these properties of microRNAs to develop novel, targeted strategies to combat complex disorders. In this review, we will focus on recent discoveries of microRNA signatures in brain of human alcoholics supporting the hypothesis that changes in gene expression and regulation by microRNAs are responsible for long-term neuroadaptations occurring during development of alcoholism. We also discuss insights into the potential modulation of epigenetic regulators by a subset of microRNAs. Taken together, microRNA activity may be controlling many of the cellular mechanisms already known to be involved in the development of alcoholism, and suggests potential targets for the development of novel therapeutic

  13. The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1.

    Science.gov (United States)

    Toczyski, D P; Steitz, J A

    1993-01-01

    EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that > 95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function. Images PMID:8380232

  14. Small catalytic RNA: Structure, function and application

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, Joseph Albert [Univ. of California, Berkeley, CA (United States)

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the ``paperclip`` and ``hammerhead`` RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a ``hammerhead,`` to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 121±s are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus_minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  15. Small catalytic RNA: Structure, function and application

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, J.A.

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the paperclip'' and hammerhead'' RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a hammerhead,'' to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  16. Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes.

    Science.gov (United States)

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2011-10-28

    The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

    International Nuclear Information System (INIS)

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2011-01-01

    Highlights: ► We use MEL-A-containing cationic liposomes for siRNA delivery. ► MEL-A-containing cationic liposomes can efficiently and rapidly deliver siRNA into the cytoplasm. ► Rapid delivery of siRNA is due to the membrane fusion between liposomes and plasma membrane. -- Abstract: The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine™ RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by

  18. Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

    Science.gov (United States)

    Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian; Wei, Taiyun

    2016-01-15

    Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an

  19. Protection against lethal Marburg virus infection mediated by lipid encapsulated small interfering RNA.

    Science.gov (United States)

    Ursic-Bedoya, Raul; Mire, Chad E; Robbins, Marjorie; Geisbert, Joan B; Judge, Adam; MacLachlan, Ian; Geisbert, Thomas W

    2014-02-15

    Marburg virus (MARV) infection causes severe morbidity and mortality in humans and nonhuman primates. Currently, there are no licensed therapeutics available for treating MARV infection. Here, we present the in vitro development and in vivo evaluation of lipid-encapsulated small interfering RNA (siRNA) as a potential therapeutic for the treatment of MARV infection. The activity of anti-MARV siRNAs was assessed using dual luciferase reporter assays followed by in vitro testing against live virus. Lead candidates were tested in lethal guinea pig models of 3 different MARV strains (Angola, Ci67, Ravn). Treatment resulted in 60%-100% survival of guinea pigs infected with MARV. Although treatment with siRNA targeting other MARV messenger RNA (mRNA) had a beneficial effect, targeting the MARV NP mRNA resulted in the highest survival rates. NP-718m siRNA in lipid nanoparticles provided 100% protection against MARV strains Angola and Ci67, and 60% against Ravn. A cocktail containing NP-718m and NP-143m provided 100% protection against MARV Ravn. These data show protective efficacy against the most pathogenic Angola strain of MARV. Further development of the lipid nanoparticle technology has the potential to yield effective treatments for MARV infection.

  20. Alpha-synuclein suppression by targeted small interfering RNA in the primate substantia nigra.

    Directory of Open Access Journals (Sweden)

    Alison L McCormack

    Full Text Available The protein alpha-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal alpha-synuclein burden. Here, feasibility and safety of alpha-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA directed against alpha-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of alpha-synuclein mRNA and protein in the infused (left vs. untreated (right hemisphere and revealed a significant 40-50% suppression of alpha-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in alpha-synuclein. Infusion with alpha-synuclein siRNA, while lowering alpha-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i the number and phenotype of nigral dopaminergic neurons, and (ii the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-alpha-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

  1. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

    Science.gov (United States)

    Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

    2018-02-13

    Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

  2. Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi.

    Science.gov (United States)

    Zhang, Xiaoyu; Gamarra, Jaime; Castro, Steven; Carrasco, Estela; Hernandez, Aaron; Mock, Thomas; Hadaegh, Ahmad R; Read, Betsy A

    2016-01-01

    Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18-24 nt and 71-252 nt, respectively), genome copy number (3-1,459), and the number of genes targeted (2-107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways.

  3. Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi.

    Directory of Open Access Journals (Sweden)

    Xiaoyu Zhang

    Full Text Available Small RNAs (smRNAs control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi. Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18-24 nt and 71-252 nt, respectively, genome copy number (3-1,459, and the number of genes targeted (2-107. Stem-loop real time reverse transcriptase (RT PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways.

  4. voomDDA: discovery of diagnostic biomarkers and classification of RNA-seq data

    Directory of Open Access Journals (Sweden)

    Gokmen Zararsiz

    2017-10-01

    Full Text Available RNA-Seq is a recent and efficient technique that uses the capabilities of next-generation sequencing technology for characterizing and quantifying transcriptomes. One important task using gene-expression data is to identify a small subset of genes that can be used to build diagnostic classifiers particularly for cancer diseases. Microarray based classifiers are not directly applicable to RNA-Seq data due to its discrete nature. Overdispersion is another problem that requires careful modeling of mean and variance relationship of the RNA-Seq data. In this study, we present voomDDA classifiers: variance modeling at the observational level (voom extensions of the nearest shrunken centroids (NSC and the diagonal discriminant classifiers. VoomNSC is one of these classifiers and brings voom and NSC approaches together for the purpose of gene-expression based classification. For this purpose, we propose weighted statistics and put these weighted statistics into the NSC algorithm. The VoomNSC is a sparse classifier that models the mean-variance relationship using the voom method and incorporates voom’s precision weights into the NSC classifier via weighted statistics. A comprehensive simulation study was designed and four real datasets are used for performance assessment. The overall results indicate that voomNSC performs as the sparsest classifier. It also provides the most accurate results together with power-transformed Poisson linear discriminant analysis, rlog transformed support vector machines and random forests algorithms. In addition to prediction purposes, the voomNSC classifier can be used to identify the potential diagnostic biomarkers for a condition of interest. Through this work, statistical learning methods proposed for microarrays can be reused for RNA-Seq data. An interactive web application is freely available at http://www.biosoft.hacettepe.edu.tr/voomDDA/.

  5. voomDDA: discovery of diagnostic biomarkers and classification of RNA-seq data.

    Science.gov (United States)

    Zararsiz, Gokmen; Goksuluk, Dincer; Klaus, Bernd; Korkmaz, Selcuk; Eldem, Vahap; Karabulut, Erdem; Ozturk, Ahmet

    2017-01-01

    RNA-Seq is a recent and efficient technique that uses the capabilities of next-generation sequencing technology for characterizing and quantifying transcriptomes. One important task using gene-expression data is to identify a small subset of genes that can be used to build diagnostic classifiers particularly for cancer diseases. Microarray based classifiers are not directly applicable to RNA-Seq data due to its discrete nature. Overdispersion is another problem that requires careful modeling of mean and variance relationship of the RNA-Seq data. In this study, we present voomDDA classifiers: variance modeling at the observational level (voom) extensions of the nearest shrunken centroids (NSC) and the diagonal discriminant classifiers. VoomNSC is one of these classifiers and brings voom and NSC approaches together for the purpose of gene-expression based classification. For this purpose, we propose weighted statistics and put these weighted statistics into the NSC algorithm. The VoomNSC is a sparse classifier that models the mean-variance relationship using the voom method and incorporates voom's precision weights into the NSC classifier via weighted statistics. A comprehensive simulation study was designed and four real datasets are used for performance assessment. The overall results indicate that voomNSC performs as the sparsest classifier. It also provides the most accurate results together with power-transformed Poisson linear discriminant analysis, rlog transformed support vector machines and random forests algorithms. In addition to prediction purposes, the voomNSC classifier can be used to identify the potential diagnostic biomarkers for a condition of interest. Through this work, statistical learning methods proposed for microarrays can be reused for RNA-Seq data. An interactive web application is freely available at http://www.biosoft.hacettepe.edu.tr/voomDDA/.

  6. A dual transcript-discovery approach to improve the delimitation of gene features from RNA-seq data in the chicken model

    Directory of Open Access Journals (Sweden)

    Mickael Orgeur

    2018-01-01

    Full Text Available The sequence of the chicken genome, like several other draft genome sequences, is presently not fully covered. Gaps, contigs assigned with low confidence and uncharacterized chromosomes result in gene fragmentation and imprecise gene annotation. Transcript abundance estimation from RNA sequencing (RNA-seq data relies on read quality, library complexity and expression normalization. In addition, the quality of the genome sequence used to map sequencing reads, and the gene annotation that defines gene features, must also be taken into account. A partially covered genome sequence causes the loss of sequencing reads from the mapping step, while an inaccurate definition of gene features induces imprecise read counts from the assignment step. Both steps can significantly bias interpretation of RNA-seq data. Here, we describe a dual transcript-discovery approach combining a genome-guided gene prediction and a de novo transcriptome assembly. This dual approach enabled us to increase the assignment rate of RNA-seq data by nearly 20% as compared to when using only the chicken reference annotation, contributing therefore to a more accurate estimation of transcript abundance. More generally, this strategy could be applied to any organism with partial genome sequence and/or lacking a manually-curated reference annotation in order to improve the accuracy of gene expression studies.

  7. Archaeal rRNA operons, intron splicing and homing endonucleases, RNA polymerase operons and phylogeny

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Aagaard, Claus Sindbjerg; Andersen, Morten

    1994-01-01

    Over the past decade our laboratory has had a strong interest in defining the phylogenetic status of the archaea. This has involved determining and analysing the sequences of operons of both rRNAs and RNA polymerases and it led to the discovery of the first archaeal rRNA intron. What follows...

  8. RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection

    International Nuclear Information System (INIS)

    Kleinschmidt, A.M.; Pederson, T.

    1990-01-01

    The authors present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32 P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells

  9. A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

    Directory of Open Access Journals (Sweden)

    Baodong Wu

    2009-03-01

    Full Text Available Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

  10. The Old and New RNA World

    Directory of Open Access Journals (Sweden)

    Zofia Szweykowska-Kulińska

    2014-12-01

    Full Text Available Among the numerous hypotheses offering a scenario for the origin of life on Earth, the one called “The RNA World” has gained the most attention. According to this hypothesis RNA acted as a genetic information storage material, as a catalyst of all metabolic reactions, and as a regulator of all processes in the primordial world. Various experiments show that RNA molecules could have been synthesized abiotically, with the potential to mediate a whole repertoire of metabolic reactions. Ribozymes carrying out aminoacyl-tRNA reactions have been found in SELEX (systematic evolution of ligands by exponential enrichment approaches and the development of a ribosome from a RNA-built protoribosome is easy to imagine. Transfer RNA aminoacylation, protoribosome origin, and the availability of amino acids on early Earth allowed the genetic code to evolve. Encoded proteins most likely stabilized RNA molecules and were able to create channels across membranes. In the modern cell, DNA replaced RNA as the main depositor of genetic information and proteins carry out almost all metabolic reactions. However, RNA is still playing versatile, crucial roles in the cell. Apart from its classical functions in the cell, a huge small RNA world is controlling gene expression, chromatin condensation, response to environmental cues, and protecting the cell against the invasion of various nucleic acids forms. Long non-coding RNAs act as crucial gene expression regulators. Riboswitches act at the level of transcription, splicing or translation and mediate feedback regulation on biosynthesis and transport of the ligand they sense. Alternative splicing generates genetic variability and increases the protein repertoire in response to developmental or environmental changes. All these regulatory functions are essential in shaping cell plasticity in the changing milieu. Recent discoveries of new, unexpected and important functions of RNA molecules support the hypothesis that we

  11. Mpn1, Mutated in Poikiloderma with Neutropenia Protein 1, Is a Conserved 3′-to-5′ RNA Exonuclease Processing U6 Small Nuclear RNA

    Directory of Open Access Journals (Sweden)

    Vadim Shchepachev

    2012-10-01

    Full Text Available Clericuzio-type poikiloderma with neutropenia (PN is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3′ end polyuridine tails that are longer than those in normal cells and lack a terminal 2′,3′ cyclic phosphate group. In mpn1Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 is a 3′-to-5′ RNA exonuclease that removes uridines from U6 3′ ends, generating terminal 2′,3′ cyclic phosphates in vitro. Finally, U6 degradation rates increase in mpn1Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3′ end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis.

  12. iSmaRT: a toolkit for a comprehensive analysis of small RNA-Seq data.

    Science.gov (United States)

    Panero, Riccardo; Rinaldi, Antonio; Memoli, Domenico; Nassa, Giovanni; Ravo, Maria; Rizzo, Francesca; Tarallo, Roberta; Milanesi, Luciano; Weisz, Alessandro; Giurato, Giorgio

    2017-03-15

    The interest in investigating the biological roles of small non-coding RNAs (sncRNAs) is increasing, due to the pleiotropic effects of these molecules exert in many biological contexts. While several methods and tools are available to study microRNAs (miRNAs), only few focus on novel classes of sncRNAs, in particular PIWI-interacting RNAs (piRNAs). To overcome these limitations, we implemented iSmaRT ( i ntegrative Sm all R NA T ool-kit), an automated pipeline to analyze smallRNA-Seq data. iSmaRT is a collection of bioinformatics tools and own algorithms, interconnected through a Graphical User Interface (GUI). In addition to performing comprehensive analyses on miRNAs, it implements specific computational modules to analyze piRNAs, predicting novel ones and identifying their RNA targets. A smallRNA-Seq dataset generated from brain samples of Huntington's Disease patients was used here to illustrate iSmaRT performances, demonstrating how the pipeline can provide, in a rapid and user friendly way, a comprehensive analysis of different classes of sncRNAs. iSmaRT is freely available on the web at ftp://labmedmolge-1.unisa.it (User: iSmart - Password: password). aweisz@unisa.it or ggiurato@unisa.it. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  13. Small Molecule Targeting of a MicroRNA Associated with Hepatocellular Carcinoma.

    Science.gov (United States)

    Childs-Disney, Jessica L; Disney, Matthew D

    2016-02-19

    Development of precision therapeutics is of immense interest, particularly as applied to the treatment of cancer. By analyzing the preferred cellular RNA targets of small molecules, we discovered that 5"-azido neomycin B binds the Drosha processing site in the microRNA (miR)-525 precursor. MiR-525 confers invasive properties to hepatocellular carcinoma (HCC) cells. Although HCC is one of the most common cancers, treatment options are limited, making the disease often fatal. Herein, we find that addition of 5"-azido neomycin B and its FDA-approved precursor, neomycin B, to an HCC cell line selectively inhibits production of the mature miRNA, boosts a downstream protein, and inhibits invasion. Interestingly, neomycin B is a second-line agent for hepatic encephalopathy (HE) and bacterial infections due to cirrhosis. Our results provocatively suggest that neomycin B, or second-generation derivatives, may be dual functioning molecules to treat both HE and HCC. Collectively, these studies show that rational design approaches can be tailored to disease-associated RNAs to afford potential lead therapeutics.

  14. Annotating RNA motifs in sequences and alignments.

    Science.gov (United States)

    Gardner, Paul P; Eldai, Hisham

    2015-01-01

    RNA performs a diverse array of important functions across all cellular life. These functions include important roles in translation, building translational machinery and maturing messenger RNA. More recent discoveries include the miRNAs and bacterial sRNAs that regulate gene expression, the thermosensors, riboswitches and other cis-regulatory elements that help prokaryotes sense their environment and eukaryotic piRNAs that suppress transposition. However, there can be a long period between the initial discovery of a RNA and determining its function. We present a bioinformatic approach to characterize RNA motifs, which are critical components of many RNA structure-function relationships. These motifs can, in some instances, provide researchers with functional hypotheses for uncharacterized RNAs. Moreover, we introduce a new profile-based database of RNA motifs--RMfam--and illustrate some applications for investigating the evolution and functional characterization of RNA. All the data and scripts associated with this work are available from: https://github.com/ppgardne/RMfam. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. A Small RNA-Based Immune System Defends Germ Cells against Mobile Genetic Elements

    Directory of Open Access Journals (Sweden)

    Astrid D. Haase

    2016-01-01

    Full Text Available Transposons are mobile genetic elements that threaten the survival of species by destabilizing the germline genomes. Limiting the spread of these selfish elements is imperative. Germ cells employ specialized small regulatory RNA pathways to restrain transposon activity. PIWI proteins and Piwi-interacting RNAs (piRNAs silence transposons at the transcriptional and posttranscriptional level with loss-of-function mutant animals universally exhibiting sterility often associated with germ cell defects. This short review aims to illustrate basic strategies of piRNA-guided defense against transposons. Mechanisms of piRNA silencing are most readily studied in Drosophila melanogaster, which serves as a model to delineate molecular concepts and as a reference for mammalian piRNA systems. PiRNA pathways utilize two major strategies to handle the challenges of transposon control: (1 the hard-wired molecular memory of prior transpositions enables recognition of mobile genetic elements and discriminates transposons from host genes; (2 a feed-forward adaptation mechanism shapes piRNA populations to selectively combat the immediate threat of transposon transcripts. In flies, maternally contributed PIWI-piRNA complexes bolster both of these lines of defense and ensure transgenerational immunity. While recent studies have provided a conceptual framework of what could be viewed as an ancient immune system, we are just beginning to appreciate its many molecular innovations.

  16. Prokaryotic Argonautes - variations on the RNA interference theme

    Science.gov (United States)

    van der Oost, John; Swarts, Daan C.; Jore, Matthijs M.

    2014-01-01

    The discovery of RNA interference (RNAi) has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago), an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo) proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo) containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo) that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted) nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases) have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes. PMID:28357239

  17. From "Cellular" RNA to "Smart" RNA: Multiple Roles of RNA in Genome Stability and Beyond.

    Science.gov (United States)

    Michelini, Flavia; Jalihal, Ameya P; Francia, Sofia; Meers, Chance; Neeb, Zachary T; Rossiello, Francesca; Gioia, Ubaldo; Aguado, Julio; Jones-Weinert, Corey; Luke, Brian; Biamonti, Giuseppe; Nowacki, Mariusz; Storici, Francesca; Carninci, Piero; Walter, Nils G; Fagagna, Fabrizio d'Adda di

    2018-03-30

    Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.

  18. Hopf Bifurcation Analysis of a Gene Regulatory Network Mediated by Small Noncoding RNA with Time Delays and Diffusion

    Science.gov (United States)

    Li, Chengxian; Liu, Haihong; Zhang, Tonghua; Yan, Fang

    2017-12-01

    In this paper, a gene regulatory network mediated by small noncoding RNA involving two time delays and diffusion under the Neumann boundary conditions is studied. Choosing the sum of delays as the bifurcation parameter, the stability of the positive equilibrium and the existence of spatially homogeneous and spatially inhomogeneous periodic solutions are investigated by analyzing the corresponding characteristic equation. It is shown that the sum of delays can induce Hopf bifurcation and the diffusion incorporated into the system can effect the amplitude of periodic solutions. Furthermore, the spatially homogeneous periodic solution always exists and the spatially inhomogeneous periodic solution will arise when the diffusion coefficients of protein and mRNA are suitably small. Particularly, the small RNA diffusion coefficient is more robust and its effect on model is much less than protein and mRNA. Finally, the explicit formulae for determining the direction of Hopf bifurcation and the stability of the bifurcating periodic solutions are derived by employing the normal form theory and center manifold theorem for partial functional differential equations. Finally, numerical simulations are carried out to illustrate our theoretical analysis.

  19. Functional specialization of the small interfering RNA pathway in response to virus infection.

    Directory of Open Access Journals (Sweden)

    Joao Trindade Marques

    Full Text Available In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA is processed into small interfering RNAs (siRNAs by Dicer-2 (Dcr-2 in association with a dsRNA-binding protein (dsRBP cofactor called Loquacious (Loqs-PD. siRNAs are then loaded onto Argonaute-2 (Ago2 by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.

  20. Insight into small RNA abundance and expression in high- and low-temperature stress response using deep sequencing in Arabidopsis.

    Science.gov (United States)

    Baev, Vesselin; Milev, Ivan; Naydenov, Mladen; Vachev, Tihomir; Apostolova, Elena; Mehterov, Nikolay; Gozmanva, Mariyana; Minkov, Georgi; Sablok, Gaurav; Yahubyan, Galina

    2014-11-01

    Small RNA profiling and assessing its dependence on changing environmental factors have expanded our understanding of the transcriptional and post-transcriptional regulation of plant stress responses. Insufficient data have been documented earlier to depict the profiling of small RNA classes in temperature-associated stress which has a wide implication for climate change biology. In the present study, we report a comparative assessment of the genome-wide profiling of small RNAs in Arabidopsis thaliana using two conditional responses, induced by high- and low-temperature. Genome-wide profiling of small RNAs revealed an abundance of 21 nt small RNAs at low temperature, while high temperature showed an abundance of 21 nt and 24 nt small RNAs. The two temperature treatments altered the expression of a specific subset of mature miRNAs and displayed differential expression of a number of miRNA isoforms (isomiRs). Comparative analysis demonstrated that a large number of protein-coding genes can give rise to differentially expressed small RNAs following temperature shifts. Low temperature caused accumulation of small RNAs, corresponding to the sense strand of a number of cold-responsive genes. In contrast, high temperature stimulated the production of small RNAs of both polarities from genes encoding functionally diverse proteins. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. Arabidopsis RNASE THREE LIKE2 Modulates the Expression of Protein-Coding Genes via 24-Nucleotide Small Interfering RNA-Directed DNA Methylation.

    Science.gov (United States)

    Elvira-Matelot, Emilie; Hachet, Mélanie; Shamandi, Nahid; Comella, Pascale; Sáez-Vásquez, Julio; Zytnicki, Matthias; Vaucheret, Hervé

    2016-02-01

    RNaseIII enzymes catalyze the cleavage of double-stranded RNA (dsRNA) and have diverse functions in RNA maturation. Arabidopsis thaliana RNASE THREE LIKE2 (RTL2), which carries one RNaseIII and two dsRNA binding (DRB) domains, is a unique Arabidopsis RNaseIII enzyme resembling the budding yeast small interfering RNA (siRNA)-producing Dcr1 enzyme. Here, we show that RTL2 modulates the production of a subset of small RNAs and that this activity depends on both its RNaseIII and DRB domains. However, the mode of action of RTL2 differs from that of Dcr1. Whereas Dcr1 directly cleaves dsRNAs into 23-nucleotide siRNAs, RTL2 likely cleaves dsRNAs into longer molecules, which are subsequently processed into small RNAs by the DICER-LIKE enzymes. Depending on the dsRNA considered, RTL2-mediated maturation either improves (RTL2-dependent loci) or reduces (RTL2-sensitive loci) the production of small RNAs. Because the vast majority of RTL2-regulated loci correspond to transposons and intergenic regions producing 24-nucleotide siRNAs that guide DNA methylation, RTL2 depletion modifies DNA methylation in these regions. Nevertheless, 13% of RTL2-regulated loci correspond to protein-coding genes. We show that changes in 24-nucleotide siRNA levels also affect DNA methylation levels at such loci and inversely correlate with mRNA steady state levels, thus implicating RTL2 in the regulation of protein-coding gene expression. © 2016 American Society of Plant Biologists. All rights reserved.

  2. 13 CFR 134.310 - Discovery.

    Science.gov (United States)

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Discovery. 134.310 Section 134.310 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION RULES OF PROCEDURE GOVERNING CASES BEFORE THE... Designations § 134.310 Discovery. Discovery will not be permitted in appeals from size determinations or NAICS...

  3. MicroRNA-mediated regulation of glutathione and methionine metabolism and its relevance for liver disease.

    Science.gov (United States)

    Lu, Shelly C; Mato, José M; Espinosa-Diez, Cristina; Lamas, Santiago

    2016-11-01

    The discovery of the microRNA (miRNA) family of small RNAs as fundamental regulators of post-transcriptional gene expression has fostered research on their importance in every area of biology and clinical medicine. In the particular area of liver metabolism and disease, miRNAs are gaining increasing importance. By focusing on two fundamental hepatic biosynthetic pathways, glutathione and methionine, we review recent advances on the comprehension of the role of miRNAs in liver pathophysiology and more specifically of models of hepatic cholestasis/fibrosis and hepatocellular carcinoma. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Recent advances in the discovery of small molecule c-Met Kinase inhibitors.

    Science.gov (United States)

    Parikh, Palak K; Ghate, Manjunath D

    2018-01-01

    c-Met is a prototype member of a subfamily of heterodimeric receptor tyrosine kinases (RTKs) and is the receptor for hepatocyte growth factor (HGF). Binding of HGF to its receptor c-Met, initiates a wide range of cellular signalling, including those involved in proliferation, motility, migration and invasion. Importantly, dysregulated HGF/c-Met signalling is a driving factor for numerous malignancies and promotes tumour growth, invasion, dissemination and/or angiogenesis. Dysregulated HGF/c-Met signalling has also been associated with poor clinical outcomes and resistance acquisition to some approved targeted therapies. Thus, c-Met kinase has emerged as a promising target for cancer drug development. Different therapeutic approaches targeting the HGF/c-Met signalling pathway are under development for targeted cancer therapy, among which small molecule inhibitors of c-Met kinase constitute the largest effort within the pharmaceutical industry. The review is an effort to summarize recent advancements in medicinal chemistry development of small molecule c-Met kinase inhibitors as potential anti-cancer agents which would certainly help future researchers to bring further developments in the discovery of small molecule c-Met kinase inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing.

    Science.gov (United States)

    Amini, Parisa; Ettlin, Julia; Opitz, Lennart; Clementi, Elena; Malbon, Alexandra; Markkanen, Enni

    2017-08-23

    Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult. We excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step. We successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well. We present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now

  6. Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection.

    Science.gov (United States)

    Zografidis, Aris; Van Nieuwerburgh, Filip; Kolliopoulou, Anna; Apostolou-Karampelis, Konstantinos; Head, Steven R; Deforce, Dieter; Smagghe, Guy; Swevers, Luc

    2015-11-01

    The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera. This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV; Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10. Copyright © 2015, American

  7. Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jin Ye

    2009-04-01

    Full Text Available Abstract Background The low pH environment of the human stomach is lethal for most microorganisms; but not Escherichia coli, which can tolerate extreme acid stress. Acid resistance in E. coli is hierarchically controlled by numerous regulators among which are small noncoding RNAs (sncRNA. Results In this study, we individually deleted seventy-nine sncRNA genes from the E. coli K12-MG1655 chromosome, and established a single-sncRNA gene knockout library. By systematically screening the sncRNA mutant library, we show that the sncRNA GcvB is a novel regulator of acid resistance in E. coli. We demonstrate that GcvB enhances the ability of E. coli to survive low pH by upregulating the levels of the alternate sigma factor RpoS. Conclusion GcvB positively regulates acid resistance by affecting RpoS expression. These data advance our understanding of the sncRNA regulatory network involved in modulating acid resistance in E. coli.

  8. Elucidating the Small Regulatory RNA Repertoire of the Sea Anemone Anemonia viridis Based on Whole Genome and Small RNA Sequencing.

    Science.gov (United States)

    Urbarova, Ilona; Patel, Hardip; Forêt, Sylvain; Karlsen, Bård Ove; Jørgensen, Tor Erik; Hall-Spencer, Jason M; Johansen, Steinar D

    2018-02-01

    Cnidarians harbor a variety of small regulatory RNAs that include microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), but detailed information is limited. Here, we report the identification and expression of novel miRNAs and putative piRNAs, as well as their genomic loci, in the symbiotic sea anemone Anemonia viridis. We generated a draft assembly of the A. viridis genome with putative size of 313 Mb that appeared to be composed of about 36% repeats, including known transposable elements. We detected approximately equal fractions of DNA transposons and retrotransposons. Deep sequencing of small RNA libraries constructed from A. viridis adults sampled at a natural CO2 gradient off Vulcano Island, Italy, identified 70 distinct miRNAs. Eight were homologous to previously reported miRNAs in cnidarians, whereas 62 appeared novel. Nine miRNAs were recognized as differentially expressed along the natural seawater pH gradient. We found a highly abundant and diverse population of piRNAs, with a substantial fraction showing ping-pong signatures. We identified nearly 22% putative piRNAs potentially targeting transposable elements within the A. viridis genome. The A. viridis genome appeared similar in size to that of other hexacorals with a very high divergence of transposable elements resembling that of the sea anemone genus Exaiptasia. The genome encodes and expresses a high number of small regulatory RNAs, which include novel miRNAs and piRNAs. Differentially expressed small RNAs along the seawater pH gradient indicated regulatory gene responses to environmental stressors. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  9. Specific Regional and Age-Related Small Noncoding RNA Expression Patterns Within Superior Temporal Gyrus of Typical Human Brains Are Less Distinct in Autism Brains.

    Science.gov (United States)

    Stamova, Boryana; Ander, Bradley P; Barger, Nicole; Sharp, Frank R; Schumann, Cynthia M

    2015-12-01

    Small noncoding RNAs play a critical role in regulating messenger RNA throughout brain development and when altered could have profound effects leading to disorders such as autism spectrum disorders (ASD). We assessed small noncoding RNAs, including microRNA and small nucleolar RNA, in superior temporal sulcus association cortex and primary auditory cortex in typical and ASD brains from early childhood to adulthood. Typical small noncoding RNA expression profiles were less distinct in ASD, both between regions and changes with age. Typical micro-RNA coexpression associations were absent in ASD brains. miR-132, miR-103, and miR-320 micro-RNAs were dysregulated in ASD and have previously been associated with autism spectrum disorders. These diminished region- and age-related micro-RNA expression profiles are in line with previously reported findings of attenuated messenger RNA and long noncoding RNA in ASD brain. This study demonstrates alterations in superior temporal sulcus in ASD, a region implicated in social impairment, and is the first to demonstrate molecular alterations in the primary auditory cortex. © The Author(s) 2015.

  10. Small Molecule Binding, Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

    Directory of Open Access Journals (Sweden)

    Mary C. Hames

    2013-11-01

    Full Text Available Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.

  11. Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

    OpenAIRE

    Zografidis, Aris; Van Nieuwerburgh, Filip; Kolliopoulou, Anna; Apostolou-Karampelis, Konstantinos; Head, Steven R.; Deforce, Dieter; Smagghe, Guy; Swevers, Luc

    2015-01-01

    The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by...

  12. Identification of small non-coding RNA classes expressed in swine whole blood during HP-PRRSV infection.

    Science.gov (United States)

    Fleming, Damarius S; Miller, Laura C

    2018-04-01

    It has been established that reduced susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) has a genetic component. This genetic component may take the form of small non-coding RNAs (sncRNA), which are molecules that function as regulators of gene expression. Various sncRNAs have emerged as having an important role in the immune system in humans. The study uses transcriptomic read counts to profile the type and quantity of both well and lesser characterized sncRNAs, such as microRNAs and small nucleolar RNAs to identify and quantify the classes of sncRNA expressed in whole blood between healthy and highly pathogenic PRRSV-infected pigs. Our results returned evidence on nine classes of sncRNA, four of which were consistently statistically significantly different based on Fisher's Exact Test, that can be detected and possibly interrogated for their effect on host dysregulation during PRRSV infections. Published by Elsevier Inc.

  13. sRNA-Mediated Regulation of P-Fimbriae Phase Variation in Uropathogenic Escherichia coli

    DEFF Research Database (Denmark)

    Khandige, Surabhi; Kronborg, Tina; Uhlin, Bernt Eric

    2015-01-01

    Uropathogenic Escherichia coli (UPEC) are capable of occupying physiologically distinct intracellular and extracellular niches within the urinary tract. This feat requires the timely regulation of gene expression and small RNAs (sRNAs) are known to mediate such rapid adjustments in response to ch...... to changing environmental cues. This study aimed to uncover sRNA-mediated gene regulation in the UPEC strain UTI89, during infection of bladder epithelial cells. Hfq is an RNA chaperone known to facilitate and stabilize sRNA and target mRNA interactions with bacterial cells. The co...... to the discovery of a novel virulence-associated trans-acting sRNA-PapR. Deletion of papR was found to enhance adhesion of UTI89 to both bladder and kidney cell lines in a manner independent of type-1 fimbriae. We demonstrate PapR mediated posttranscriptional repression of the P-fimbriae phase regulator gene pap...

  14. The Regulatory Small RNA MarS Supports Virulence of Streptococcus pyogenes.

    Science.gov (United States)

    Pappesch, Roberto; Warnke, Philipp; Mikkat, Stefan; Normann, Jana; Wisniewska-Kucper, Aleksandra; Huschka, Franziska; Wittmann, Maja; Khani, Afsaneh; Schwengers, Oliver; Oehmcke-Hecht, Sonja; Hain, Torsten; Kreikemeyer, Bernd; Patenge, Nadja

    2017-09-25

    Small regulatory RNAs (sRNAs) play a role in the control of bacterial virulence gene expression. In this study, we investigated an sRNA that was identified in Streptococcus pyogenes (group A Streptococcus, GAS) but is conserved throughout various streptococci. In a deletion strain, expression of mga, the gene encoding the multiple virulence gene regulator, was reduced. Accordingly, transcript and proteome analyses revealed decreased expression of several Mga-activated genes. Therefore, and because the sRNA was shown to interact with the 5' UTR of the mga transcript in a gel-shift assay, we designated it MarS for m ga-activating regulatory sRNA. Down-regulation of important virulence factors, including the antiphagocytic M-protein, led to increased susceptibility of the deletion strain to phagocytosis and reduced adherence to human keratinocytes. In a mouse infection model, the marS deletion mutant showed reduced dissemination to the liver, kidney, and spleen. Additionally, deletion of marS led to increased tolerance towards oxidative stress. Our in vitro and in vivo results indicate a modulating effect of MarS on virulence gene expression and on the pathogenic potential of GAS.

  15. Associations of mRNA:microRNA for the shared downstream molecules of EGFR and alternative tyrosine kinase receptors in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Fengfeng Wang

    2016-10-01

    Full Text Available Lung cancer is the top cancer killer worldwide with high mortality rate. Majority belong to non-small cell lung cancers (NSCLCs. The epidermal growth factor receptor (EGFR has been broadly explored as a drug target for therapy. However, the drug responses are not durable due to the acquired resistance. MicroRNAs (miRNAs are small noncoding and endogenous molecules that can inhibit mRNA translation initiation and degrade mRNAs. We wonder if some downstream molecules shared by EGFR and the other tyrosine kinase receptors (TKRs further transduce the signals alternatively, and some miRNAs play the key roles in affecting the expression of these downstream molecules. In this study, we investigated the mRNA:miRNA associations for the direct EGFR downstream molecules in the EGFR signaling pathway shared with the other TKRs, including c-MET (hepatocyte growth factor receptor, Ron (a protein tyrosine kinase related to c-MET, PDGFR (platelet-derived growth factor receptor, and IGF-1R (insulin-like growth factor receptor-1. The multiple linear regression and support vector regression (SVR models were used to discover the statistically significant and the best weighted miRNAs regulating the mRNAs of these downstream molecules. These two models revealed the similar mRNA:miRNA associations. It was found that the miRNAs significantly affecting the mRNA expressions in the multiple regression model were also those with the largest weights in the SVR model. To conclude, we effectively identified a list of meaningful mRNA:miRNA associations: phospholipase C, gamma 1 (PLCG1 with miR-34a, phosphoinositide-3-kinase, regulatory subunit 2 (PIK3R2 with miR-30a-5p, growth factor receptor-bound protein 2 (GRB2 with miR-27a, and Janus kinase 1 (JAK1 with miR-302b and miR-520e. These associations could make great contributions to explore new mechanism in NSCLCs. These candidate miRNAs may be regarded as the potential drug targets for treating NSCLCs with acquired drug

  16. Extraction of low molecular weight RNA from Citrus trifolita tissues ...

    African Journals Online (AJOL)

    Jane

    2010-12-20

    Dec 20, 2010 ... A critical prerequisite in miRNA studies is acquisition of high quality LMW RNA. LMW RNA is ..... air-dried for a few minutes and then exposed to BIOMAX X-ray film for 48 h using an .... Approaches to microRNA discovery. Nat.

  17. Modular arrangement of regulatory RNA elements.

    Science.gov (United States)

    Roßmanith, Johanna; Narberhaus, Franz

    2017-03-04

    Due to their simple architecture and control mechanism, regulatory RNA modules are attractive building blocks in synthetic biology. This is especially true for riboswitches, which are natural ligand-binding regulators of gene expression. The discovery of various tandem riboswitches inspired the design of combined RNA modules with activities not yet found in nature. Riboswitches were placed in tandem or in combination with a ribozyme or temperature-responsive RNA thermometer resulting in new functionalities. Here, we compare natural examples of tandem riboswitches with recently designed artificial RNA regulators suggesting substantial modularity of regulatory RNA elements. Challenges associated with modular RNA design are discussed.

  18. Small RNA fragments in complex culture media cause alterations in protein profiles of three species of bacteria.

    Science.gov (United States)

    Pavankumar, Asalapuram R; Ayyappasamy, Sudalaiyadum Perumal; Sankaran, Krishnan

    2012-03-01

    Efforts to delineate the basis for variations in protein profiles of different membrane fractions from various bacterial pathogens led to the finding that even the same medium [e.g., Luria Bertani (LB) broth] purchased from different commercial sources generates remarkably dissimilar protein profiles despite similar growth characteristics. Given the pervasive roles small RNAs play in regulating gene expression, we inquired if these source-specific differences due to media arise from disparities in the presence of small RNAs. Indeed, LB media components from two different commercial suppliers contained varying, yet significant, amounts of 10-80 bp small RNAs. Removal of small RNA from LB using RNaseA during media preparation resulted in significant changes in bacterial protein expression profiles. Our studies underscore the fact that seemingly identical growth media can lead to dramatic alterations in protein expression patterns, highlighting the importance of utilizing media free of small RNA during bacteriological studies. Finally, these results raise the intriguing possibility that similar pools of small RNAs in the environment can influence bacterial adaptation.

  19. Small Rna Regulatory Networks In Pseudomonas Putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara; Long, Katherine

    2015-01-01

    chemicals and has a potential to be used as an efficient cell factory for various products. P. putida KT2240 is a genome-sequenced strain and a well characterized pseudomonad. Our major aim is to identify small RNA molecules (sRNAs) and their regulatory networks. A previous study has identified 37 sRNAs...... in this strain, while in other pseudomonads many more sRNAs have been found so far.P. putida KT2440 has been grown in different conditions which are likely to be encountered in industrial fermentations with the aim of using sRNAs for generation of improved cell factories. For that, cells have been grown in LB......Pseudomonas putida is a ubiquitous Gram-negative soil bacterium with a versatile metabolism and ability to degrade various toxic compounds. It has a high tolerance to different future biobased building blocks and various other stringent conditions. It is used in industry to produce some important...

  20. De novo transcriptome and small RNA analysis of two Chinese willow cultivars reveals stress response genes in Salix matsudana.

    Directory of Open Access Journals (Sweden)

    Guodong Rao

    Full Text Available Salix matsudana Koidz. is a deciduous, rapidly growing, and drought resistant tree and is one of the most widely distributed and commonly cultivated willow species in China. Currently little transcriptomic and small RNAomic data are available to reveal the genes involve in the stress resistant in S. matsudana. Here, we report the RNA-seq analysis results of both transcriptome and small RNAome data using Illumina deep sequencing of shoot tips from two willow variants(Salix. matsudana and Salix matsudana Koidz. cultivar 'Tortuosa'. De novo gene assembly was used to generate the consensus transcriptome and small RNAome, which contained 106,403 unique transcripts with an average length of 944 bp and a total length of 100.45 MB, and 166 known miRNAs representing 35 miRNA families. Comparison of transcriptomes and small RNAomes combined with quantitative real-time PCR from the two Salix libraries revealed a total of 292 different expressed genes(DEGs and 36 different expressed miRNAs (DEMs. Among the DEGs and DEMs, 196 genes and 24 miRNAs were up regulated, 96 genes and 12 miRNA were down regulated in S. matsudana. Functional analysis of DEGs and miRNA targets showed that many genes were involved in stress resistance in S. matsudana. Our global gene expression profiling presents a comprehensive view of the transcriptome and small RNAome which provide valuable information and sequence resources for uncovering the stress response genes in S. matsudana. Moreover the transcriptome and small RNAome data provide a basis for future study of genetic resistance in Salix.

  1. cWords - systematic microRNA regulatory motif discovery from mRNA expression data

    DEFF Research Database (Denmark)

    Rasmussen, Simon Horskjær; Jacobsen, Anders; Krogh, Anders

    2013-01-01

    and statistical methods of cWords, resulting in at least a factor 100 speed gain over the previous implementation. On a benchmark dataset of 19 microRNA (miRNA) perturbation experiments cWords showed equal or better performance than two comparable methods, miReduce and Sylamer. We have developed rigorous motif...... that demonstrate comparable or better performance than other existing methods. Rich visualization of results promotes intuitive and efficient interpretation of data. cWords is available as a stand-alone Open Source program at Github https://github.com/simras/cWords webcite and as a web-service at: http...

  2. Data Mining of Small RNA-Seq Suggests an Association Between Prostate Cancer and Altered Abundance of 5′ Transfer RNA Halves in Seminal Fluid and Prostatic Tissues

    Directory of Open Access Journals (Sweden)

    Joseph M Dhahbi

    2018-02-01

    Full Text Available Extracellular RNAs are gaining clinical interest as biofluid-based noninvasive markers for diseases, especially cancer. In particular, derivatives of transfer RNA (tRNA are emerging as a new class of small-noncoding RNAs with high biomarker potential. We and others previously reported alterations in serum levels of specific tRNA halves in disease states including cancer. Here, we explored seminal fluid for tRNA halves as potential markers of prostate cancer. We found that 5′ tRNA halves are abundant in seminal fluid and are elevated in prostate cancer relative to noncancer patients. Importantly, most of these tRNA halves are also detectable in prostatic tissues, and a subset were increased in malignant relative to adjacent normal tissue. These findings emphasize the potential of 5′ tRNA halves as noninvasive markers for prostate cancer screening and diagnosis and provide leads for future work to elucidate a putative role of the 5′ tRNA halves in carcinogenesis.

  3. Analysis of small RNA production patterns among the two potato spindle tuber viroid variants in tomato plants

    Directory of Open Access Journals (Sweden)

    Charith Raj Adkar-Purushothama

    2015-12-01

    Full Text Available In order to analyze the production of small RNA (sRNA by viroids upon infecting the plants, the tomato plants (Solanum lycopersicum cultivar Rutgers were inoculated with the variants of Potato spindle tuber viroid (PSTVd. After 21-days of postinoculation, total RNA was extracted and subjected for deep-sequencing using Illumina HiSeq platform. The primers were trimmed and only 21- to 24-nt long sRNAs were filtered after quality check of the raw data. The filtered sRNA population was then mapped against both the genomic (+ and antigenomic (− strands of the respective PSTVd variants using standard pattern-matching algorithm. The profiling of viroid derived sRNA (vd-sRNA revealed that the viroids are susceptible to host RNA silencing mechanism. High-throughput sequence data linked to this project have been deposited in the Gene Expression Omnibus (GEO database under accession number GSE69225.

  4. The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

    Science.gov (United States)

    Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P; Pino, Karla; Tischler, Nicole D; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

    2012-02-01

    The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

  5. Identification of the base-pairing requirements for repression of hctA translation by the small RNA IhtA leads to the discovery of a new mRNA target in Chlamydia trachomatis.

    Directory of Open Access Journals (Sweden)

    Nicole A Grieshaber

    Full Text Available The non-coding small RNA, IhtA expressed by the obligate intracellular human pathogen Chlamydia trachomatis modulates the translation of HctA, a key protein involved in replicative to infectious cell type differentiation. Using a combination of bioinformatics and mutagenesis we sought to identify the base pairing requirement for functional repression of HctA protein expression, with an eye to applying our findings towards the identification of additional targets. IhtA is predicted to fold into a three stem:loop structure. We found that loop 1 occludes the initiation codon of hctA, while loop 2 and 3 are not required for function. This 7 nucleotide region forms G/C rich interactions surrounding the AUG of hctA. Two additional genes in the chlamydial genome, CTL0322 and CTL0097, contained some elements of the hctA:IhtA recognition sequence. The mRNA of both CTL0322and CTL0097 interacted with IhtA in vitro as measured by biolayer interferometry. However, using a CheZ reporter expression system, IhtA only inhibited the translation of CTL0322. The proposed IhtA recognition site in the CTL0322 message contains significant G/C base pairing on either side of the initiation codon while CTL0097 only contains G/C base pairing 3' to the AUG initiation codon. These data suggest that as the functional interacting region is only 6-7nt in length that full translation repression is dependent on the degree of G/C base pairing. Additionally our results indicate that IhtA may regulate multiple mRNAs involved in the chlamydial infectious cycle.

  6. Ancient Origin of the U2 Small Nuclear RNA Gene-Targeting Non-LTR Retrotransposons Utopia.

    Science.gov (United States)

    Kojima, Kenji K; Jurka, Jerzy

    2015-01-01

    Most non-long terminal repeat (non-LTR) retrotransposons encoding a restriction-like endonuclease show target-specific integration into repetitive sequences such as ribosomal RNA genes and microsatellites. However, only a few target-specific lineages of non-LTR retrotransposons are distributed widely and no lineage is found across the eukaryotic kingdoms. Here we report the most widely distributed lineage of target sequence-specific non-LTR retrotransposons, designated Utopia. Utopia is found in three supergroups of eukaryotes: Amoebozoa, SAR, and Opisthokonta. Utopia is inserted into a specific site of U2 small nuclear RNA genes with different strength of specificity for each family. Utopia families from oomycetes and wasps show strong target specificity while only a small number of Utopia copies from reptiles are flanked with U2 snRNA genes. Oomycete Utopia families contain an "archaeal" RNase H domain upstream of reverse transcriptase (RT), which likely originated from a plant RNase H gene. Analysis of Utopia from oomycetes indicates that multiple lineages of Utopia have been maintained inside of U2 genes with few copy numbers. Phylogenetic analysis of RT suggests the monophyly of Utopia, and it likely dates back to the early evolution of eukaryotes.

  7. Discovery of inhibitors of bacterial histidine kinases

    NARCIS (Netherlands)

    Velikova, N.R.

    2014-01-01

    Discovery of Inhibitors of Bacterial Histidine Kinases Summary

    The thesis is on novel antibacterial drug discovery (http://youtu.be/NRMWOGgeysM). Using structure-based and fragment-based drug discovery approach, we have identified small-molecule histidine-kinase

  8. MicroRNA-dependent regulation of transcription in non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Sonia Molina-Pinelo

    Full Text Available Squamous cell lung cancer (SCC and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC, and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA and mRNA profiling (Whole Genome 44 K array G112A, Agilent was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708 and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1 were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

  9. Single Nucleotide Polymorphism Discovery in Bovine Pituitary Gland Using RNA-Seq Technology.

    Science.gov (United States)

    Pareek, Chandra Shekhar; Smoczyński, Rafał; Kadarmideen, Haja N; Dziuba, Piotr; Błaszczyk, Paweł; Sikora, Marcin; Walendzik, Paulina; Grzybowski, Tomasz; Pierzchała, Mariusz; Horbańczuk, Jarosław; Szostak, Agnieszka; Ogluszka, Magdalena; Zwierzchowski, Lech; Czarnik, Urszula; Fraser, Leyland; Sobiech, Przemysław; Wąsowicz, Krzysztof; Gelfand, Brian; Feng, Yaping; Kumar, Dibyendu

    2016-01-01

    Examination of bovine pituitary gland transcriptome by strand-specific RNA-seq allows detection of putative single nucleotide polymorphisms (SNPs) within potential candidate genes (CGs) or QTLs regions as well as to understand the genomics variations that contribute to economic trait. Here we report a breed-specific model to successfully perform the detection of SNPs in the pituitary gland of young growing bulls representing Polish Holstein-Friesian (HF), Polish Red, and Hereford breeds at three developmental ages viz., six months, nine months, and twelve months. A total of 18 bovine pituitary gland polyA transcriptome libraries were prepared and sequenced using the Illumina NextSeq 500 platform. Sequenced FastQ databases of all 18 young bulls were submitted to NCBI-SRA database with NCBI-SRA accession numbers SRS1296732. For the investigated young bulls, a total of 113,882,3098 raw paired-end reads with a length of 156 bases were obtained, resulting in an approximately 63 million paired-end reads per library. Breed-wise, a total of 515.38, 215.39, and 408.04 million paired-end reads were obtained for Polish HF, Polish Red, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed 93.04%, 94.39%, and 83.46% of the mapped sequencing reads were properly paired to the Polish HF, Polish Red, and Hereford breeds, respectively. Constructed breed-specific SNP-db of three cattle breeds yielded at 13,775,885 SNPs. On an average 765,326 breed-specific SNPs per young bull were identified. Using two stringent filtering parameters, i.e., a minimum 10 SNP reads per base with an accuracy ≥ 90% and a minimum 10 SNP reads per base with an accuracy = 100%, SNP-db records were trimmed to construct a highly reliable SNP-db. This resulted in a reduction of 95,7% and 96,4% cut-off mark of constructed raw SNP-db. Finally, SNP discoveries using RNA-Seq data were validated by KASP™ SNP genotyping assay. The comprehensive QTLs/CGs analysis of 76 QTLs

  10. Single Nucleotide Polymorphism Discovery in Bovine Pituitary Gland Using RNA-Seq Technology.

    Directory of Open Access Journals (Sweden)

    Chandra Shekhar Pareek

    Full Text Available Examination of bovine pituitary gland transcriptome by strand-specific RNA-seq allows detection of putative single nucleotide polymorphisms (SNPs within potential candidate genes (CGs or QTLs regions as well as to understand the genomics variations that contribute to economic trait. Here we report a breed-specific model to successfully perform the detection of SNPs in the pituitary gland of young growing bulls representing Polish Holstein-Friesian (HF, Polish Red, and Hereford breeds at three developmental ages viz., six months, nine months, and twelve months. A total of 18 bovine pituitary gland polyA transcriptome libraries were prepared and sequenced using the Illumina NextSeq 500 platform. Sequenced FastQ databases of all 18 young bulls were submitted to NCBI-SRA database with NCBI-SRA accession numbers SRS1296732. For the investigated young bulls, a total of 113,882,3098 raw paired-end reads with a length of 156 bases were obtained, resulting in an approximately 63 million paired-end reads per library. Breed-wise, a total of 515.38, 215.39, and 408.04 million paired-end reads were obtained for Polish HF, Polish Red, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA read alignments showed 93.04%, 94.39%, and 83.46% of the mapped sequencing reads were properly paired to the Polish HF, Polish Red, and Hereford breeds, respectively. Constructed breed-specific SNP-db of three cattle breeds yielded at 13,775,885 SNPs. On an average 765,326 breed-specific SNPs per young bull were identified. Using two stringent filtering parameters, i.e., a minimum 10 SNP reads per base with an accuracy ≥ 90% and a minimum 10 SNP reads per base with an accuracy = 100%, SNP-db records were trimmed to construct a highly reliable SNP-db. This resulted in a reduction of 95,7% and 96,4% cut-off mark of constructed raw SNP-db. Finally, SNP discoveries using RNA-Seq data were validated by KASP™ SNP genotyping assay. The comprehensive QTLs/CGs analysis

  11. Mouse nucleolin binds to 4.5S RNAH, a small noncoding RNA

    International Nuclear Information System (INIS)

    Hirose, Yutaka; Harada, Fumio

    2008-01-01

    4.5S RNAH is a rodent-specific small noncoding RNA that exhibits extensive homology to the B1 short interspersed element. Although 4.5S RNAH is known to associate with cellular poly(A)-terminated RNAs and retroviral genomic RNAs, its function remains unclear. In this study, we analyzed 4.5S RNAH-binding proteins in mouse nuclear extracts using gel mobility shift and RNA-protein UV cross-linking assays. We found that at least nine distinct polypeptides (p170, p110, p93, p70, p48, p40, p34, p20, and p16.5) specifically interacted with 4.5S RNAHin vitro. Using anti-La antibody, p48 was identified as mouse La protein. To identify the other 4.5S RNAH-binding proteins, we performed expression cloning from a mouse cDNA library and obtained cDNA clones derived from nucleolin mRNA. We identified p110 as nucleolin using nucleolin-specific antibodies. UV cross-linking analysis using various deletion mutants of nucleolin indicated that the third of four tandem RNA recognition motifs is a major determinant for 4.5S RNAH recognition. Immunoprecipitation of nucleolin from the subcellular fractions of mouse cell extracts revealed that a portion of the endogenous 4.5S RNAH was associated with nucleolin and that this complex was located in both the nucleoplasm and nucleolus

  12. Small RNA analysis in Petunia hybrida identifies unusual tissue-specific expression patterns of conserved miRNAs and of a 24mer RNA

    Science.gov (United States)

    Tedder, Philip; Zubko, Elena; Westhead, David R.; Meyer, Peter

    2009-01-01

    Two pools of small RNAs were cloned from inflorescences of Petunia hybrida using a 5′-ligation dependent and a 5′-ligation independent approach. The two libraries were integrated into a public website that allows the screening of individual sequences against 359,769 unique clones. The library contains 15 clones with 100% identity and 53 clones with one mismatch to miRNAs described for other plant species. For two conserved miRNAs, miR159 and miR390, we find clear differences in tissue-specific distribution, compared with other species. This shows that evolutionary conservation of miRNA sequences does not necessarily include a conservation of the miRNA expression profile. Almost 60% of all clones in the database are 24-nucleotide clones. In accordance with the role of 24mers in marking repetitive regions, we find them distributed across retroviral and transposable element sequences but other 24mers map to promoter regions and to different transcript regions. For one target region we observe tissue-specific variation of matching 24mers, which demonstrates that, as for 21mers, 24mer concentrations are not necessarily identical in different tissues. Asymmetric distribution of a putative novel miRNA in the two libraries suggests that the cloning method can be selective for the representation of certain small RNAs in a collection. PMID:19369427

  13. The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs.

    Directory of Open Access Journals (Sweden)

    Hongtao Hu

    Full Text Available MicroRNAs (miRNAs and secondary small interfering RNAs (principally phased siRNAs or trans-acting siRNAs are two distinct subfamilies of small RNAs (sRNAs that are emerging as key regulators of posttranscriptional gene expression in plants. Both miRNAs and secondary-siRNAs (sec-siRNAs are processed from longer RNA precursors by DICER-LIKE proteins (DCLs. Gossypium arboreum L., also known as tree cotton or Asian cotton, is a diploid, possibly ancestral relative of tetraploid Gossypium hirsutum L., the predominant type of commercially grown cotton worldwide known as upland cotton. To understand the biological significance of these gene regulators in G. arboreum, a bioinformatics analysis was performed on G. arboreum small RNAs produced from G. arboreum leaf, flower, and boll tissues. Consequently, 263 miRNAs derived from 353 precursors, including 155 conserved miRNAs (cs-miRNAs and 108 novel lineage-specific miRNAs (ls-miRNAs. Along with miRNAs, 2,033 miRNA variants (isomiRNAs were identified as well. Those isomiRNAs with variation at the 3'-miRNA end were expressed at the highest levels, compared to other types of variants. In addition, 755 pha-siRNAs derived 319 pha-siRNA gene transcripts (PGTs were identified, and the potential pha-siRNA initiators were predicted. Also, 2,251 non-phased siRNAs were found as well, of which 1,088 appeared to be produced by so-called cis- or trans-cleavage of the PGTs observed at positions differing from pha-siRNAs. Of those sRNAs, 148 miRNAs/isomiRNAs and 274 phased/non-phased siRNAs were differentially expressed in one or more pairs of tissues examined. Target analysis revealed that target genes for both miRNAs and pha-siRNAs are involved a broad range of metabolic and enzymatic activities. We demonstrate that secondary siRNA production could result from initial cleavage of precursors by both miRNAs or isomiRNAs, and that subsequently produced phased and unphased siRNAs could result that also serve as triggers

  14. The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs.

    Science.gov (United States)

    Hu, Hongtao; Rashotte, Aaron M; Singh, Narendra K; Weaver, David B; Goertzen, Leslie R; Singh, Shree R; Locy, Robert D

    2015-01-01

    MicroRNAs (miRNAs) and secondary small interfering RNAs (principally phased siRNAs or trans-acting siRNAs) are two distinct subfamilies of small RNAs (sRNAs) that are emerging as key regulators of posttranscriptional gene expression in plants. Both miRNAs and secondary-siRNAs (sec-siRNAs) are processed from longer RNA precursors by DICER-LIKE proteins (DCLs). Gossypium arboreum L., also known as tree cotton or Asian cotton, is a diploid, possibly ancestral relative of tetraploid Gossypium hirsutum L., the predominant type of commercially grown cotton worldwide known as upland cotton. To understand the biological significance of these gene regulators in G. arboreum, a bioinformatics analysis was performed on G. arboreum small RNAs produced from G. arboreum leaf, flower, and boll tissues. Consequently, 263 miRNAs derived from 353 precursors, including 155 conserved miRNAs (cs-miRNAs) and 108 novel lineage-specific miRNAs (ls-miRNAs). Along with miRNAs, 2,033 miRNA variants (isomiRNAs) were identified as well. Those isomiRNAs with variation at the 3'-miRNA end were expressed at the highest levels, compared to other types of variants. In addition, 755 pha-siRNAs derived 319 pha-siRNA gene transcripts (PGTs) were identified, and the potential pha-siRNA initiators were predicted. Also, 2,251 non-phased siRNAs were found as well, of which 1,088 appeared to be produced by so-called cis- or trans-cleavage of the PGTs observed at positions differing from pha-siRNAs. Of those sRNAs, 148 miRNAs/isomiRNAs and 274 phased/non-phased siRNAs were differentially expressed in one or more pairs of tissues examined. Target analysis revealed that target genes for both miRNAs and pha-siRNAs are involved a broad range of metabolic and enzymatic activities. We demonstrate that secondary siRNA production could result from initial cleavage of precursors by both miRNAs or isomiRNAs, and that subsequently produced phased and unphased siRNAs could result that also serve as triggers of a second

  15. Integrated mRNA and microRNA analysis identifies genes and small miRNA molecules associated with transcriptional and post-transcriptional-level responses to both drought stress and re-watering treatment in tobacco.

    Science.gov (United States)

    Chen, Qiansi; Li, Meng; Zhang, Zhongchun; Tie, Weiwei; Chen, Xia; Jin, Lifeng; Zhai, Niu; Zheng, Qingxia; Zhang, Jianfeng; Wang, Ran; Xu, Guoyun; Zhang, Hui; Liu, Pingping; Zhou, Huina

    2017-01-10

    Drought stress is one of the most severe problem limited agricultural productivity worldwide. It has been reported that plants response to drought-stress by sophisticated mechanisms at both transcriptional and post-transcriptional levels. However, the precise molecular mechanisms governing the responses of tobacco leaves to drought stress and water status are not well understood. To identify genes and miRNAs involved in drought-stress responses in tobacco, we performed both mRNA and small RNA sequencing on tobacco leaf samples from the following three treatments: untreated-control (CL), drought stress (DL), and re-watering (WL). In total, we identified 798 differentially expressed genes (DEGs) between the DL and CL (DL vs. CL) treatments and identified 571 DEGs between the WL and DL (WL vs. DL) treatments. Further analysis revealed 443 overlapping DEGs between the DL vs. CL and WL vs. DL comparisons, and, strikingly, all of these genes exhibited opposing expression trends between these two comparisons, strongly suggesting that these overlapping DEGs are somehow involved in the responses of tobacco leaves to drought stress. Functional annotation analysis showed significant up-regulation of genes annotated to be involved in responses to stimulus and stress, (e.g., late embryogenesis abundant proteins and heat-shock proteins) antioxidant defense (e.g., peroxidases and glutathione S-transferases), down regulation of genes related to the cell cycle pathway, and photosynthesis processes. We also found 69 and 56 transcription factors (TFs) among the DEGs in, respectively, the DL vs. CL and the WL vs. DL comparisons. In addition, small RNA sequencing revealed 63 known microRNAs (miRNA) from 32 families and 368 novel miRNA candidates in tobacco. We also found that five known miRNA families (miR398, miR390, miR162, miR166, and miR168) showed differential regulation under drought conditions. Analysis to identify negative correlations between the differentially expressed mi

  16. DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Willson Richard C

    2010-12-01

    Full Text Available Abstract Background Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Results Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. Conclusions The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use

  17. Novel microRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection.

    Science.gov (United States)

    Shi, Jiandong; Sun, Jing; Wang, Bin; Wu, Meini; Zhang, Jing; Duan, Zhiqing; Wang, Haixuan; Hu, Ningzhu; Hu, Yunzhang

    2014-10-01

    MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host-virus interactions. DNA viruses encode miRNAs that regulate the viral life cycle. However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery. Here, we provide a comprehensive genome-wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV; Hu/China/H2/1982), which is a typical cytoplasmic RNA virus. Using deep-sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav-miR-1-5p and hav-miR-2-5p. Both of the novel virally encoded miRNAs were clearly detected in infected cells. Analysis of Dicer enzyme silencing demonstrated that HAV-derived miRNA biogenesis is Dicer dependent. Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre-miRNAs) in host cells. Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post-transcriptional gene silencing (PTGS). Genomic location analysis revealed novel miRNAs located in the coding region of the viral genome. Overall, our results show that HAV naturally generates functional miRNA-like small regulatory RNAs during infection. This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus. These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do. These findings also contribute to improved understanding of host-RNA virus interactions mediated by RNA virus-derived miRNAs. © FASEB.

  18. Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes.

    Science.gov (United States)

    Sittka, Alexandra; Sharma, Cynthia M; Rolle, Katarzyna; Vogel, Jörg

    2009-01-01

    The bacterial Sm-like protein, Hfq, is a key factor for the stability and function of small non-coding RNAs (sRNAs) in Escherichia coli. Homologues of this protein have been predicted in many distantly related organisms yet their functional conservation as sRNA-binding proteins has not entirely been clear. To address this, we expressed in Salmonella the Hfq proteins of two eubacteria (Neisseria meningitides, Aquifex aeolicus) and an archaeon (Methanocaldococcus jannaschii), and analyzed the associated RNA by deep sequencing. This in vivo approach identified endogenous Salmonella sRNAs as a major target of the foreign Hfq proteins. New Salmonella sRNA species were also identified, and some of these accumulated specifically in the presence of a foreign Hfq protein. In addition, we observed specific RNA processing defects, e.g., suppression of precursor processing of SraH sRNA by Methanocaldococcus Hfq, or aberrant accumulation of extracytoplasmic target mRNAs of the Salmonella GcvB, MicA or RybB sRNAs. Taken together, our study provides evidence of a conserved inherent sRNA-binding property of Hfq, which may facilitate the lateral transmission of regulatory sRNAs among distantly related species. It also suggests that the expression of heterologous RNA-binding proteins combined with deep sequencing analysis of RNA ligands can be used as a molecular tool to dissect individual steps of RNA metabolism in vivo.

  19. Endogenous MCM7 microRNA cluster as a novel platform to multiplex small interfering and nucleolar RNAs for combinational HIV-1 gene therapy.

    Science.gov (United States)

    Chung, Janet; Zhang, Jane; Li, Haitang; Ouellet, Dominique L; DiGiusto, David L; Rossi, John J

    2012-11-01

    Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications.

  20. RNA Profiling for Biomarker Discovery: Practical Considerations for Limiting Sample Sizes

    Directory of Open Access Journals (Sweden)

    Danny J. Kelly

    2005-01-01

    Full Text Available We have compared microarray data generated on Affymetrix™ chips from standard (8 micrograms or low (100 nanograms amounts of total RNA. We evaluated the gene signals and gene fold-change estimates obtained from the two methods and validated a subset of the results by real time, polymerase chain reaction assays. The correlation of low RNA derived gene signals to gene signals obtained from standard RNA was poor for less to moderately abundant genes. Genes with high abundance showed better correlation in signals between the two methods. The signal correlation between the low RNA and standard RNA methods was improved by including a reference sample in the microarray analysis. In contrast, the fold-change estimates for genes were better correlated between the two methods regardless of the magnitude of gene signals. A reference sample based method is suggested for studies that would end up comparing gene signal data from a combination of low and standard RNA templates; no such referencing appears to be necessary when comparing fold-changes of gene expression between standard and low template reactions.

  1. Comprehensive processing of high-throughput small RNA sequencing data including quality checking, normalization, and differential expression analysis using the UEA sRNA Workbench.

    Science.gov (United States)

    Beckers, Matthew; Mohorianu, Irina; Stocks, Matthew; Applegate, Christopher; Dalmay, Tamas; Moulton, Vincent

    2017-06-01

    Recently, high-throughput sequencing (HTS) has revealed compelling details about the small RNA (sRNA) population in eukaryotes. These 20 to 25 nt noncoding RNAs can influence gene expression by acting as guides for the sequence-specific regulatory mechanism known as RNA silencing. The increase in sequencing depth and number of samples per project enables a better understanding of the role sRNAs play by facilitating the study of expression patterns. However, the intricacy of the biological hypotheses coupled with a lack of appropriate tools often leads to inadequate mining of the available data and thus, an incomplete description of the biological mechanisms involved. To enable a comprehensive study of differential expression in sRNA data sets, we present a new interactive pipeline that guides researchers through the various stages of data preprocessing and analysis. This includes various tools, some of which we specifically developed for sRNA analysis, for quality checking and normalization of sRNA samples as well as tools for the detection of differentially expressed sRNAs and identification of the resulting expression patterns. The pipeline is available within the UEA sRNA Workbench, a user-friendly software package for the processing of sRNA data sets. We demonstrate the use of the pipeline on a H. sapiens data set; additional examples on a B. terrestris data set and on an A. thaliana data set are described in the Supplemental Information A comparison with existing approaches is also included, which exemplifies some of the issues that need to be addressed for sRNA analysis and how the new pipeline may be used to do this. © 2017 Beckers et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  2. CoLIde: A bioinformatics tool for CO-expression based small RNA Loci Identification using high-throughput sequencing data

    OpenAIRE

    Mohorianu, Irina; Stocks, Matthew Benedict; Wood, John; Dalmay, Tamas; Moulton, Vincent

    2013-01-01

    Small RNAs (sRNAs) are 20–25 nt non-coding RNAs that act as guides for the highly sequence-specific regulatory mechanism known as RNA silencing. Due to the recent increase in sequencing depth, a highly complex and diverse population of sRNAs in both plants and animals has been revealed. However, the exponential increase in sequencing data has also made the identification of individual sRNA transcripts corresponding to biological units (sRNA loci) more challenging when based exclusively on the...

  3. A Convenient In Vivo Model Using Small Interfering RNA Silencing to Rapidly Assess Skeletal Gene Function.

    Directory of Open Access Journals (Sweden)

    Wen Zhang

    Full Text Available It is difficult to study bone in vitro because it contains various cell types that engage in cross-talk. Bone biologically links various organs, and it has thus become increasingly evident that skeletal physiology must be studied in an integrative manner in an intact animal. We developed a model using local intraosseous small interfering RNA (siRNA injection to rapidly assess the effects of a target gene on the local skeletal environment. In this model, 160-g male Sprague-Dawley rats were treated for 1-2 weeks. The left tibia received intraosseous injection of a parathyroid hormone 1 receptor (Pth1r or insulin-like growth factor 1 receptor (Igf-1r siRNA transfection complex loaded in poloxamer 407 hydrogel, and the right tibia received the same volume of control siRNA. All the tibias received an intraosseous injection of recombinant human parathyroid hormone (1-34 (rhPTH (1-34 or insulin-like growth factor-1 (IGF-1. Calcein green and alizarin red were injected 6 and 2 days before euthanasia, respectively. IGF-1R and PTH1R expression levels were detected via RT-PCR assays and immunohistochemistry. Bone mineral density (BMD, microstructure, mineral apposition rates (MARs, and strength were determined by dual-energy X-ray absorptiometry, micro-CT, histology and biomechanical tests. The RT-PCR and immunohistochemistry results revealed that IGF-1R and PTH1R expression levels were dramatically diminished in the siRNA-treated left tibias compared to the right tibias (both p<0.05. Using poloxamer 407 hydrogel as a controlled-release system prolonged the silencing effect of a single dose of siRNA; the mRNA expression levels of IGF-1R were lower at two weeks than at one week (p<0.01. The BMD, bone microstructure parameters, MAR and bone strength were significantly decreased in the left tibias compared to the right tibias (all p<0.05. This simple and convenient local intraosseous siRNA injection model achieved gene silencing with very small quantities of

  4. The genome of the Hi5 germ cell line from Trichoplusia ni, an agricultural pest and novel model for small RNA biology.

    Science.gov (United States)

    Fu, Yu; Yang, Yujing; Zhang, Han; Farley, Gwen; Wang, Junling; Quarles, Kaycee A; Weng, Zhiping; Zamore, Phillip D

    2018-01-29

    We report a draft assembly of the genome of Hi5 cells from the lepidopteran insect pest, Trichoplusia ni , assigning 90.6% of bases to one of 28 chromosomes and predicting 14,037 protein-coding genes. Chemoreception and detoxification gene families reveal T. ni -specific gene expansions that may explain its widespread distribution and rapid adaptation to insecticides. Transcriptome and small RNA data from thorax, ovary, testis, and the germline-derived Hi5 cell line show distinct expression profiles for 295 microRNA- and >393 piRNA-producing loci, as well as 39 genes encoding small RNA pathway proteins. Nearly all of the W chromosome is devoted to piRNA production, and T. ni siRNAs are not 2´- O -methylated. To enable use of Hi5 cells as a model system, we have established genome editing and single-cell cloning protocols. The T. ni genome provides insights into pest control and allows Hi5 cells to become a new tool for studying small RNAs ex vivo. © 2018, Fu et al.

  5. U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

    Science.gov (United States)

    Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

    2014-04-25

    The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Small activating RNA induces myogenic differentiation of rat adipose-derived stem cells by upregulating MyoD

    Directory of Open Access Journals (Sweden)

    Chenghe Wang

    2015-08-01

    Full Text Available ABSTRACTPurpose:RNA activation (RNAa is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs, also known as small activating RNAs (saRNAs. Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI–a condition primarily resulted from urethral sphincter deficiency.

  7. Development of a software tool and criteria evaluation for efficient design of small interfering RNA

    International Nuclear Information System (INIS)

    Chaudhary, Aparna; Srivastava, Sonam; Garg, Sanjeev

    2011-01-01

    Research highlights: → The developed tool predicted siRNA constructs with better thermodynamic stability and total score based on positional and other criteria. → Off-target silencing below score 30 were observed for the best siRNA constructs for different genes. → Immunostimulation and cytotoxicity motifs considered and penalized in the developed tool. → Both positional and compositional criteria were observed to be important. -- Abstract: RNA interference can be used as a tool for gene silencing mediated by small interfering RNAs (siRNA). The critical step in effective and specific RNAi processing is the selection of suitable constructs. Major design criteria, i.e., Reynolds's design rules, thermodynamic stability, internal repeats, immunostimulatory motifs were emphasized and implemented in the siRNA design tool. The tool provides thermodynamic stability score, GC content and a total score based on other design criteria in the output. The viability of the tool was established with different datasets. In general, the siRNA constructs produced by the tool had better thermodynamic score and positional properties. Comparable thermodynamic scores and better total scores were observed with the existing tools. Moreover, the results generated had comparable off-target silencing effect. Criteria evaluations with additional criteria were achieved in WEKA.

  8. Discovery of precursor and mature microRNAs and their putative gene targets using high-throughput sequencing in pineapple (Ananas comosus var. comosus).

    Science.gov (United States)

    Yusuf, Noor Hydayaty Md; Ong, Wen Dee; Redwan, Raimi Mohamed; Latip, Mariam Abd; Kumar, S Vijay

    2015-10-15

    MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Signatures of RNA binding proteins globally coupled to effective microRNA target sites

    DEFF Research Database (Denmark)

    Jacobsen, Anders; Wen, Jiayu; Marks, Debora S

    2010-01-01

    MicroRNAs (miRNAs) and small interfering RNAs (siRNAs), bound to Argonaute proteins (RISC), destabilize mRNAs through base-pairing with the mRNA. However, the gene expression changes after perturbations of these small RNAs are only partially explained by predicted miRNA/siRNA targeting. Targeting...

  10. RNA 3D modules in genome-wide predictions of RNA 2D structure

    DEFF Research Database (Denmark)

    Theis, Corinna; Zirbel, Craig L; Zu Siederdissen, Christian Höner

    2015-01-01

    . These modules can, for example, occur inside structural elements which in RNA 2D predictions appear as internal loops. Hence one question is if the use of such RNA 3D information can improve the prediction accuracy of RNA secondary structure at a genome-wide level. Here, we use RNAz in combination with 3D......Recent experimental and computational progress has revealed a large potential for RNA structure in the genome. This has been driven by computational strategies that exploit multiple genomes of related organisms to identify common sequences and secondary structures. However, these computational...... approaches have two main challenges: they are computationally expensive and they have a relatively high false discovery rate (FDR). Simultaneously, RNA 3D structure analysis has revealed modules composed of non-canonical base pairs which occur in non-homologous positions, apparently by independent evolution...

  11. Control of the gut microbiome by fecal microRNA

    Directory of Open Access Journals (Sweden)

    Shirong Liu

    2016-03-01

    Full Text Available Since their discovery in the early 90s, microRNAs (miRNAs, small non-coding RNAs, have mainly been associated with posttranscriptional regulation of gene expression on a cell-autonomous level. Recent evidence has extended this role by adding inter-species communication to the manifold functional range. In our latest study [Liu S, et al., 2016, Cell Host & Microbe], we identified miRNAs in gut lumen and feces of both mice and humans. We found that intestinal epithelial cells (IEC and Hopx+ cells were the two main sources of fecal miRNA. Deficiency of IEC-miRNA resulted in gut dysbiosis and WT fecal miRNA transplantation restored the gut microbiota. We investigated potential mechanisms for this effect and found that miRNAs were able to regulate the gut microbiome. By culturing bacteria with miRNAs, we found that host miRNAs were able to enter bacteria, specifically regulate bacterial gene transcripts and affect bacterial growth. Oral administration of synthetic miRNA mimics affected specific bacteria in the gut. Our findings describe a previously unknown pathway by which the gut microbiome is regulated by the host and raises the possibility that miRNAs may be used therapeutically to manipulate the microbiome for the treatment of disease.

  12. Bioinformatics for discovery of microbiome variation

    DEFF Research Database (Denmark)

    Brejnrod, Asker Daniel

    of various molecular methods to build hypotheses about the impact of a copper contaminated soil. The introduction is a broad introduction to the field of microbiome research with a focus on the technologies that enable these discoveries and how some of the broader issues have related to this thesis......Sequencing based tools have revolutionized microbiology in recent years. Highthroughput DNA sequencing have allowed high-resolution studies on microbial life in many different environments and at unprecedented low cost. These culture-independent methods have helped discovery of novel bacteria...... 1 ,“Large-scale benchmarking reveals false discoveries and count transformation sensitivity in 16S rRNA gene amplicon data analysis methods used in microbiome studies”, benchmarked the performance of a variety of popular statistical methods for discovering differentially abundant bacteria . between...

  13. Discovery of Protein–lncRNA Interactions by Integrating Large-Scale CLIP-Seq and RNA-Seq Datasets

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jun-Hao; Liu, Shun; Zheng, Ling-Ling; Wu, Jie; Sun, Wen-Ju; Wang, Ze-Lin; Zhou, Hui; Qu, Liang-Hu, E-mail: lssqlh@mail.sysu.edu.cn; Yang, Jian-Hua, E-mail: lssqlh@mail.sysu.edu.cn [RNA Information Center, Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Sun Yat-sen University, Guangzhou (China)

    2015-01-14

    Long non-coding RNAs (lncRNAs) are emerging as important regulatory molecules in developmental, physiological, and pathological processes. However, the precise mechanism and functions of most of lncRNAs remain largely unknown. Recent advances in high-throughput sequencing of immunoprecipitated RNAs after cross-linking (CLIP-Seq) provide powerful ways to identify biologically relevant protein–lncRNA interactions. In this study, by analyzing millions of RNA-binding protein (RBP) binding sites from 117 CLIP-Seq datasets generated by 50 independent studies, we identified 22,735 RBP–lncRNA regulatory relationships. We found that one single lncRNA will generally be bound and regulated by one or multiple RBPs, the combination of which may coordinately regulate gene expression. We also revealed the expression correlation of these interaction networks by mining expression profiles of over 6000 normal and tumor samples from 14 cancer types. Our combined analysis of CLIP-Seq data and genome-wide association studies data discovered hundreds of disease-related single nucleotide polymorphisms resided in the RBP binding sites of lncRNAs. Finally, we developed interactive web implementations to provide visualization, analysis, and downloading of the aforementioned large-scale datasets. Our study represented an important step in identification and analysis of RBP–lncRNA interactions and showed that these interactions may play crucial roles in cancer and genetic diseases.

  14. Discovery of Protein–lncRNA Interactions by Integrating Large-Scale CLIP-Seq and RNA-Seq Datasets

    International Nuclear Information System (INIS)

    Li, Jun-Hao; Liu, Shun; Zheng, Ling-Ling; Wu, Jie; Sun, Wen-Ju; Wang, Ze-Lin; Zhou, Hui; Qu, Liang-Hu; Yang, Jian-Hua

    2015-01-01

    Long non-coding RNAs (lncRNAs) are emerging as important regulatory molecules in developmental, physiological, and pathological processes. However, the precise mechanism and functions of most of lncRNAs remain largely unknown. Recent advances in high-throughput sequencing of immunoprecipitated RNAs after cross-linking (CLIP-Seq) provide powerful ways to identify biologically relevant protein–lncRNA interactions. In this study, by analyzing millions of RNA-binding protein (RBP) binding sites from 117 CLIP-Seq datasets generated by 50 independent studies, we identified 22,735 RBP–lncRNA regulatory relationships. We found that one single lncRNA will generally be bound and regulated by one or multiple RBPs, the combination of which may coordinately regulate gene expression. We also revealed the expression correlation of these interaction networks by mining expression profiles of over 6000 normal and tumor samples from 14 cancer types. Our combined analysis of CLIP-Seq data and genome-wide association studies data discovered hundreds of disease-related single nucleotide polymorphisms resided in the RBP binding sites of lncRNAs. Finally, we developed interactive web implementations to provide visualization, analysis, and downloading of the aforementioned large-scale datasets. Our study represented an important step in identification and analysis of RBP–lncRNA interactions and showed that these interactions may play crucial roles in cancer and genetic diseases.

  15. nRC: non-coding RNA Classifier based on structural features.

    Science.gov (United States)

    Fiannaca, Antonino; La Rosa, Massimo; La Paglia, Laura; Rizzo, Riccardo; Urso, Alfonso

    2017-01-01

    Non-coding RNA (ncRNA) are small non-coding sequences involved in gene expression regulation of many biological processes and diseases. The recent discovery of a large set of different ncRNAs with biologically relevant roles has opened the way to develop methods able to discriminate between the different ncRNA classes. Moreover, the lack of knowledge about the complete mechanisms in regulative processes, together with the development of high-throughput technologies, has required the help of bioinformatics tools in addressing biologists and clinicians with a deeper comprehension of the functional roles of ncRNAs. In this work, we introduce a new ncRNA classification tool, nRC (non-coding RNA Classifier). Our approach is based on features extraction from the ncRNA secondary structure together with a supervised classification algorithm implementing a deep learning architecture based on convolutional neural networks. We tested our approach for the classification of 13 different ncRNA classes. We obtained classification scores, using the most common statistical measures. In particular, we reach an accuracy and sensitivity score of about 74%. The proposed method outperforms other similar classification methods based on secondary structure features and machine learning algorithms, including the RNAcon tool that, to date, is the reference classifier. nRC tool is freely available as a docker image at https://hub.docker.com/r/tblab/nrc/. The source code of nRC tool is also available at https://github.com/IcarPA-TBlab/nrc.

  16. QNB: differential RNA methylation analysis for count-based small-sample sequencing data with a quad-negative binomial model.

    Science.gov (United States)

    Liu, Lian; Zhang, Shao-Wu; Huang, Yufei; Meng, Jia

    2017-08-31

    As a newly emerged research area, RNA epigenetics has drawn increasing attention recently for the participation of RNA methylation and other modifications in a number of crucial biological processes. Thanks to high throughput sequencing techniques, such as, MeRIP-Seq, transcriptome-wide RNA methylation profile is now available in the form of count-based data, with which it is often of interests to study the dynamics at epitranscriptomic layer. However, the sample size of RNA methylation experiment is usually very small due to its costs; and additionally, there usually exist a large number of genes whose methylation level cannot be accurately estimated due to their low expression level, making differential RNA methylation analysis a difficult task. We present QNB, a statistical approach for differential RNA methylation analysis with count-based small-sample sequencing data. Compared with previous approaches such as DRME model based on a statistical test covering the IP samples only with 2 negative binomial distributions, QNB is based on 4 independent negative binomial distributions with their variances and means linked by local regressions, and in the way, the input control samples are also properly taken care of. In addition, different from DRME approach, which relies only the input control sample only for estimating the background, QNB uses a more robust estimator for gene expression by combining information from both input and IP samples, which could largely improve the testing performance for very lowly expressed genes. QNB showed improved performance on both simulated and real MeRIP-Seq datasets when compared with competing algorithms. And the QNB model is also applicable to other datasets related RNA modifications, including but not limited to RNA bisulfite sequencing, m 1 A-Seq, Par-CLIP, RIP-Seq, etc.

  17. Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions

    DEFF Research Database (Denmark)

    Orum, H; Nielsen, Henrik; Engberg, J

    1991-01-01

    We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T....... thermophila snRNAs all have unique 5' ends, which start with an adenine residue. In contrast, with the exception of U6, their 3' ends show some size heterogeneity. The primary sequences of the T. thermophila snRNAs contain the sequence motifs shown, or proposed, to be of functional importance in other...

  18. Assessing Specific Oligonucleotides and Small Molecule Antibiotics for the Ability to Inhibit the CRD-BP-CD44 RNA Interaction

    Science.gov (United States)

    Thomsen, Dana; Lee, Chow H.

    2014-01-01

    Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions. PMID:24622399

  19. RNA graph partitioning for the discovery of RNA modularity: a novel application of graph partition algorithm to biology.

    Directory of Open Access Journals (Sweden)

    Namhee Kim

    Full Text Available Graph representations have been widely used to analyze and design various economic, social, military, political, and biological networks. In systems biology, networks of cells and organs are useful for understanding disease and medical treatments and, in structural biology, structures of molecules can be described, including RNA structures. In our RNA-As-Graphs (RAG framework, we represent RNA structures as tree graphs by translating unpaired regions into vertices and helices into edges. Here we explore the modularity of RNA structures by applying graph partitioning known in graph theory to divide an RNA graph into subgraphs. To our knowledge, this is the first application of graph partitioning to biology, and the results suggest a systematic approach for modular design in general. The graph partitioning algorithms utilize mathematical properties of the Laplacian eigenvector (µ2 corresponding to the second eigenvalues (λ2 associated with the topology matrix defining the graph: λ2 describes the overall topology, and the sum of µ2's components is zero. The three types of algorithms, termed median, sign, and gap cuts, divide a graph by determining nodes of cut by median, zero, and largest gap of µ2's components, respectively. We apply these algorithms to 45 graphs corresponding to all solved RNA structures up through 11 vertices (∼ 220 nucleotides. While we observe that the median cut divides a graph into two similar-sized subgraphs, the sign and gap cuts partition a graph into two topologically-distinct subgraphs. We find that the gap cut produces the best biologically-relevant partitioning for RNA because it divides RNAs at less stable connections while maintaining junctions intact. The iterative gap cuts suggest basic modules and assembly protocols to design large RNA structures. Our graph substructuring thus suggests a systematic approach to explore the modularity of biological networks. In our applications to RNA structures, subgraphs

  20. CoLIde: a bioinformatics tool for CO-expression-based small RNA Loci Identification using high-throughput sequencing data.

    Science.gov (United States)

    Mohorianu, Irina; Stocks, Matthew Benedict; Wood, John; Dalmay, Tamas; Moulton, Vincent

    2013-07-01

    Small RNAs (sRNAs) are 20-25 nt non-coding RNAs that act as guides for the highly sequence-specific regulatory mechanism known as RNA silencing. Due to the recent increase in sequencing depth, a highly complex and diverse population of sRNAs in both plants and animals has been revealed. However, the exponential increase in sequencing data has also made the identification of individual sRNA transcripts corresponding to biological units (sRNA loci) more challenging when based exclusively on the genomic location of the constituent sRNAs, hindering existing approaches to identify sRNA loci. To infer the location of significant biological units, we propose an approach for sRNA loci detection called CoLIde (Co-expression based sRNA Loci Identification) that combines genomic location with the analysis of other information such as variation in expression levels (expression pattern) and size class distribution. For CoLIde, we define a locus as a union of regions sharing the same pattern and located in close proximity on the genome. Biological relevance, detected through the analysis of size class distribution, is also calculated for each locus. CoLIde can be applied on ordered (e.g., time-dependent) or un-ordered (e.g., organ, mutant) series of samples both with or without biological/technical replicates. The method reliably identifies known types of loci and shows improved performance on sequencing data from both plants (e.g., A. thaliana, S. lycopersicum) and animals (e.g., D. melanogaster) when compared with existing locus detection techniques. CoLIde is available for use within the UEA Small RNA Workbench which can be downloaded from: http://srna-workbench.cmp.uea.ac.uk.

  1. Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs.

    Science.gov (United States)

    Miesen, Pascal; Ivens, Alasdair; Buck, Amy H; van Rij, Ronald P

    2016-02-01

    In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species.

  2. Discovery of Fragment-Derived Small Molecules for in Vivo Inhibition of Ketohexokinase (KHK)

    Energy Technology Data Exchange (ETDEWEB)

    Huard, Kim; Ahn, Kay; Amor, Paul; Beebe, David A.; Borzilleri, Kris A.; Chrunyk, Boris A.; Coffey, Steven B.; Cong, Yang; Conn, Edward L.; Culp, Jeffrey S.; Dowling, Matthew S.; Gorgoglione, Matthew F.; Gutierrez, Jemy A.; Knafels, John D.; Lachapelle, Erik A.; Pandit, Jayvardhan; Parris, Kevin D.; Perez, Sylvie; Pfefferkorn, Jeffrey A.; Price, David A.; Raymer, Brian; Ross, Trenton T.; Shavnya, Andre; Smith, Aaron C.; Subashi, Timothy A.; Tesz, Gregory J.; Thuma, Benjamin A.; Tu, Meihua; Weaver, John D.; Weng, Yan; Withka, Jane M.; Xing, Gang; Magee, Thomas V. (Pfizer)

    2017-05-23

    Increased fructose consumption and its subsequent metabolism have been implicated in hepatic steatosis, dyslipidemia, obesity, and insulin resistance in humans. Since ketohexokinase (KHK) is the principal enzyme responsible for fructose metabolism, identification of a selective KHK inhibitor may help to further elucidate the effect of KHK inhibition on these metabolic disorders. Until now, studies on KHK inhibition with small molecules have been limited due to the lack of viable in vivo pharmacological tools. Herein we report the discovery of 12, a selective KHK inhibitor with potency and properties suitable for evaluating KHK inhibition in rat models. Key structural features interacting with KHK were discovered through fragment-based screening and subsequent optimization using structure-based drug design, and parallel medicinal chemistry led to the identification of pyridine 12.

  3. A novel albumin nanocomplex containing both small interfering RNA and gold nanorods for synergetic anticancer therapy

    Science.gov (United States)

    Choi, Jin-Ha; Hwang, Hai-Jin; Shin, Seung Won; Choi, Jeong-Woo; Um, Soong Ho; Oh, Byung-Keun

    2015-05-01

    Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine.Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au

  4. Small RNA and A-to-I Editing in Autism Spectrum Disorders

    Science.gov (United States)

    Eran, Alal

    One in every 88 children is diagnosed with Autism Spectrum Disorders (ASDs), a set of neurodevelopmental conditions characterized by social impairments, communication deficits, and repetitive behavior. ASDs have a substantial genetic component, but the specific cause of most cases remains unknown. Understanding gene-environment interactions underlying ASD is essential for improving early diagnosis and identifying critical targets for intervention and prevention. Towards this goal, we surveyed adenosine-to-inosine (A-to-I) RNA editing in autistic brains. A-to-I editing is an epigenetic mechanism that fine-tunes synaptic function in response to environmental stimuli, shown to modulate complex behavior in animals. We used ultradeep sequencing to quantify A-to-I receding of candidate synaptic genes in postmortem cerebella from individuals with ASD and neurotypical controls. We found unexpectedly wide distributions of human A-to-I editing levels, whose extremes were consistently populated by individuals with ASD. We correlated A-to-I editing with isoform usage, identified clusters of correlated sites, and examined differential editing patterns. Importantly, we found that individuals with ASD commonly use a dysfunctional form of the editing enzyme ADARB1. We next profiled small RNAs thought to regulate A-to-I editing, which originate from one of the most commonly altered loci in ASD, 15q11. Deep targeted sequencing of SNORD115 and SNORD116 transcripts enabled their high-resolution detection in human brains, and revealed a strong gender bias underlying their expression. The consistent 2-fold upregulation of 15q11 small RNAs in male vs. female cerebella could be important in delineating the role of this locus in ASD, a male dominant disorder. Overall, these studies provide an accurate population-level view of small RNA and A-to-I editing in human cerebella, and suggest that A-to-I editing of synaptic genes may be informative for assessing the epigenetic risk for autism

  5. Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA.

    Science.gov (United States)

    Prats, A C; Sarih, L; Gabus, C; Litvak, S; Keith, G; Darlix, J L

    1988-06-01

    Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5' end of the RNA genome. An RSV mutant with a Val-Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way.

  6. Design of a Bioactive Small Molecule that Targets the Myotonic Dystrophy Type 1 RNA Via an RNA Motif-Ligand Database & Chemical Similarity Searching

    Science.gov (United States)

    Parkesh, Raman; Childs-Disney, Jessica L.; Nakamori, Masayuki; Kumar, Amit; Wang, Eric; Wang, Thomas; Hoskins, Jason; Tran, Tuan; Housman, David; Thornton, Charles A.; Disney, Matthew D.

    2012-01-01

    Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5′CUG/3′GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, Sarco(endo)plasmic reticulum Ca2+ ATPase 1 (Serca1/Atp2a1), and cardiac troponin T (cTNT). Based on these observations, the development of small molecule ligands that target specifically expanded DM1 repeats could serve as therapeutics. In the present study, computational screening was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of inhibitors of the RNA-protein complex with low micromolar IC50’s, which are >20-fold more potent than the query compounds, were identified. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with virtual screening. PMID:22300544

  7. Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

    Science.gov (United States)

    Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

    2017-10-01

    Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

  8. Next Generation Sequencing Analysis of Human Platelet PolyA+ mRNAs and rRNA-Depleted Total RNA

    Science.gov (United States)

    Kissopoulou, Antheia; Jonasson, Jon; Lindahl, Tomas L.; Osman, Abdimajid

    2013-01-01

    Background Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. Materials and Methods We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT) isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. Results Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. Conclusion The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components compared with other cell and

  9. Next generation sequencing analysis of human platelet PolyA+ mRNAs and rRNA-depleted total RNA.

    Directory of Open Access Journals (Sweden)

    Antheia Kissopoulou

    Full Text Available BACKGROUND: Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. MATERIALS AND METHODS: We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. RESULTS: Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. CONCLUSION: The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components

  10. 5S rRNA and ribosome.

    Science.gov (United States)

    Gongadze, G M

    2011-12-01

    5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

  11. Potential of small-molecule fungal metabolites in antiviral chemotherapy.

    Science.gov (United States)

    Roy, Biswajit G

    2017-08-01

    Various viral diseases, such as acquired immunodeficiency syndrome, influenza, and hepatitis, have emerged as leading causes of human death worldwide. Scientific endeavor since invention of DNA-dependent RNA polymerase of pox virus in 1967 resulted in better understanding of virus replication and development of various novel therapeutic strategies. Despite considerable advancement in every facet of drug discovery process, development of commercially viable, safe, and effective drugs for these viruses still remains a big challenge. Decades of intense research yielded a handful of natural and synthetic therapeutic options. But emergence of new viruses and drug-resistant viral strains had made new drug development process a never-ending battle. Small-molecule fungal metabolites due to their vast diversity, stereochemical complexity, and preapproved biocompatibility always remain an attractive source for new drug discovery. Though, exploration of therapeutic importance of fungal metabolites has started early with discovery of penicillin, recent prediction asserted that only a small percentage (5-10%) of fungal species have been identified and much less have been scientifically investigated. Therefore, exploration of new fungal metabolites, their bioassay, and subsequent mechanistic study bears huge importance in new drug discovery endeavors. Though no fungal metabolites so far approved for antiviral treatment, many of these exhibited high potential against various viral diseases. This review comprehensively discussed about antiviral activities of fungal metabolites of diverse origin against some important viral diseases. This also highlighted the mechanistic details of inhibition of viral replication along with structure-activity relationship of some common and important classes of fungal metabolites.

  12. NCI RNA Biology 2017 symposium recap | Center for Cancer Research

    Science.gov (United States)

    The recent discovery of new classes of RNAs and the demonstration that alterations in RNA metabolism underlie numerous human cancers have resulted in enormous interest among CCR investigators in RNA biology. In order to share the latest research in this exciting field, the CCR Initiative in RNA Biology held its second international symposium April 23-24, 2017, in Natcher

  13. A contig-based strategy for the genome-wide discovery of microRNAs without complete genome resources.

    Directory of Open Access Journals (Sweden)

    Jun-Zhi Wen

    Full Text Available MicroRNAs (miRNAs are important regulators of many cellular processes and exist in a wide range of eukaryotes. High-throughput sequencing is a mainstream method of miRNA identification through which it is possible to obtain the complete small RNA profile of an organism. Currently, most approaches to miRNA identification rely on a reference genome for the prediction of hairpin structures. However, many species of economic and phylogenetic importance are non-model organisms without complete genome sequences, and this limits miRNA discovery. Here, to overcome this limitation, we have developed a contig-based miRNA identification strategy. We applied this method to a triploid species of edible banana (GCTCV-119, Musa spp. AAA group and identified 180 pre-miRNAs and 314 mature miRNAs, which is three times more than those were predicted by the available dataset-based methods (represented by EST+GSS. Based on the recently published miRNA data set of Musa acuminate, the recall rate and precision of our strategy are estimated to be 70.6% and 92.2%, respectively, significantly better than those of EST+GSS-based strategy (10.2% and 50.0%, respectively. Our novel, efficient and cost-effective strategy facilitates the study of the functional and evolutionary role of miRNAs, as well as miRNA-based molecular breeding, in non-model species of economic or evolutionary interest.

  14. Small RNA-seq during acute maximal exercise reveal RNAs involved in vascular inflammation and cardiometabolic health: brief report.

    Science.gov (United States)

    Shah, Ravi; Yeri, Ashish; Das, Avash; Courtright-Lim, Amanda; Ziegler, Olivia; Gervino, Ernest; Ocel, Jeffrey; Quintero-Pinzon, Pablo; Wooster, Luke; Bailey, Cole Shields; Tanriverdi, Kahraman; Beaulieu, Lea M; Freedman, Jane E; Ghiran, Ionita; Lewis, Gregory D; Van Keuren-Jensen, Kendall; Das, Saumya

    2017-12-01

    Exercise improves cardiometabolic and vascular function, although the mechanisms remain unclear. Our objective was to demonstrate the diversity of circulating extracellular RNA (ex-RNA) release during acute exercise in humans and its relevance to exercise-mediated benefits on vascular inflammation. We performed plasma small RNA sequencing in 26 individuals undergoing symptom-limited maximal treadmill exercise, with replication of our top candidate miRNA in a separate cohort of 59 individuals undergoing bicycle ergometry. We found changes in miRNAs and other ex-RNAs with exercise (e.g., Y RNAs and tRNAs) implicated in cardiovascular disease. In two independent cohorts of acute maximal exercise, we identified miR-181b-5p as a key ex-RNA increased in plasma after exercise, with validation in a separate cohort. In a mouse model of acute exercise, we found significant increases in miR-181b-5p expression in skeletal muscle after acute exercise in young (but not older) mice. Previous work revealed a strong role for miR-181b-5p in vascular inflammation in obesity, insulin resistance, sepsis, and cardiovascular disease. We conclude that circulating ex-RNAs were altered in plasma after acute exercise target pathways involved in inflammation, including miR-181b-5p. Further investigation into the role of known (e.g., miRNA) and novel (e.g., Y RNAs) RNAs is warranted to uncover new mechanisms of vascular inflammation on exercise-mediated benefits on health. NEW & NOTEWORTHY How exercise provides benefits to cardiometabolic health remains unclear. We performed RNA sequencing in plasma during exercise to identify the landscape of small noncoding circulating transcriptional changes. Our results suggest a link between inflammation and exercise, providing rich data on circulating noncoding RNAs for future studies by the scientific community. Copyright © 2017 the American Physiological Society.

  15. Single nucleotide polymorphism discovery in bovine liver using RNA-seq technology

    DEFF Research Database (Denmark)

    Pareek, Chandra Shekhar; Błaszczyk, Paweł; Dziuba, Piotr

    2017-01-01

    Background RNA-seq is a useful next-generation sequencing (NGS) technology that has been widely used to understand mammalian transcriptome architecture and function. In this study, a breed-specific RNA-seq experiment was utilized to detect putative single nucleotide polymorphisms (SNPs) in liver...

  16. Measuring RNA-Ligand Interactions with Microscale Thermophoresis.

    Science.gov (United States)

    Moon, Michelle H; Hilimire, Thomas A; Sanders, Allix M; Schneekloth, John S

    2018-01-31

    In recent years, there has been dramatic growth in the study of RNA. RNA has gone from being known as an intermediate in the central dogma of molecular biology to a molecule with a large diversity of structure and function that is involved in all aspects of biology. As new functions are rapidly discovered, it has become clear that there is a need for RNA-targeting small molecule probes to investigate RNA biology and clarify the potential for therapeutics based on RNA-small molecule interactions. While a host of techniques exist to measure RNA-small molecule interactions, many of these have drawbacks that make them intractable for routine use and are often not broadly applicable. A newer technology called microscale thermophoresis (MST), which measures the directed migration of a molecule and/or molecule-ligand complex along a temperature gradient, can be used to measure binding affinities using very small amounts of sample. The high sensitivity of this technique enables measurement of affinity constants in the nanomolar and micromolar range. Here, we demonstrate how MST can be used to study a range of biologically relevant RNA interactions, including peptide-RNA interactions, RNA-small molecule interactions, and displacement of an RNA-bound peptide by a small molecule.

  17. Synthetic biology of antimicrobial discovery.

    Science.gov (United States)

    Zakeri, Bijan; Lu, Timothy K

    2013-07-19

    Antibiotic discovery has a storied history. From the discovery of penicillin by Sir Alexander Fleming to the relentless quest for antibiotics by Selman Waksman, the stories have become like folklore used to inspire future generations of scientists. However, recent discovery pipelines have run dry at a time when multidrug-resistant pathogens are on the rise. Nature has proven to be a valuable reservoir of antimicrobial agents, which are primarily produced by modularized biochemical pathways. Such modularization is well suited to remodeling by an interdisciplinary approach that spans science and engineering. Herein, we discuss the biological engineering of small molecules, peptides, and non-traditional antimicrobials and provide an overview of the growing applicability of synthetic biology to antimicrobials discovery.

  18. Novel Small Molecule Inhibitors of Choline Kinase Identified by Fragment-Based Drug Discovery.

    Science.gov (United States)

    Zech, Stephan G; Kohlmann, Anna; Zhou, Tianjun; Li, Feng; Squillace, Rachel M; Parillon, Lois E; Greenfield, Matthew T; Miller, David P; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Xu, Yongjin; Miret, Juan J; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

    2016-01-28

    Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.

  19. DNA-encoded libraries - an efficient small molecule discovery technology for the biomedical sciences.

    Science.gov (United States)

    Kunig, Verena; Potowski, Marco; Gohla, Anne; Brunschweiger, Andreas

    2018-06-27

    DNA-encoded compound libraries are a highly attractive technology for the discovery of small molecule protein ligands. These compound collections consist of small molecules covalently connected to individual DNA sequences carrying readable information about the compound structure. DNA-tagging allows for efficient synthesis, handling and interrogation of vast numbers of chemically synthesized, drug-like compounds. They are screened on proteins by an efficient, generic assay based on Darwinian principles of selection. To date, selection of DNA-encoded libraries allowed for the identification of numerous bioactive compounds. Some of these compounds uncovered hitherto unknown allosteric binding sites on target proteins; several compounds proved their value as chemical biology probes unraveling complex biology; and the first examples of clinical candidates that trace their ancestry to a DNA-encoded library were reported. Thus, DNA-encoded libraries proved their value for the biomedical sciences as a generic technology for the identification of bioactive drug-like molecules numerous times. However, large scale experiments showed that even the selection of billions of compounds failed to deliver bioactive compounds for the majority of proteins in an unbiased panel of target proteins. This raises the question of compound library design.

  20. Development and Mechanism of Small Activating RNA Targeting CEBPA, a Novel Therapeutic in Clinical Trials for Liver Cancer.

    Science.gov (United States)

    Voutila, Jon; Reebye, Vikash; Roberts, Thomas C; Protopapa, Pantelitsa; Andrikakou, Pinelopi; Blakey, David C; Habib, Robert; Huber, Hans; Saetrom, Pal; Rossi, John J; Habib, Nagy A

    2017-12-06

    Small activating RNAs (saRNAs) are short double-stranded oligonucleotides that selectively increase gene transcription. Here, we describe the development of an saRNA that upregulates the transcription factor CCATT/enhancer binding protein alpha (CEBPA), investigate its mode of action, and describe its development into a clinical candidate. A bioinformatically directed nucleotide walk around the CEBPA gene identified an saRNA sequence that upregulates CEBPA mRNA 2.5-fold in human hepatocellular carcinoma cells. A nuclear run-on assay confirmed that this upregulation is a transcriptionally driven process. Mechanistic experiments demonstrate that Argonaute-2 (Ago2) is required for saRNA activity, with the guide strand of the saRNA shown to be associated with Ago2 and localized at the CEBPA genomic locus using RNA chromatin immunoprecipitation (ChIP) assays. The data support a sequence-specific on-target saRNA activity that leads to enhanced CEBPA mRNA transcription. Chemical modifications were introduced in the saRNA duplex to prevent activation of the innate immunity. This modified saRNA retains activation of CEBPA mRNA and downstream targets and inhibits growth of liver cancer cell lines in vitro. This novel drug has been encapsulated in a liposomal formulation for liver delivery, is currently in a phase I clinical trial for patients with liver cancer, and represents the first human study of an saRNA therapeutic. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Identification of rat lung-specific microRNAs by microRNA microarray: valuable discoveries for the facilitation of lung research

    Directory of Open Access Journals (Sweden)

    Chintagari Narendranath

    2007-01-01

    Full Text Available Abstract Background An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs. These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. Results In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM and Tukey Honestly Significant Difference (HSD revealed 2 miRNAs (miR-195 and miR-200c expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis. Conclusion The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.

  2. Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research.

    Science.gov (United States)

    Wang, Yang; Weng, Tingting; Gou, Deming; Chen, Zhongming; Chintagari, Narendranath Reddy; Liu, Lin

    2007-01-24

    An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis. The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.

  3. Deep sequencing of small RNAs identifies canonical and non-canonical miRNA and endogenous siRNAs in mammalian somatic tissues.

    Science.gov (United States)

    Castellano, Leandro; Stebbing, Justin

    2013-03-01

    MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression. They are characterized by specific maturation processes defined by canonical and non-canonical biogenic pathways. Analysis of ∼0.5 billion sequences from mouse data sets derived from different tissues, developmental stages and cell types, partly characterized by either ablation or mutation of the main proteins belonging to miRNA processor complexes, reveals 66 high-confidence new genomic loci coding for miRNAs that could be processed in a canonical or non-canonical manner. A proportion of the newly discovered miRNAs comprises mirtrons, for which we define a new sub-class. Notably, some of these newly discovered miRNAs are generated from untranslated and open reading frames of coding genes, and we experimentally validate these. We also show that many annotated miRNAs do not present miRNA-like features, as they are neither processed by known processing complexes nor loaded on AGO2; this indicates that the current miRNA miRBase database list should be refined and re-defined. Accordingly, a group of them map on ribosomal RNA molecules, whereas others cannot undergo genuine miRNA biogenesis. Notably, a group of annotated miRNAs are Dgcr8 independent and DICER dependent endogenous small interfering RNAs that derive from a unique hairpin formed from a short interspersed nuclear element.

  4. NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs

    Directory of Open Access Journals (Sweden)

    Shobbir Hussain

    2013-07-01

    Full Text Available Autosomal-recessive loss of the NSUN2 gene has been identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNAs, yet the identification of cytosine methylation in other RNA species has been hampered by the lack of sensitive and reliable molecular techniques. Here, we describe miCLIP as an additional approach for identifying RNA methylation sites in transcriptomes. miCLIP is a customized version of the individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP method. We confirm site-specific methylation in tRNAs and additional messenger and noncoding RNAs (ncRNAs. Among these, vault ncRNAs contained six NSun2-methylated cytosines, three of which were confirmed by RNA bisulfite sequencing. Using patient cells lacking the NSun2 protein, we further show that loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders.

  5. Identification of small non-coding RNA classes expressed in swine whole blood during HP-PRRSV infection

    Science.gov (United States)

    It has been established that reduced susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) has a genetic component. This genetic component may take the form of small non-coding RNAs (sncRNA), which are molecules that function as regulators of gene expression. Various sncRNAs ...

  6. NCI RNA Biology 2017 symposium recap | Center for Cancer Research

    Science.gov (United States)

    The recent discovery of new classes of RNAs and the demonstration that alterations in RNA metabolism underlie numerous human cancers have resulted in enormous interest among CCR investigators in RNA biology. In order to share the latest research in this exciting field, the CCR Initiative in RNA Biology held its second international symposium April 23-24, 2017, in Natcher Auditorium. Learn more...

  7. Ribonucleic artefacts: are some extracellular RNA discoveries driven by cell culture medium components?

    Science.gov (United States)

    Tosar, Juan Pablo; Cayota, Alfonso; Eitan, Erez; Halushka, Marc K; Witwer, Kenneth W

    2017-01-01

    In a recently published study, Anna Krichevsky and colleagues raise the important question of whether results of in vitro extracellular RNA (exRNA) studies, including extracellular vesicle (EV) investigations, are confounded by the presence of RNA in cell culture medium components such as foetal bovine serum (FBS). The answer, according to their data, is a resounding "yes". Even after lengthy ultracentrifugation to remove bovine EVs from FBS, the majority of exRNA in FBS remained. Although technical factors may affect the degree of depletion, residual EVs and exRNA in FBS could influence the conclusions of in vitro studies: certainly, for secreted RNA, and possibly also for cell-associated RNA. In this commentary, we critically examine some of the literature in this field, including a recent study from some of the authors of this piece, in light of the Wei et al. study and explore how cell culture-derived RNAs may affect what we think we know about EV RNAs. These findings hold particular consequence as the field moves towards a deeper understanding of EV-RNA associations and potential functions.

  8. A complex small RNA repertoire is generated by a plant/fungal-like machinery and effected by a metazoan-like Argonaute in the single-cell human parasite Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Laurence Braun

    2010-05-01

    Full Text Available In RNA silencing, small RNAs produced by the RNase-III Dicer guide Argonaute-like proteins as part of RNA-induced silencing complexes (RISC to regulate gene expression transcriptionally or post-transcriptionally. Here, we have characterized the RNA silencing machinery and exhaustive small RNAome of Toxoplasma gondii, member of the Apicomplexa, a phylum of animal- and human-infecting parasites that cause extensive health and economic damages to human populations worldwide. Remarkably, the small RNA-generating machinery of Toxoplasma is phylogenetically and functionally related to that of plants and fungi, and accounts for an exceptionally diverse array of small RNAs. This array includes conspicuous populations of repeat-associated small interfering RNA (siRNA, which, as in plants, likely generate and maintain heterochromatin at DNA repeats and satellites. Toxoplasma small RNAs also include many microRNAs with clear metazoan-like features whose accumulation is sometimes extremely high and dynamic, an unexpected finding given that Toxoplasma is a unicellular protist. Both plant-like heterochromatic small RNAs and metazoan-like microRNAs bind to a single Argonaute protein, Tg-AGO. Toxoplasma miRNAs co-sediment with polyribosomes, and thus, are likely to act as translational regulators, consistent with the lack of catalytic residues in Tg-AGO. Mass spectrometric analyses of the Tg-AGO protein complex revealed a common set of virtually all known RISC components so far characterized in human and Drosophila, as well as novel proteins involved in RNA metabolism. In agreement with its loading with heterochromatic small RNAs, Tg-AGO also associates substoichiometrically with components of known chromatin-repressing complexes. Thus, a puzzling patchwork of silencing processor and effector proteins from plant, fungal and metazoan origin accounts for the production and action of an unsuspected variety of small RNAs in the single-cell parasite Toxoplasma and

  9. Evaluating Quality of Aged Archival Formalin-Fixed Paraffin-Embedded Samples for RNA-Sequencing

    Science.gov (United States)

    Archival formalin-fixed paraffin-embedded (FFPE) samples offer a vast, untapped source of genomic data for biomarker discovery. However, the quality of FFPE samples is often highly variable, and conventional methods to assess RNA quality for RNA-sequencing (RNA-seq) are not infor...

  10. Delivery of small interfering RNA for inhibition of endothelial cell apoptosis by hypoxia and serum deprivation

    International Nuclear Information System (INIS)

    Cho, Seung-Woo; Hartle, Lauren; Son, Sun Mi; Yang, Fan; Goldberg, Michael; Xu, Qiaobing; Langer, Robert; Anderson, Daniel G.

    2008-01-01

    RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-α (TNF-α). siRNA was designed and synthesized targeting tumor necrosis factor-α receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5% and SHP-1; 97.2%) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-α expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-α expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia

  11. RNA modifications by oxidation

    DEFF Research Database (Denmark)

    Poulsen, Henrik E; Specht, Elisabeth; Broedbaek, Kasper

    2012-01-01

    to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation......The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found......, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas...

  12. Inhibition of endothelial cell proliferation by targeting Rac1 GTPase with small interference RNA in tumor cells

    International Nuclear Information System (INIS)

    Xue Yan; Bi Feng; Zhang Xueyong; Pan Yanglin; Liu Na; Zheng Yi; Fan Daiming

    2004-01-01

    Hypoxia-induced angiogenesis plays an important role in the malignancy of solid tumors. A number of recent studies including our own have suggested that Rho family small GTPases are involved in this process, and Rac1, a prominent member of the Rho family, may be critical in regulating hypoxia-induced gene activation of several angiogenesis factors and tumor suppressors. To further define Rac1 function in angiogenesis and to explore novel approaches to modulate angiogenesis, we employed the small interference RNA technique to knock down gene expression of Rac1 in gastric cancer cell line AGS that expresses a high level of Rac1. Both the mRNA and protein levels of Rac1 in the AGS cells were decreased dramatically after transfection with a Rac1-specific siRNA vector. When the conditioned medium derived from the Rac1 downregulated AGS cells was applied to the human endothelial cells, it could significantly inhibit the cell proliferation. Further study proved that, VEGF and HIF-1α, two angiogenesis promoting factors, were found to be downregulated whereas p53 and VHL, which are tumor suppressors and angiogenesis inhibitors, were upregulated in the Rac1 siRNA transfected cells. Our results suggest that Rac1 may be involved in angiogenesis by controlling the expression of angiogenesis-related factors and provide a possible strategy for the treatment of tumor angiogenesis by targeting the Rac1 GTPase

  13. Synthetic biology of antimicrobial discovery

    Science.gov (United States)

    Zakeri, Bijan; Lu, Timothy K.

    2012-01-01

    Antibiotic discovery has a storied history. From the discovery of penicillin by Sir Alexander Fleming to the relentless quest for antibiotics by Selman Waksman, the stories have become like folklore, used to inspire future generations of scientists. However, recent discovery pipelines have run dry at a time when multidrug resistant pathogens are on the rise. Nature has proven to be a valuable reservoir of antimicrobial agents, which are primarily produced by modularized biochemical pathways. Such modularization is well suited to remodeling by an interdisciplinary approach that spans science and engineering. Herein, we discuss the biological engineering of small molecules, peptides, and non-traditional antimicrobials and provide an overview of the growing applicability of synthetic biology to antimicrobials discovery. PMID:23654251

  14. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...... and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II m......RNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF...

  15. Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

    Science.gov (United States)

    Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

    2003-01-01

    Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

  16. Riboregulation of the bacterial actin-homolog MreB by DsrA small noncoding RNA.

    Science.gov (United States)

    Cayrol, Bastien; Fortas, Emilie; Martret, Claire; Cech, Grzegorz; Kloska, Anna; Caulet, Stephane; Barbet, Marion; Trépout, Sylvain; Marco, Sergio; Taghbalout, Aziz; Busi, Florent; Wegrzyn, Grzegorz; Arluison, Véronique

    2015-01-01

    The bacterial actin-homolog MreB is a key player in bacterial cell-wall biosynthesis and is required for the maintenance of the rod-like morphology of Escherichia coli. However, how MreB cellular levels are adjusted to growth conditions is poorly understood. Here, we show that DsrA, an E. coli small noncoding RNA (sRNA), is involved in the post-transcriptional regulation of mreB. DsrA is required for the downregulation of MreB cellular concentration during environmentally induced slow growth-rates, mainly growth at low temperature and during the stationary phase. DsrA interacts in an Hfq-dependent manner with the 5' region of mreB mRNA, which contains signals for translation initiation and thereby affects mreB translation and stability. Moreover, as DsrA is also involved in the regulation of two transcriptional regulators, σ(S) and the nucleoid associated protein H-NS, which negatively regulate mreB transcription, it also indirectly contributes to mreB transcriptional down-regulation. By using quantitative analyses, our results evidence the complexity of this regulation and the tangled interplay between transcriptional and post-transcriptional control. As transcription factors and sRNA-mediated post-transcriptional regulators use different timescales, we propose that the sRNA pathway helps to adapt to changes in temperature, but also indirectly mediates long-term regulation of MreB concentration. The tight regulation and fine-tuning of mreB gene expression in response to cellular stresses is discussed in regard to the effect of the MreB protein on cell elongation.

  17. The Gozo discovery bus : a successful experiment

    OpenAIRE

    Vella, Maryrose

    2008-01-01

    The introduction of a tourist discovery bus in Gozo came about as a result of an EU Project which is part of the Interreg III B Archimed programmes in which the Islands and Small States Institute of the University of Malta participated. Other countries participating in this programme besides Malta, represented by the Islands and Small States Institute, are Italy, Cyprus and Greece. The discovery bus service was aimed at encouraging more tourists to come to Gozo and enabling them to visit stra...

  18. microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein.

    Science.gov (United States)

    Pinder, Benjamin D; Smibert, Craig A

    2013-01-01

    Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.

  19. MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

    International Nuclear Information System (INIS)

    Severino, Patricia; Mathor, Monica Beatriz; Nunes, Fabio Daumas; Ragoussis, Jiannis; Tajara, Eloiza Helena; Brüggemann, Holger; Andreghetto, Flavia Maziero; Camps, Carme; Klingbeil, Maria de Fatima Garrido; Pereira, Welbert Oliveira de; Soares, Renata Machado; Moyses, Raquel; Wünsch-Filho, Victor

    2013-01-01

    Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA

  20. Sequencing of Isotope-Labeled Small RNA Using Femtosecond Laser Ablation Time-of-Flight Mass Spectrometry

    Science.gov (United States)

    Kurata-Nishimura, Mizuki; Ando, Yoshinari; Kobayashi, Tohru; Matsuo, Yukari; Suzuki, Harukazu; Hayashizaki, Yoshihide; Kawai, Jun

    2010-04-01

    A novel method for the analysis of sequences of small RNAs using nucleotide triphosphates labeled with stable isotopes has been developed using time-of-flight mass spectroscopy combined with femtosecond laser ablation (fsLA-TOF-MS). Small RNAs synthesized with nucleotides enriched in 13C and 15N were efficiently atomized and ionized by single-shot fsLA and the isotope ratios 13C/12C and 15N/14N were evaluated using the TOF-MS method. By comparing the isotope ratios among four different configurations, the number of nucleotide contents of the control RNA sample were successfully reproduced.

  1. MicroRNA-directed siRNA biogenesis in Caenorhabditis elegans.

    Science.gov (United States)

    Corrêa, Régis L; Steiner, Florian A; Berezikov, Eugene; Ketting, René F

    2010-04-08

    RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi-related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.

  2. Deep sequencing uncovers commonality in small RNA profiles between transgene-induced and naturally occurring RNA silencing of chalcone synthase-A gene in petunia.

    Science.gov (United States)

    Kasai, Megumi; Matsumura, Hideo; Yoshida, Kentaro; Terauchi, Ryohei; Taneda, Akito; Kanazawa, Akira

    2013-01-30

    Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs. We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved. The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these silencing systems especially in the

  3. Reconstruction and analysis of the lncRNA-miRNA-mRNA network based on competitive endogenous RNA reveal functional lncRNAs in rheumatoid arthritis.

    Science.gov (United States)

    Jiang, Hui; Ma, Rong; Zou, Shubiao; Wang, Yongzhong; Li, Zhuqing; Li, Weiping

    2017-06-01

    Rheumatoid arthritis (RA) is an autoimmune disease with an unknown etiology, occurring in approximately 1.0% of general population. More and more studies have suggested that long non-coding RNAs (lncRNAs) could play important roles in various biological processes and be associated with the pathogenesis of different kinds of diseases including RA. Although a large number of lncRNAs have been found, our knowledge of their function and physiological/pathological significance is still in its infancy. In order to reveal functional lncRNAs and identify the key lncRNAs in RA, we reconstructed a global triple network based on the competitive endogenous RNA (ceRNA) theory using the data from National Center for Biotechnology Information Gene Expression Omnibus and our previous paper. Meanwhile, Gene Ontology (GO) and pathway analysis were performed using Cytoscape plug-in BinGO and Database for Annotation, Visualization, and Integration Discovery (DAVID), respectively. We found that the lncRNA-miRNA-mRNA network was composed of 7 lncRNA nodes, 90 mRNA nodes, 24 miRNA nodes, and 301 edges. The functional assay showed that 147 GO terms and 23 pathways were enriched. In addition, three lncRNAs (S5645.1, XR_006437.1, J01878) were highly related to RA, and therefore, were selected as key lncRNAs. This study suggests that specific lncRNAs are associated with the development of RA, and three lncRNAs (S5645.1, XR_006437.1, J01878) could be used as potential diagnostic biomarkers and therapeutic targets.

  4. Preclinical and clinical development of siRNA-based therapeutics.

    Science.gov (United States)

    Ozcan, Gulnihal; Ozpolat, Bulent; Coleman, Robert L; Sood, Anil K; Lopez-Berestein, Gabriel

    2015-06-29

    The discovery of RNA interference, first in plants and Caenorhabditis elegans and later in mammalian cells, led to the emergence of a transformative view in biomedical research. Knowledge of the multiple actions of non-coding RNAs has truly allowed viewing DNA, RNA and proteins in novel ways. Small interfering RNAs (siRNAs) can be used as tools to study single gene function both in vitro and in vivo and are an attractive new class of therapeutics, especially against undruggable targets for the treatment of cancer and other diseases. Despite the potential of siRNAs in cancer therapy, many challenges remain, including rapid degradation, poor cellular uptake and off-target effects. Rational design strategies, selection algorithms, chemical modifications and nanocarriers offer significant opportunities to overcome these challenges. Here, we review the development of siRNAs as therapeutic agents from early design to clinical trial, with special emphasis on the development of EphA2-targeting siRNAs for ovarian cancer treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

    Directory of Open Access Journals (Sweden)

    Ekaterina Smirnova

    2015-05-01

    Full Text Available Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3, a movement protein (ORF4, and a carboxy-terminal extension to the coat protein (ORF5. These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV, a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

  6. Discovery of a Small Non-AUG-Initiated ORF in Poleroviruses and Luteoviruses That Is Required for Long-Distance Movement.

    Science.gov (United States)

    Smirnova, Ekaterina; Firth, Andrew E; Miller, W Allen; Scheidecker, Danièle; Brault, Véronique; Reinbold, Catherine; Rakotondrafara, Aurélie M; Chung, Betty Y-W; Ziegler-Graff, Véronique

    2015-05-01

    Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.

  7. DISCOVERY OF X-RAY EMISSION FROM YOUNG SUNS IN THE SMALL MAGELLANIC CLOUD

    International Nuclear Information System (INIS)

    Oskinova, L. M.; Hainich, R.; Sun, W.; Chen, Y.; Evans, C. J.; Hénault-Brunet, V.; Chu, Y.-H.; Gruendl, R. A.; Gallagher, J. S. III; Guerrero, M. A.; Güdel, M.; Silich, S.; Nazé, Y.; Reyes-Iturbide, J.

    2013-01-01

    We report the discovery of extended X-ray emission within the young star cluster NGC 602a in the Wing of the Small Magellanic Cloud (SMC) based on observations obtained with the Chandra X-Ray Observatory. X-ray emission is detected from the cluster core area with the highest stellar density and from a dusty ridge surrounding the H II region. We use a census of massive stars in the cluster to demonstrate that a cluster wind or wind-blown bubble is unlikely to provide a significant contribution to the X-ray emission detected from the central area of the cluster. We therefore suggest that X-ray emission at the cluster core originates from an ensemble of low- and solar-mass pre-main-sequence (PMS) stars, each of which would be too weak in X-rays to be detected individually. We attribute the X-ray emission from the dusty ridge to the embedded tight cluster of the newborn stars known in this area from infrared studies. Assuming that the levels of X-ray activity in young stars in the low-metallicity environment of NGC 602a are comparable to their Galactic counterparts, then the detected spatial distribution, spectral properties, and level of X-ray emission are largely consistent with those expected from low- and solar-mass PMS stars and young stellar objects (YSOs). This is the first discovery of X-ray emission attributable to PMS stars and YSOs in the SMC, which suggests that the accretion and dynamo processes in young, low-mass objects in the SMC resemble those in the Galaxy.

  8. Upregulation of Long Noncoding RNA Small Nucleolar RNA Host Gene 18 Promotes Radioresistance of Glioma by Repressing Semaphorin 5A

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Rong [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong (China); Department of Radiation Oncology, Fujian Medical University Union Hospital, Fuzhou, Fujian (China); Yao, Qiwei [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong (China); Department of Radiation Oncology, Teaching Hospital of Fujian Medical University, Fujian Provincial Cancer Hospital, Fuzhou, Fujian (China); Ren, Chen; Liu, Ying; Yang, Hongli; Xie, Guozhu; Du, Shasha [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong (China); Yang, Kaijun [Department of Neurosurgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong (China); Yuan, Yawei, E-mail: yuanyawei2015@outlook.com [Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong (China); Department of Radiation Oncology, Cancer Hospital Center of Guangzhou Medical University, Guangzhou, Guangdong (China)

    2016-11-15

    Purpose: Although increasing evidence has shown that long noncoding RNAs play an important regulatory role in carcinogenesis and tumor progression, little is known about the role of small nucleolar RNA host gene 18 (SNHG18) in cancer. The goal of this study was to investigate the expression of SNHG18 and its clinical significance in glioma. Methods and Materials: Differences in the lncRNA expression profile between M059K and M059J cells were assessed by lncRNA expression microarray analysis. The expression and localization of SNHG18 in glioma cells or tissues was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH), respectively. the clinical associations of SNHG18 in glioma was evaluated by qRT-PCR, ISH and immunohistochemistry. The role of SNHG18 in glioma radiosensitivity was evaluated by colony formation assays, immunofluorescence, Western blot and tumor growth inhibition study. Results: The present study investigated the clinical associations of SNHG18 and its role in glioma. Our results showed that the expression of SNHG18 was remarkably upregulated in clinical glioma tissues compared with normal brain tissues. SNHG18 expression was associated with the clinical tumor grade and correlated negatively with isocitrate dehydrogenase 1 mutation. In addition, knockdown of SNHG18 with short hairpin RNA suppressed the radioresistance of glioma cells, and transgenic expression of SNHG18 had the opposite effect. Furthermore, xenograft tumors grown from cells with SNHG18 deletion were more radiosensitive than tumors grown from control cells. Further studies revealed that SNHG18 promotes radioresistance by inhibiting semaphorin 5A and that inhibition of semaphorin 5A expression abrogated the radiosensitizing effect caused by SNHG18 deletion. Conclusions: Our findings provide new insights into the role of SNHG18 in glioma and suggest its potential as a target for glioma therapy.

  9. Upregulation of Long Noncoding RNA Small Nucleolar RNA Host Gene 18 Promotes Radioresistance of Glioma by Repressing Semaphorin 5A

    International Nuclear Information System (INIS)

    Zheng, Rong; Yao, Qiwei; Ren, Chen; Liu, Ying; Yang, Hongli; Xie, Guozhu; Du, Shasha; Yang, Kaijun; Yuan, Yawei

    2016-01-01

    Purpose: Although increasing evidence has shown that long noncoding RNAs play an important regulatory role in carcinogenesis and tumor progression, little is known about the role of small nucleolar RNA host gene 18 (SNHG18) in cancer. The goal of this study was to investigate the expression of SNHG18 and its clinical significance in glioma. Methods and Materials: Differences in the lncRNA expression profile between M059K and M059J cells were assessed by lncRNA expression microarray analysis. The expression and localization of SNHG18 in glioma cells or tissues was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH), respectively. the clinical associations of SNHG18 in glioma was evaluated by qRT-PCR, ISH and immunohistochemistry. The role of SNHG18 in glioma radiosensitivity was evaluated by colony formation assays, immunofluorescence, Western blot and tumor growth inhibition study. Results: The present study investigated the clinical associations of SNHG18 and its role in glioma. Our results showed that the expression of SNHG18 was remarkably upregulated in clinical glioma tissues compared with normal brain tissues. SNHG18 expression was associated with the clinical tumor grade and correlated negatively with isocitrate dehydrogenase 1 mutation. In addition, knockdown of SNHG18 with short hairpin RNA suppressed the radioresistance of glioma cells, and transgenic expression of SNHG18 had the opposite effect. Furthermore, xenograft tumors grown from cells with SNHG18 deletion were more radiosensitive than tumors grown from control cells. Further studies revealed that SNHG18 promotes radioresistance by inhibiting semaphorin 5A and that inhibition of semaphorin 5A expression abrogated the radiosensitizing effect caused by SNHG18 deletion. Conclusions: Our findings provide new insights into the role of SNHG18 in glioma and suggest its potential as a target for glioma therapy.

  10. The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus.

    Science.gov (United States)

    Danger, Jessica L; Makthal, Nishanth; Kumaraswami, Muthiah; Sumby, Paul

    2015-12-01

    The group A Streptococcus (GAS; Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to the prtF1 and prtF2 mRNAs within their 5' untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and the prtF1 and prtF2 mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection. More than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small

  11. Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion.

    Science.gov (United States)

    Zhao, Shanrong; Zhang, Ying; Gamini, Ramya; Zhang, Baohong; von Schack, David

    2018-03-19

    To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.

  12. Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

    DEFF Research Database (Denmark)

    Kruhøffer, Mogens; Andersen, Lars Dyrskjøt; Voss, Thorsten

    2007-01-01

    We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood...... and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated micro......RNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis....

  13. High-throughput sequencing of small RNA transcriptome reveals salt stress regulated microRNAs in sugarcane.

    Directory of Open Access Journals (Sweden)

    Mariana Carnavale Bottino

    Full Text Available Salt stress is a primary cause of crop losses worldwide, and it has been the subject of intense investigation to unravel the complex mechanisms responsible for salinity tolerance. MicroRNA is implicated in many developmental processes and in responses to various abiotic stresses, playing pivotal roles in plant adaptation. Deep sequencing technology was chosen to determine the small RNA transcriptome of Saccharum sp cultivars grown on saline conditions. We constructed four small RNAs libraries prepared from plants grown on hydroponic culture submitted to 170 mM NaCl and harvested after 1 h, 6 hs and 24 hs. Each library was sequenced individually and together generated more than 50 million short reads. Ninety-eight conserved miRNAs and 33 miRNAs* were identified by bioinformatics. Several of the microRNA showed considerable differences of expression in the four libraries. To confirm the results of the bioinformatics-based analysis, we studied the expression of the 10 most abundant miRNAs and 1 miRNA* in plants treated with 170 mM NaCl and in plants with a severe treatment of 340 mM NaCl. The results showed that 11 selected miRNAs had higher expression in samples treated with severe salt treatment compared to the mild one. We also investigated the regulation of the same miRNAs in shoots of four cultivars grown on soil treated with 170 mM NaCl. Cultivars could be grouped according to miRNAs expression in response to salt stress. Furthermore, the majority of the predicted target genes had an inverse regulation with their correspondent microRNAs. The targets encode a wide range of proteins, including transcription factors, metabolic enzymes and genes involved in hormone signaling, probably assisting the plants to develop tolerance to salinity. Our work provides insights into the regulatory functions of miRNAs, thereby expanding our knowledge on potential salt-stressed regulated genes.

  14. Radioactivity. Centenary of radioactivity discovery

    International Nuclear Information System (INIS)

    Charpak, G.; Tubiana, M.; Bimbot, R.

    1997-01-01

    This small booklet was edited for the occasion of the exhibitions of the celebration of the centenary of radioactivity discovery which took place in various locations in France from 1996 to 1998. It recalls some basic knowledge concerning radioactivity and its applications: history of discovery, atoms and isotopes, radiations, measurement of ionizing radiations, natural and artificial radioactivity, isotope dating and labelling, radiotherapy, nuclear power and reactors, fission and fusion, nuclear wastes, dosimetry, effects and radioprotection. (J.S.)

  15. RNAi pathways in Mucor: A tale of proteins, small RNAs and functional diversity.

    Science.gov (United States)

    Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M

    2016-05-01

    The existence of an RNA-mediated silencing mechanism in the opportunistic fungal pathogen Mucor circinelloides was first described in the early 2000. Since then, Mucor has reached an outstanding position within the fungal kingdom as a model system to achieve a deeper understanding of regulation of endogenous functions by the RNA interference (RNAi) machinery. M. circinelloides combines diverse components of its RNAi machinery to carry out functions not only limited to the defense against invasive nucleic acids, but also to regulate expression of its own genes by producing different classes of endogenous small RNA molecules (esRNAs). The recent discovery of a novel RNase that participates in a new RNA degradation pathway adds more elements to the gene silencing-mediated regulation. This review focuses on esRNAs in M. circinelloides, the different pathways involved in their biogenesis, and their roles in regulating specific physiological and developmental processes in response to environmental signals, highlighting the complexity of silencing-mediated regulation in fungi. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. The Dynamic Radio Sky: An Opportunity for Discovery

    Science.gov (United States)

    2010-01-01

    giant pulses from the Crab pulsar , a small number of dedicated radio transient surveys, and the serendipitous discovery of transient radio sources...used in the discovery of over 1800 radio pulsars . In 2003, the Parkes Multibeam Survey had covered the entire Galactic plane visible from Parkes... pulsars , and confirmed the neutron star nature of these sources, dubbed Rotating Radio Transients (RRATs). Since the discovery of the original 11 RRATs

  17. Small RNA sequence analysis of adenovirus VA RNA-derived miRNAs reveals an unexpected serotype-specific difference in structure and abundance.

    Directory of Open Access Journals (Sweden)

    Wael Kamel

    Full Text Available Human adenoviruses (HAds encode for one or two highly abundant virus-associated RNAs, designated VA RNAI and VA RNAII, which fold into stable hairpin structures resembling miRNA precursors. Here we show that the terminal stem of the VA RNAs originating from Ad4, Ad5, Ad11 and Ad37, all undergo Dicer dependent processing into virus-specific miRNAs (so-called mivaRNAs. We further show that the mivaRNA duplex is subjected to a highly asymmetric RISC loading with the 3'-strand from all VA RNAs being the favored strand, except for the Ad37 VA RNAII, where the 5'-mivaRNAII strand was preferentially assembled into RISC. Although the mivaRNA seed sequences are not fully conserved between the HAds a bioinformatics prediction approach suggests that a large fraction of the VA RNAII-, but not the VA RNAI-derived mivaRNAs still are able to target the same cellular genes. Using small RNA deep sequencing we demonstrate that the Dicer processing event in the terminal stem of the VA RNAs is not unique and generates 3'-mivaRNAs with a slight variation of the position of the 5' terminal nucleotide in the RISC loaded guide strand. Also, we show that all analyzed VA RNAs, except Ad37 VA RNAI and Ad5 VA RNAII, utilize an alternative upstream A start site in addition to the classical +1 G start site. Further, the 5'-mivaRNAs with an A start appears to be preferentially incorporated into RISC. Although the majority of mivaRNA research has been done using Ad5 as the model system our analysis demonstrates that the mivaRNAs expressed in Ad11- and Ad37-infected cells are the most abundant mivaRNAs associated with Ago2-containing RISC. Collectively, our results show an unexpected variability in Dicer processing of the VA RNAs and a serotype-specific loading of mivaRNAs into Ago2-based RISC.

  18. Comprehensive discovery of noncoding RNAs in acute myeloid leukemia cell transcriptomes.

    Science.gov (United States)

    Zhang, Jin; Griffith, Malachi; Miller, Christopher A; Griffith, Obi L; Spencer, David H; Walker, Jason R; Magrini, Vincent; McGrath, Sean D; Ly, Amy; Helton, Nichole M; Trissal, Maria; Link, Daniel C; Dang, Ha X; Larson, David E; Kulkarni, Shashikant; Cordes, Matthew G; Fronick, Catrina C; Fulton, Robert S; Klco, Jeffery M; Mardis, Elaine R; Ley, Timothy J; Wilson, Richard K; Maher, Christopher A

    2017-11-01

    To detect diverse and novel RNA species comprehensively, we compared deep small RNA and RNA sequencing (RNA-seq) methods applied to a primary acute myeloid leukemia (AML) sample. We were able to discover previously unannotated small RNAs using deep sequencing of a library method using broader insert size selection. We analyzed the long noncoding RNA (lncRNA) landscape in AML by comparing deep sequencing from multiple RNA-seq library construction methods for the sample that we studied and then integrating RNA-seq data from 179 AML cases. This identified lncRNAs that are completely novel, differentially expressed, and associated with specific AML subtypes. Our study revealed the complexity of the noncoding RNA transcriptome through a combined strategy of strand-specific small RNA and total RNA-seq. This dataset will serve as an invaluable resource for future RNA-based analyses. Copyright © 2017 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  19. How the RNA isolation method can affect microRNA microarray results

    DEFF Research Database (Denmark)

    Podolska, Agnieszka; Kaczkowski, Bogumil; Litman, Thomas

    2011-01-01

    RNA microarray analysis on porcine brain tissue. One method is a phenol-guanidine isothiocyanate-based procedure that permits isolation of total RNA. The second method, miRVana™ microRNA isolation, is column based and recovers the small RNA fraction alone. We found that microarray analyses give different results...... that depend on the RNA fraction used, in particular because some microRNAs appear very sensitive to the RNA isolation method. We conclude that precautions need to be taken when comparing microarray studies based on RNA isolated with different methods.......The quality of RNA is crucial in gene expression experiments. RNA degradation interferes in the measurement of gene expression, and in this context, microRNA quantification can lead to an incorrect estimation. In the present study, two different RNA isolation methods were used to perform micro...

  20. Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.).

    Science.gov (United States)

    Asha, Srinivasan; Soniya, Eppurath V

    2016-01-01

    Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5'tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5'tRFs in the infected leaf and root. The abundance of 5'tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5'Ala(CGC) tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5'Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper.

  1. Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells.

    Science.gov (United States)

    Slanina, H; Schmutzler, M; Christodoulides, M; Kim, K S; Schubert-Unkmeir, A

    2012-01-01

    Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin in Neisseria meningitidis pathogenesis. Copyright © 2012 S. Karger AG, Basel.

  2. The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System.

    Science.gov (United States)

    Lloyd, Chelsea R; Park, Seongjin; Fei, Jingyi; Vanderpool, Carin K

    2017-06-01

    The bacterial small RNA (sRNA) SgrS has been a fruitful model for discovery of novel RNA-based regulatory mechanisms and new facets of bacterial physiology and metabolism. SgrS is one of only a few characterized dual-function sRNAs. SgrS can control gene expression posttranscriptionally via sRNA-mRNA base-pairing interactions. Its second function is coding for the small protein SgrT. Previous work demonstrated that both functions contribute to relief of growth inhibition caused by glucose-phosphate stress, a condition characterized by disrupted glycolytic flux and accumulation of sugar phosphates. The base-pairing activity of SgrS has been the subject of numerous studies, but the activity of SgrT is less well characterized. Here, we provide evidence that SgrT acts to specifically inhibit the transport activity of the major glucose permease PtsG. Superresolution microscopy demonstrated that SgrT localizes to the cell membrane in a PtsG-dependent manner. Mutational analysis determined that residues in the N-terminal domain of PtsG are important for conferring sensitivity to SgrT-mediated inhibition of transport activity. Growth assays support a model in which SgrT-mediated inhibition of PtsG transport activity reduces accumulation of nonmetabolizable sugar phosphates and promotes utilization of alternative carbon sources by modulating carbon catabolite repression. The results of this study expand our understanding of a basic and well-studied biological problem, namely, how cells coordinate carbohydrate transport and metabolism. Further, this work highlights the complex activities that can be carried out by sRNAs and small proteins in bacteria. IMPORTANCE Sequencing, annotation and investigation of hundreds of bacterial genomes have identified vast numbers of small RNAs and small proteins, the majority of which have no known function. In this study, we explore the function of a small protein that acts in tandem with a well-characterized small RNA during metabolic

  3. Machine Learning Approaches Toward Building Predictive Models for Small Molecule Modulators of miRNA and Its Utility in Virtual Screening of Molecular Databases.

    Science.gov (United States)

    Periwal, Vinita; Scaria, Vinod

    2017-01-01

    The ubiquitous role of microRNAs (miRNAs) in a number of pathological processes has suggested that they could act as potential drug targets. RNA-binding small molecules offer an attractive means for modulating miRNA function. The availability of bioassay data sets for a variety of biological assays and molecules in public domain provides a new opportunity toward utilizing them to create models and further utilize them for in silico virtual screening approaches to prioritize or assign potential functions for small molecules. Here, we describe a computational strategy based on machine learning for creation of predictive models from high-throughput biological screens for virtual screening of small molecules with the potential to inhibit microRNAs. Such models could be potentially used for computational prioritization of small molecules before performing high-throughput biological assay.

  4. Small Bodies, Big Discoveries: NASA's Small Bodies Education Program

    Science.gov (United States)

    Mayo, L.; Erickson, K. J.

    2014-12-01

    2014 is turning out to be a watershed year for celestial events involving the solar system's unsung heroes, small bodies. This includes the close flyby of comet C/2013 A1 / Siding Spring with Mars in October and the historic Rosetta mission with its Philae lander to comet 67P/Churyumov-Gerasimenko. Beyond 2014, the much anticipated 2015 Pluto flyby by New Horizons and the February Dawn Mission arrival at Ceres will take center stage. To deliver the excitement and wonder of our solar system's small bodies to worldwide audiences, NASA's JPL and GSFC education teams in partnership with NASA EDGE will reach out to the public through multiple venues including broadcast media, social media, science and math focused educational activities, observing challenges, interactive visualization tools like "Eyes on the Solar System" and more. This talk will highlight NASA's focused education effort to engage the public in small bodies mission science and the role these objects play in our understanding of the formation and evolution of the solar system.

  5. RNA viruses in the sea.

    Science.gov (United States)

    Lang, Andrew S; Rise, Matthew L; Culley, Alexander I; Steward, Grieg F

    2009-03-01

    Viruses are ubiquitous in the sea and appear to outnumber all other forms of marine life by at least an order of magnitude. Through selective infection, viruses influence nutrient cycling, community structure, and evolution in the ocean. Over the past 20 years we have learned a great deal about the diversity and ecology of the viruses that constitute the marine virioplankton, but until recently the emphasis has been on DNA viruses. Along with expanding knowledge about RNA viruses that infect important marine animals, recent isolations of RNA viruses that infect single-celled eukaryotes and molecular analyses of the RNA virioplankton have revealed that marine RNA viruses are novel, widespread, and genetically diverse. Discoveries in marine RNA virology are broadening our understanding of the biology, ecology, and evolution of viruses, and the epidemiology of viral diseases, but there is still much that we need to learn about the ecology and diversity of RNA viruses before we can fully appreciate their contributions to the dynamics of marine ecosystems. As a step toward making sense of how RNA viruses contribute to the extraordinary viral diversity in the sea, we summarize in this review what is currently known about RNA viruses that infect marine organisms.

  6. Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation.

    Science.gov (United States)

    Tao, Wenjing; Sun, Lina; Shi, Hongjuan; Cheng, Yunying; Jiang, Dongneng; Fu, Beide; Conte, Matthew A; Gammerdinger, William J; Kocher, Thomas D; Wang, Deshou

    2016-05-04

    MicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia. We identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish. The sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.

  7. An integrated computational validation approach for potential novel miRNA prediction

    Directory of Open Access Journals (Sweden)

    Pooja Viswam

    2017-12-01

    Full Text Available MicroRNAs (miRNAs are short, non-coding RNAs between 17bp-24bp length that regulate gene expression by targeting mRNA molecules. The regulatory functions of miRNAs are known to be majorly associated with disease phenotypes such as cancer, cell signaling, cell division, growth and other metabolisms. Novel miRNAs are defined as sequences which does not have any similarity with the existing known sequences and void of any experimental evidences. In recent decades, the advent of next-generation sequencing allows us to capture the small RNA molecules form the cells and developing methods to estimate their expression levels. Several computational algorithms are available to predict the novel miRNAs from the deep sequencing data. In this work, we integrated three novel miRNA prediction programs miRDeep, miRanalyzer and miRPRo to compare and validate their prediction efficiency. The dicer cleavage sites, alignment density, seed conservation, minimum free energy, AU-GC percentage, secondary loop scores, false discovery rates and confidence scores will be considered for comparison and evaluation. Efficiency to identify isomiRs and base pair mismatches in a strand specific manner will also be considered for the computational validation. Further, the criteria and parameters for the identification of the best possible novel miRNA with minimal false positive rates were deduced.

  8. MicroRNA Expression Profile in Penile Cancer Revealed by Next-Generation Small RNA Sequencing.

    Directory of Open Access Journals (Sweden)

    Li Zhang

    Full Text Available Penile cancer (PeCa is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also

  9. MicroRNA expression in benign breast tissue and risk of subsequent invasive breast cancer.

    Science.gov (United States)

    Rohan, Thomas; Ye, Kenny; Wang, Yihong; Glass, Andrew G; Ginsberg, Mindy; Loudig, Olivier

    2018-01-01

    MicroRNAs are endogenous, small non-coding RNAs that control gene expression by directing their target mRNAs for degradation and/or posttranscriptional repression. Abnormal expression of microRNAs is thought to contribute to the development and progression of cancer. A history of benign breast disease (BBD) is associated with increased risk of subsequent breast cancer. However, no large-scale study has examined the association between microRNA expression in BBD tissue and risk of subsequent invasive breast cancer (IBC). We conducted discovery and validation case-control studies nested in a cohort of 15,395 women diagnosed with BBD in a large health plan between 1971 and 2006 and followed to mid-2015. Cases were women with BBD who developed subsequent IBC; controls were matched 1:1 to cases on age, age at diagnosis of BBD, and duration of plan membership. The discovery stage (316 case-control pairs) entailed use of the Illumina MicroRNA Expression Profiling Assay (in duplicate) to identify breast cancer-associated microRNAs. MicroRNAs identified at this stage were ranked by the strength of the correlation between Illumina array and quantitative PCR results for 15 case-control pairs. The top ranked 14 microRNAs entered the validation stage (165 case-control pairs) which was conducted using quantitative PCR (in triplicate). In both stages, linear regression was used to evaluate the association between the mean expression level of each microRNA (response variable) and case-control status (independent variable); paired t-tests were also used in the validation stage. None of the 14 validation stage microRNAs was associated with breast cancer risk. The results of this study suggest that microRNA expression in benign breast tissue does not influence the risk of subsequent IBC.

  10. A mathematical model and quantitative comparison of the small RNA circuit in the Vibrio harveyi and Vibrio cholerae quorum sensing systems

    International Nuclear Information System (INIS)

    Hunter, G A M; Vasquez, F Guevara; Keener, J P

    2013-01-01

    Quorum sensing is the process by which bacteria regulate their gene expression based on the local cell-population density. The quorum sensing systems of Vibrio harveyi and Vibrio cholerae are comprised of a phosphorelay cascade coupled to a small RNA (sRNA) circuit. The sRNA circuit contains multiple quorum regulated small RNA (Qrr) that regulate expression of the homologous master transcriptional regulators LuxR (in V. harveyi) and HapR (in V. cholerae). Their quorum sensing systems are topologically similar and homologous thereby making it difficult to understand why repression of HapR is more robust than LuxR to changes in Qrr. In this work we formulate and parameterize a novel mathematical model of the V. harveyi and V. cholerae sRNA circuit. We parameterize the model by fitting it to a variety of empirical data from both species. We show that we can distinguish all of the parameters and that the parameterizations (one for each species) are robust to errors in the data. We then use our model to propose some experiments to identify and explain kinetic differences between the species. We find that V. cholerae Qrr are more abundant and more sensitive to changes in LuxO than V. harveyi Qrr and argue that this is why expression of HapR is more robust than LuxR to changes in Qrr. (paper)

  11. Topical Anti-Nuclear Factor-Kappa B Small Interfering RNA with Functional Peptides Containing Sericin-Based Hydrogel for Atopic Dermatitis

    Directory of Open Access Journals (Sweden)

    Takanori Kanazawa

    2015-09-01

    Full Text Available The small interfering RNA (siRNA is suggested to offer a novel means of treating atopic dermatitis (AD because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA siRNA and functional peptides and determined the intradermal permeation and the anti-AD effects in an AD mouse model. We selected the silk protein, sericin (SC, which is a versatile biocompatible biomaterial to prepare hydrogel as an aqueous gel base. We found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel containing functional peptides than via the preparation without functional peptides. In addition, the ear thickness and clinical skin severity of the AD-induced mice treated with hydrogel containing anti-RelA siRNA with functional peptides improved more than that of mice treated with the preparation formulated with negative siRNA.

  12. tRNA-like structure regulates translation of Brome mosaic virus RNA.

    Science.gov (United States)

    Barends, Sharief; Rudinger-Thirion, Joëlle; Florentz, Catherine; Giegé, Richard; Pleij, Cornelis W A; Kraal, Barend

    2004-04-01

    For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS(TYMV) of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLS(BMV) (about 200 nucleotides) with determinants for tyrosylation. We discovered TLS(BMV)-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS(BMV) tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS(BMV) in trans. Intriguingly, a subdomain of the TLS(BMV) could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS(BMV) during the BMV infection cycle.

  13. The Non-Coding RNA Ontology (NCRO): a comprehensive resource for the unification of non-coding RNA biology.

    Science.gov (United States)

    Huang, Jingshan; Eilbeck, Karen; Smith, Barry; Blake, Judith A; Dou, Dejing; Huang, Weili; Natale, Darren A; Ruttenberg, Alan; Huan, Jun; Zimmermann, Michael T; Jiang, Guoqian; Lin, Yu; Wu, Bin; Strachan, Harrison J; He, Yongqun; Zhang, Shaojie; Wang, Xiaowei; Liu, Zixing; Borchert, Glen M; Tan, Ming

    2016-01-01

    In recent years, sequencing technologies have enabled the identification of a wide range of non-coding RNAs (ncRNAs). Unfortunately, annotation and integration of ncRNA data has lagged behind their identification. Given the large quantity of information being obtained in this area, there emerges an urgent need to integrate what is being discovered by a broad range of relevant communities. To this end, the Non-Coding RNA Ontology (NCRO) is being developed to provide a systematically structured and precisely defined controlled vocabulary for the domain of ncRNAs, thereby facilitating the discovery, curation, analysis, exchange, and reasoning of data about structures of ncRNAs, their molecular and cellular functions, and their impacts upon phenotypes. The goal of NCRO is to serve as a common resource for annotations of diverse research in a way that will significantly enhance integrative and comparative analysis of the myriad resources currently housed in disparate sources. It is our belief that the NCRO ontology can perform an important role in the comprehensive unification of ncRNA biology and, indeed, fill a critical gap in both the Open Biological and Biomedical Ontologies (OBO) Library and the National Center for Biomedical Ontology (NCBO) BioPortal. Our initial focus is on the ontological representation of small regulatory ncRNAs, which we see as the first step in providing a resource for the annotation of data about all forms of ncRNAs. The NCRO ontology is free and open to all users, accessible at: http://purl.obolibrary.org/obo/ncro.owl.

  14. Discovery of a Parenteral Small Molecule Coagulation Factor XIa Inhibitor Clinical Candidate (BMS-962212).

    Science.gov (United States)

    Pinto, Donald J P; Orwat, Michael J; Smith, Leon M; Quan, Mimi L; Lam, Patrick Y S; Rossi, Karen A; Apedo, Atsu; Bozarth, Jeffrey M; Wu, Yiming; Zheng, Joanna J; Xin, Baomin; Toussaint, Nathalie; Stetsko, Paul; Gudmundsson, Olafur; Maxwell, Brad; Crain, Earl J; Wong, Pancras C; Lou, Zhen; Harper, Timothy W; Chacko, Silvi A; Myers, Joseph E; Sheriff, Steven; Zhang, Huiping; Hou, Xiaoping; Mathur, Arvind; Seiffert, Dietmar A; Wexler, Ruth R; Luettgen, Joseph M; Ewing, William R

    2017-12-14

    Factor XIa (FXIa) is a blood coagulation enzyme that is involved in the amplification of thrombin generation. Mounting evidence suggests that direct inhibition of FXIa can block pathologic thrombus formation while preserving normal hemostasis. Preclinical studies using a variety of approaches to reduce FXIa activity, including direct inhibitors of FXIa, have demonstrated good antithrombotic efficacy without increasing bleeding. On the basis of this potential, we targeted our efforts at identifying potent inhibitors of FXIa with a focus on discovering an acute antithrombotic agent for use in a hospital setting. Herein we describe the discovery of a potent FXIa clinical candidate, 55 (FXIa K i = 0.7 nM), with excellent preclinical efficacy in thrombosis models and aqueous solubility suitable for intravenous administration. BMS-962212 is a reversible, direct, and highly selective small molecule inhibitor of FXIa.

  15. OP17MICRORNA PROFILING USING SMALL RNA-SEQ IN PAEDIATRIC LOW GRADE GLIOMAS

    Science.gov (United States)

    Jeyapalan, Jennie N.; Jones, Tania A.; Tatevossian, Ruth G.; Qaddoumi, Ibrahim; Ellison, David W.; Sheer, Denise

    2014-01-01

    INTRODUCTION: MicroRNAs regulate gene expression by targeting mRNAs for translational repression or degradation at the post-transcriptional level. In paediatric low-grade gliomas a few key genetic mutations have been identified, including BRAF fusions, FGFR1 duplications and MYB rearrangements. Our aim in the current study is to profile aberrant microRNA expression in paediatric low-grade gliomas and determine the role of epigenetic changes in the aetiology and behaviour of these tumours. METHOD: MicroRNA profiling of tumour samples (6 pilocytic, 2 diffuse, 2 pilomyxoid astrocytomas) and normal brain controls (4 adult normal brain samples and a primary glial progenitor cell-line) was performed using small RNA sequencing. Bioinformatic analysis included sequence alignment, analysis of the number of reads (CPM, counts per million) and differential expression. RESULTS: Sequence alignment identified 695 microRNAs, whose expression was compared in tumours v. normal brain. PCA and hierarchical clustering showed separate groups for tumours and normal brain. Computational analysis identified approximately 400 differentially expressed microRNAs in the tumours compared to matched location controls. Our findings will then be validated and integrated with extensive genetic and epigenetic information we have previously obtained for the full tumour cohort. CONCLUSION: We have identified microRNAs that are differentially expressed in paediatric low-grade gliomas. As microRNAs are known to target genes involved in the initiation and progression of cancer, they provide critical information on tumour pathogenesis and are an important class of biomarkers.

  16. Intrinsic noise of microRNA-regulated genes and the ceRNA hypothesis.

    Directory of Open Access Journals (Sweden)

    Javad Noorbakhsh

    Full Text Available MicroRNAs are small noncoding RNAs that regulate genes post-transciptionally by binding and degrading target eukaryotic mRNAs. We use a quantitative model to study gene regulation by inhibitory microRNAs and compare it to gene regulation by prokaryotic small non-coding RNAs (sRNAs. Our model uses a combination of analytic techniques as well as computational simulations to calculate the mean-expression and noise profiles of genes regulated by both microRNAs and sRNAs. We find that despite very different molecular machinery and modes of action (catalytic vs stoichiometric, the mean expression levels and noise profiles of microRNA-regulated genes are almost identical to genes regulated by prokaryotic sRNAs. This behavior is extremely robust and persists across a wide range of biologically relevant parameters. We extend our model to study crosstalk between multiple mRNAs that are regulated by a single microRNA and show that noise is a sensitive measure of microRNA-mediated interaction between mRNAs. We conclude by discussing possible experimental strategies for uncovering the microRNA-mRNA interactions and testing the competing endogenous RNA (ceRNA hypothesis.

  17. Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

    Directory of Open Access Journals (Sweden)

    Mario Gustavo Mayer

    2012-06-01

    Full Text Available The addition of a capped mini-exon [spliced leader (SL] through trans-splicing is essential for the maturation of RNA polymerase (pol II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1 in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin, we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

  18. Inhibition of virus replication by RNA interference

    NARCIS (Netherlands)

    Haasnoot, P. C. Joost; Cupac, Daniel; Berkhout, Ben

    2003-01-01

    RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene

  19. Identification of unique microRNA signature associated with lupus nephritis.

    Directory of Open Access Journals (Sweden)

    Jeannie L Te

    2010-05-01

    Full Text Available MicroRNAs (miRNA have emerged as an important new class of modulators of gene expression. In this study we investigated miRNA that are differentially expressed in lupus nephritis. Microarray technology was used to investigate differentially expressed miRNA in peripheral blood mononuclear cells (PBMCs and Epstein-Barr Virus (EBV-transformed cell lines obtained from lupus nephritis affected patients and unaffected controls. TaqMan-based stem-loop real-time polymerase chain reaction was used for validation. Microarray analysis of miRNA expressed in both African American (AA and European American (EA derived lupus nephritis samples revealed 29 and 50 differentially expressed miRNA, respectively, of 850 tested. There were 18 miRNA that were differentially expressed in both racial groups. When samples from both racial groups and different specimen types were considered, there were 5 primary miRNA that were differentially expressed. We have identified 5 miRNA; hsa-miR-371-5P, hsa-miR-423-5P, hsa-miR-638, hsa-miR-1224-3P and hsa-miR-663 that were differentially expressed in lupus nephritis across different racial groups and all specimen types tested. Hsa-miR-371-5P, hsa-miR-1224-3P and hsa-miR-423-5P, are reported here for the first time to be associated with lupus nephritis. Our work establishes EBV-transformed B cell lines as a useful model for the discovery of miRNA as biomarkers for SLE. Based on these findings, we postulate that these differentially expressed miRNA may be potential novel biomarkers for SLE as well as help elucidate pathogenic mechanisms of lupus nephritis. The investigation of miRNA profiles in SLE may lead to the discovery and development of novel methods to diagnosis, treat and prevent SLE.

  20. Small RNA Sequencing Uncovers New miRNAs and moRNAs Differentially Expressed in Normal and Primary Myelofibrosis CD34+ Cells.

    Directory of Open Access Journals (Sweden)

    Paola Guglielmelli

    Full Text Available Myeloproliferative neoplasms (MPN are chronic myeloid cancers thought to arise at the level of CD34+ hematopoietic stem/progenitor cells. They include essential thrombocythemia (ET, polycythemia vera (PV and primary myelofibrosis (PMF. All can progress to acute leukemia, but PMF carries the worst prognosis. Increasing evidences indicate that deregulation of microRNAs (miRNAs might plays an important role in hematologic malignancies, including MPN. To attain deeper knowledge of short RNAs (sRNAs expression pattern in CD34+ cells and of their possible role in mediating post-transcriptional regulation in PMF, we sequenced with Illumina HiSeq2000 technology CD34+ cells from healthy subjects and PMF patients. We detected the expression of 784 known miRNAs, with a prevalence of miRNA up-regulation in PMF samples, and discovered 34 new miRNAs and 99 new miRNA-offset RNAs (moRNAs, in CD34+ cells. Thirty-seven small RNAs were differentially expressed in PMF patients compared with healthy subjects, according to microRNA sequencing data. Five miRNAs (miR-10b-5p, miR-19b-3p, miR-29a-3p, miR-379-5p, and miR-543 were deregulated also in PMF granulocytes. Moreover, 3'-moR-128-2 resulted consistently downregulated in PMF according to RNA-seq and qRT-PCR data both in CD34+ cells and granulocytes. Target predictions of these validated small RNAs de-regulated in PMF and functional enrichment analyses highlighted many interesting pathways involved in tumor development and progression, such as signaling by FGFR and DAP12 and Oncogene Induced Senescence. As a whole, data obtained in this study deepened the knowledge of miRNAs and moRNAs altered expression in PMF CD34+ cells and allowed to identify and validate a specific small RNA profile that distinguishes PMF granulocytes from those of normal subjects. We thus provided new information regarding the possible role of miRNAs and, specifically, of new moRNAs in this disease.

  1. A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown

    DEFF Research Database (Denmark)

    Higgins, Geoff S; Prevo, Remko; Lee, Yin-Fai

    2010-01-01

    The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor...... radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B......) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA...

  2. Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma

    DEFF Research Database (Denmark)

    Baraniskin, Alexander; Nöpel-Dünnebacke, Stefanie; Ahrens, Maike

    2013-01-01

    Improved non-invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non-coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non-invasive cancer diagnosis. Analyzing serum derived from nude mice implanted...... with primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR-1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR-1246 assays with RNA fragments derived from U...... that hsa-miR-1246 is likely a pseudo microRNA. In a next step, RNU2-1f was measured by qRT-PCR and normalized to cel-54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity...

  3. Next-generation small RNA sequencing for microRNAs profiling in the honey bee Apis mellifera.

    Science.gov (United States)

    Chen, X; Yu, X; Cai, Y; Zheng, H; Yu, D; Liu, G; Zhou, Q; Hu, S; Hu, F

    2010-12-01

    MicroRNAs (miRNAs) are key regulators in various physiological and pathological processes via post-transcriptional regulation of gene expression. The honey bee (Apis mellifera) is a key model for highly social species, and its complex social behaviour can be interpreted theoretically as changes in gene regulation, in which miRNAs are thought to be involved. We used the SOLiD sequencing system to identify the repertoire of miRNAs in the honey bee by sequencing a mixed small RNA library from different developmental stages. We obtained a total of 36,796,459 raw sequences; of which 5,491,100 short sequences were fragments of mRNA and other noncoding RNAs (ncRNA), and 1,759,346 reads mapped to the known miRNAs. We predicted 267 novel honey bee miRNAs representing 380,182 short reads, including eight miRNAs of other insects in 14,107,583 genome-mapped sequences. We verified 50 of them using stem-loop reverse-transcription PCR (RT-PCR), in which 35 yielded PCR products. Cross-species analyses showed 81 novel miRNAs with homologues in other insects, suggesting that they were authentic miRNAs and have similar functions. The results of this study provide a basis for studies of the miRNA-modulating networks in development and some intriguing phenomena such as caste differentiation in A. mellifera. © 2010 The Authors. Insect Molecular Biology © 2010 The Royal Entomological Society.

  4. Paraspeckles: Where Long Noncoding RNA Meets Phase Separation.

    Science.gov (United States)

    Fox, Archa H; Nakagawa, Shinichi; Hirose, Tetsuro; Bond, Charles S

    2018-02-01

    Long noncoding RNA (lncRNA) molecules are some of the newest and least understood players in gene regulation. Hence, we need good model systems with well-defined RNA and protein components. One such system is paraspeckles - protein-rich nuclear organelles built around a specific lncRNA scaffold. New discoveries show how paraspeckles are formed through multiple RNA-protein and protein-protein interactions, some of which involve extensive polymerization, and others with multivalent interactions driving phase separation. Once formed, paraspeckles influence gene regulation through sequestration of component proteins and RNAs, with subsequent depletion in other compartments. Here we focus on the dual aspects of paraspeckle structure and function, revealing an emerging role for these dynamic bodies in a multitude of cellular settings. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages.

    Directory of Open Access Journals (Sweden)

    Nan Li

    Full Text Available Non-coding small RNAs (sRNAs are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.

  6. Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines

    Science.gov (United States)

    Tosar, Juan Pablo; Gámbaro, Fabiana; Sanguinetti, Julia; Bonilla, Braulio; Witwer, Kenneth W.; Cayota, Alfonso

    2015-01-01

    Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19–60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5′ tRNA halves and 5′ RNA Y4-derived fragments of 31–33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ∼45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space. PMID:25940616

  7. Conditional estimation of local pooled dispersion parameter in small-sample RNA-Seq data improves differential expression test.

    Science.gov (United States)

    Gim, Jungsoo; Won, Sungho; Park, Taesung

    2016-10-01

    High throughput sequencing technology in transcriptomics studies contribute to the understanding of gene regulation mechanism and its cellular function, but also increases a need for accurate statistical methods to assess quantitative differences between experiments. Many methods have been developed to account for the specifics of count data: non-normality, a dependence of the variance on the mean, and small sample size. Among them, the small number of samples in typical experiments is still a challenge. Here we present a method for differential analysis of count data, using conditional estimation of local pooled dispersion parameters. A comprehensive evaluation of our proposed method in the aspect of differential gene expression analysis using both simulated and real data sets shows that the proposed method is more powerful than other existing methods while controlling the false discovery rates. By introducing conditional estimation of local pooled dispersion parameters, we successfully overcome the limitation of small power and enable a powerful quantitative analysis focused on differential expression test with the small number of samples.

  8. Discovering the first microRNA-targeted drug

    DEFF Research Database (Denmark)

    Lindow, Morten; Kauppinen, Sakari

    2012-01-01

    MicroRNAs (miRNAs) are important post-transcriptional regulators of nearly every biological process in the cell and play key roles in the pathogenesis of human disease. As a result, there are many drug discovery programs that focus on developing miRNA-based therapeutics. The most advanced...

  9. RNA folding: structure prediction, folding kinetics and ion electrostatics.

    Science.gov (United States)

    Tan, Zhijie; Zhang, Wenbing; Shi, Yazhou; Wang, Fenghua

    2015-01-01

    Beyond the "traditional" functions such as gene storage, transport and protein synthesis, recent discoveries reveal that RNAs have important "new" biological functions including the RNA silence and gene regulation of riboswitch. Such functions of noncoding RNAs are strongly coupled to the RNA structures and proper structure change, which naturally leads to the RNA folding problem including structure prediction and folding kinetics. Due to the polyanionic nature of RNAs, RNA folding structure, stability and kinetics are strongly coupled to the ion condition of solution. The main focus of this chapter is to review the recent progress in the three major aspects in RNA folding problem: structure prediction, folding kinetics and ion electrostatics. This chapter will introduce both the recent experimental and theoretical progress, while emphasize the theoretical modelling on the three aspects in RNA folding.

  10. Genome-Wide Identification, Characterization, and Expression Analysis of Small RNA Biogenesis Purveyors Reveal Their Role in Regulation of Biotic Stress Responses in Three Legume Crops

    Directory of Open Access Journals (Sweden)

    Rajeev K. Varshney

    2017-04-01

    Full Text Available Biotic stress in legume crops is one of the major threats to crop yield and productivity. Being sessile organisms, plants have evolved a myriad of mechanisms to combat different stresses imposed on them. One such mechanism, deciphered in the last decade, is small RNA (sRNA mediated defense in plants. Small RNAs (sRNAs have emerged as one of the major players in gene expression regulation in plants during developmental stages and under stress conditions. They are known to act both at transcriptional and post-transcriptional levels. Dicer-like (DCL, Argonaute (AGO, and RNA dependent RNA polymerase (RDR constitute the major components of sRNA biogenesis machinery and are known to play a significant role in combating biotic and abiotic stresses. This study is, therefore, focused on identification and characterization of sRNA biogenesis proteins in three important legume crops, namely chickpea, pigeonpea, and groundnut. Phylogenetic analysis of these proteins between legume species classified them into distinct clades and suggests the evolutionary conservation of these genes across the members of Papillionidoids subfamily. Variable expression of sRNA biogenesis genes in response to the biotic stresses among the three legumes indicate the possible existence of specialized regulatory mechanisms in different legumes. This is the first ever study to understand the role of sRNA biogenesis genes in response to pathogen attacks in the studied legumes.

  11. Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination.

    Science.gov (United States)

    Tataruch-Weinert, Dorota; Musante, Luca; Kretz, Oliver; Holthofer, Harry

    2016-01-01

    Urinary extracellular vesicles (UEVs) represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters) and chemical pretreatment (water purification tablet). For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a) Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b) UEVs were enriched with small RNA, (c) Ribosomal RNA peaks were not observed for any RNA extraction method used and (d) RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA isolation method must be selected in conjunction with

  12. Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination

    Directory of Open Access Journals (Sweden)

    Dorota Tataruch-Weinert

    2016-06-01

    Full Text Available Background: Urinary extracellular vesicles (UEVs represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established. Methods: UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis. Results: The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters and chemical pretreatment (water purification tablet. For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b UEVs were enriched with small RNA, (c Ribosomal RNA peaks were not observed for any RNA extraction method used and (d RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method. Conclusion: Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA

  13. Therapeutic effects of protein kinase N3 small interfering RNA and doxorubicin combination therapy on liver and lung metastases

    Science.gov (United States)

    Hattori, Yoshiyuki; Kikuchi, Takuto; Nakamura, Mari; Ozaki, Kei-Ichi; Onishi, Hiraku

    2017-01-01

    It has been reported that suppression of protein kinase N3 (PKN3) expression in vascular and lymphatic endothelial cells results in the inhibition of tumor progression and lymph node metastasis formation. The present study investigated whether combination therapy of small interfering RNA (siRNA) against PKN3 and doxorubicin (DXR) could increase therapeutic efficacy against liver and lung metastases. In vitro transfection of PKN3 siRNA into PKN3-positive MDA-MB-231, LLC, and Colon 26 cells and PKN3-negative MCF-7 cells did not inhibit cell growth and did not increase sensitivity to DXR. However, following in vivo treatment, PKN3 siRNA suppressed the growth of liver MDA-MB-231 and lung LLC and MCF-7 metastases, although combination therapy with DXR did not increase the therapeutic efficacy. By contrast, in liver MCF-7 metastases, PKN3 siRNA or DXR alone did not exhibit significant inhibition of tumor growth, but their combination significantly improved therapeutic efficacy. Treatment of liver MDA-MB-231 metastases with PKN3 siRNA induced a change in vasculature structure via suppression of PKN3 mRNA expression. PKN3 siRNA may induce antitumor effects in lung and liver metastases by suppression of PKN3 expression in stroma cells, such as endothelial cells. From these findings, PKN3 siRNA alone or in combination with DXR may reduce the tumor growth of liver and lung metastases regardless of PKN3 expression in tumor cells. PMID:29098022

  14. Alteration of RNA splicing by small molecule inhibitors of the interaction between NHP2L1 and U4

    Science.gov (United States)

    Diouf, Barthelemy; Lin, Wenwei; Goktug, Asli; Grace, Christy R. R.; Waddell, Michael Brett; Bao, Ju; Shao, Youming; Heath, Richard J.; Zheng, Jie J.; Shelat, Anang A.; Relling, Mary V.; Chen, Taosheng; Evans, William E.

    2018-01-01

    Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remains a challenge. We developed an assay based on time-resolved FRET (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs which could facilitate the development of therapeutic strategies to modify splicing and alter gene function. PMID:28985478

  15. Systematic Identification and Assessment of Therapeutic Targets for Breast Cancer Based on Genome-Wide RNA Interference Transcriptomes

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2017-02-01

    Full Text Available With accumulating public omics data, great efforts have been made to characterize the genetic heterogeneity of breast cancer. However, identifying novel targets and selecting the best from the sizeable lists of candidate targets is still a key challenge for targeted therapy, largely owing to the lack of economical, efficient and systematic discovery and assessment to prioritize potential therapeutic targets. Here, we describe an approach that combines the computational evaluation and objective, multifaceted assessment to systematically identify and prioritize targets for biological validation and therapeutic exploration. We first establish the reference gene expression profiles from breast cancer cell line MCF7 upon genome-wide RNA interference (RNAi of a total of 3689 genes, and the breast cancer query signatures using RNA-seq data generated from tissue samples of clinical breast cancer patients in the Cancer Genome Atlas (TCGA. Based on gene set enrichment analysis, we identified a set of 510 genes that when knocked down could significantly reverse the transcriptome of breast cancer state. We then perform multifaceted assessment to analyze the gene set to prioritize potential targets for gene therapy. We also propose drug repurposing opportunities and identify potentially druggable proteins that have been poorly explored with regard to the discovery of small-molecule modulators. Finally, we obtained a small list of candidate therapeutic targets for four major breast cancer subtypes, i.e., luminal A, luminal B, HER2+ and triple negative breast cancer. This RNAi transcriptome-based approach can be a helpful paradigm for relevant researches to identify and prioritize candidate targets for experimental validation.

  16. Exploration of miRNA families for hypotheses generation.

    KAUST Repository

    Kamanu, T.K.

    2013-10-15

    Technological improvements have resulted in increased discovery of new microRNAs (miRNAs) and refinement and enrichment of existing miRNA families. miRNA families are important because they suggest a common sequence or structure configuration in sets of genes that hint to a shared function. Exploratory tools to enhance investigation of characteristics of miRNA families and the functions of family-specific miRNA genes are lacking. We have developed, miRNAVISA, a user-friendly web-based tool that allows customized interrogation and comparisons of miRNA families for hypotheses generation, and comparison of per-species chromosomal distribution of miRNA genes in different families. This study illustrates hypothesis generation using miRNAVISA in seven species. Our results unveil a subclass of miRNAs that may be regulated by genomic imprinting, and also suggest that some miRNA families may be species-specific, as well as chromosome- and/or strand-specific.

  17. A negative regulation loop of long noncoding RNA HOTAIR and p53 in non-small-cell lung cancer

    Directory of Open Access Journals (Sweden)

    Zhai N

    2016-09-01

    Full Text Available Nailiang Zhai,1 Yongfu Xia,1 Rui Yin,2 Jinping Liu,3 Fuquan Gao1 1Department of Respiratory Medicine, Affiliated Hospital of Binzhou Medical University, 2Department of Respiratory Medicine, People’s Hospital of Binzhou City, 3Department of Pharmacology, Binzhou Medical University, Binzhou, Shandong, People’s Republic of China Abstract: Non-small-cell lung cancer (NSCLC is one of the leading causes of cancer-related death worldwide, and the 5-year survival rate is still low despite advances in diagnosis and therapeutics. A long noncoding RNA (lncRNA HOX antisense intergenic RNA (HOTAIR has been revealed to play important roles in NSCLC carcinogenesis but the detailed mechanisms are still unclear. In the current study, we aimed to investigate the regulation between the lncRNA HOTAIR and p53 in the NSCLC patient samples and cell lines. Our results showed that HOTAIR expression was significantly higher in the cancer tissues than that in the adjacent normal tissue, and was negatively correlated with p53 functionality rather than expression. When p53 was overexpressed in A549 cells, the lncRNA HOTAIR expression was downregulated, and the cell proliferation rate and cell invasion capacity decreased as a consequence. We identified two binding sites of p53 on the promoter region of HOTAIR, where the p53 protein would bind to and suppress the HOTAIR mRNA transcription. Inversely, overexpression of lncRNA HOTAIR inhibited the expression of p53 in A549 cells. Mechanistic studies revealed that HOTAIR modified the promoter of p53 and enhanced histone H3 lysine 27 trimethylation (H3K27me3. These studies identified a specific negative regulation loop of lncRNA HOTAIR and p53 in NSCLC cells, which revealed a new understanding of tumorigenesis in p53 dysfunction NSCLC cells. Keywords: NSCLC, LncRNA HOTAIR, p53, negative loop

  18. Simultaneous sequencing of coding and noncoding RNA reveals a human transcriptome dominated by a small number of highly expressed noncoding genes.

    Science.gov (United States)

    Boivin, Vincent; Deschamps-Francoeur, Gabrielle; Couture, Sonia; Nottingham, Ryan M; Bouchard-Bourelle, Philia; Lambowitz, Alan M; Scott, Michelle S; Abou-Elela, Sherif

    2018-07-01

    Comparing the abundance of one RNA molecule to another is crucial for understanding cellular functions but most sequencing techniques can target only specific subsets of RNA. In this study, we used a new fragmented ribodepleted TGIRT sequencing method that uses a thermostable group II intron reverse transcriptase (TGIRT) to generate a portrait of the human transcriptome depicting the quantitative relationship of all classes of nonribosomal RNA longer than 60 nt. Comparison between different sequencing methods indicated that FRT is more accurate in ranking both mRNA and noncoding RNA than viral reverse transcriptase-based sequencing methods, even those that specifically target these species. Measurements of RNA abundance in different cell lines using this method correlate with biochemical estimates, confirming tRNA as the most abundant nonribosomal RNA biotype. However, the single most abundant transcript is 7SL RNA, a component of the signal recognition particle. S tructured n on c oding RNAs (sncRNAs) associated with the same biological process are expressed at similar levels, with the exception of RNAs with multiple functions like U1 snRNA. In general, sncRNAs forming RNPs are hundreds to thousands of times more abundant than their mRNA counterparts. Surprisingly, only 50 sncRNA genes produce half of the non-rRNA transcripts detected in two different cell lines. Together the results indicate that the human transcriptome is dominated by a small number of highly expressed sncRNAs specializing in functions related to translation and splicing. © 2018 Boivin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

    Science.gov (United States)

    Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

    1998-10-01

    Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

  20. Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

    Science.gov (United States)

    Schnettler, Esther; Hemmes, Hans; Huismann, Rik; Goldbach, Rob; Prins, Marcel; Kormelink, Richard

    2010-11-01

    The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

  1. Genome-wide identification of novel small RNAs in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Gómez Lozano, María

    and RNA sequencing (RNAseq) technologies. The latter approach, in particular, has revolutionized sRNA discovery by enabling interrogation of the transcriptome at unprecedented depths. The size and complexity of the P. aeruginosa genome suggests that it encodes many hitherto undetected sRNAs. In this study...

  2. Steric restrictions of RISC in RNA interference identified with size-expanded RNA nucleobases.

    Science.gov (United States)

    Hernández, Armando R; Peterson, Larryn W; Kool, Eric T

    2012-08-17

    Understanding the interactions between small interfering RNAs (siRNAs) and the RNA-induced silencing complex (RISC), the key protein complex of RNA interference (RNAi), is of great importance to the development of siRNAs with improved biological and potentially therapeutic function. Although various chemically modified siRNAs have been reported, relatively few studies with modified nucleobases exist. Here we describe the synthesis and hybridization properties of siRNAs bearing size-expanded RNA (xRNA) nucleobases and their use as a novel and systematic set of steric probes in RNAi. xRNA nucleobases are expanded by 2.4 Å using benzo-homologation and retain canonical Watson-Crick base-pairing groups. Our data show that the modified siRNA duplexes display small changes in melting temperature (+1.4 to -5.0 °C); substitutions near the center are somewhat destabilizing to the RNA duplex, while substitutions near the ends are stabilizing. RNAi studies in a dual-reporter luciferase assay in HeLa cells revealed that xRNA nucleobases in the antisense strand reduce activity at some central positions near the seed region but are generally well tolerated near the ends. Most importantly, we observed that xRNA substitutions near the 3'-end increased activity over that of wild-type siRNAs. The data are analyzed in terms of site-dependent steric effects in RISC. Circular dichroism experiments show that single xRNA substitutions do not significantly distort the native A-form helical structure of the siRNA duplex, and serum stability studies demonstrated that xRNA substitutions protect siRNAs against nuclease degradation.

  3. Peptidomimetics with beta-peptoid resudies as carriers for intracellular delivery of small interfering RNA (siRNA)

    DEFF Research Database (Denmark)

    Foged, Camilla

    cytometry. We conclude that simple complex formation via electrostatic interactions between siRNA and the cationic peptidomimetics is not sufficient for the delivery of siRNA to the RNA interference (RNAi) pathway in the cytoplasm. We are currently testing chemical conjugates of si...... prepared by mixing and characterized with respect to size and surface charge. At ratios of peptide nitrogen to siRNA phosphate (N/P) of 1 and below, particles with narrow size distributions (poly dispersity indexes lower than 0.11) ranging from approximately 100 to 350 nm were formed, and they showed...... a negative zeta potential (-24 to -31 mV). At higher N/P ratios, larger aggregates with zeta potential close to neutral were formed. However, the complexes were not able to silence the expression of enhanced green fluorescent protein (EGFP) in HeLa-cells stably expressing EGFP, which was measured by flow...

  4. Dampening Enthusiasm for Circulating MicroRNA in Breast Cancer

    OpenAIRE

    Leidner, Rom S.; Li, Li; Thompson, Cheryl L.

    2013-01-01

    Genome-wide platforms for high-throughput profiling of circulating miRNA (oligoarray or miR-Seq) offer enormous promise for agnostic discovery of circulating miRNA biomarkers as a pathway for development in breast cancer detection. By harmonizing data from 15 previous reports, we found widespread inconsistencies across prior studies. Whether this arises from differences in study design, such as sample source or profiling platform, is unclear. As a reproducibility experiment, we generated a ge...

  5. Applying genetics in inflammatory disease drug discovery

    DEFF Research Database (Denmark)

    Folkersen, Lasse; Biswas, Shameek; Frederiksen, Klaus Stensgaard

    2015-01-01

    , with several notable exceptions, the journey from a small-effect genetic variant to a functional drug has proven arduous, and few examples of actual contributions to drug discovery exist. Here, we discuss novel approaches of overcoming this hurdle by using instead public genetics resources as a pragmatic guide...... alongside existing drug discovery methods. Our aim is to evaluate human genetic confidence as a rationale for drug target selection....

  6. LR1: a candidate RNA virus of Leishmania.

    OpenAIRE

    Tarr, P I; Aline, R F; Smiley, B L; Scholler, J; Keithly, J; Stuart, K

    1988-01-01

    Although viruses are important biological agents and useful molecular tools, little is known about the viruses of parasites. We report here the discovery of a candidate for an RNA virus in a kinetoplastid parasite. This potential virus, which we term LR1, is present in the promastigote form of the human pathogen Leishmania braziliensis guyanensis CUMC1-1A but not in 11 other stocks of Leishmania that were examined nor in Trypanosoma brucei. The candidate viral RNA has a size of approximately ...

  7. Arthropods as a source of new RNA viruses.

    Science.gov (United States)

    Bichaud, L; de Lamballerie, X; Alkan, C; Izri, A; Gould, E A; Charrel, R N

    2014-12-01

    The discovery and development of methods for isolation, characterisation and taxonomy of viruses represents an important milestone in the study, treatment and control of virus diseases during the 20th century. Indeed, by the late-1950s, it was becoming common belief that most human and veterinary pathogenic viruses had been discovered. However, at that time, knowledge of the impact of improved commercial transportation, urbanisation and deforestation, on disease emergence, was in its infancy. From the late 1960s onwards viruses, such as hepatitis virus (A, B and C) hantavirus, HIV, Marburg virus, Ebola virus and many others began to emerge and it became apparent that the world was changing, at least in terms of virus epidemiology, largely due to the influence of anthropological activities. Subsequently, with the improvement of molecular biotechnologies, for amplification of viral RNA, genome sequencing and proteomic analysis the arsenal of available tools for virus discovery and genetic characterization opened up new and exciting possibilities for virological discovery. Many recently identified but "unclassified" viruses are now being allocated to existing genera or families based on whole genome sequencing, bioinformatic and phylogenetic analysis. New species, genera and families are also being created following the guidelines of the International Committee for the Taxonomy of Viruses. Many of these newly discovered viruses are vectored by arthropods (arboviruses) and possess an RNA genome. This brief review will focus largely on the discovery of new arthropod-borne viruses. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Characterization of novel precursor miRNAs using next generation sequencing and prediction of miRNA targets in Atlantic halibut.

    Directory of Open Access Journals (Sweden)

    Teshome Tilahun Bizuayehu

    Full Text Available BACKGROUND: microRNAs (miRNAs are implicated in regulation of many cellular processes. miRNAs are processed to their mature functional form in a step-wise manner by multiple proteins and cofactors in the nucleus and cytoplasm. Many miRNAs are conserved across vertebrates. Mature miRNAs have recently been characterized in Atlantic halibut (Hippoglossus hippoglossus L.. The aim of this study was to identify and characterize precursor miRNA (pre-miRNAs and miRNA targets in this non-model flatfish. Discovery of miRNA precursor forms and targets in non-model organisms is difficult because of limited source information available. Therefore, we have developed a methodology to overcome this limitation. METHODS: Genomic DNA and small transcriptome of Atlantic halibut were sequenced using Roche 454 pyrosequencing and SOLiD next generation sequencing (NGS, respectively. Identified pre- miRNAs were further validated with reverse-transcription PCR. miRNA targets were identified using miRanda and RNAhybrid target prediction tools using sequences from public databases. Some of miRNA targets were also identified using RACE-PCR. miRNA binding sites were validated with luciferase assay using the RTS34st cell line. RESULTS: We obtained more than 1.3 M and 92 M sequence reads from 454 genomic DNA sequencing and SOLiD small RNA sequencing, respectively. We identified 34 known and 9 novel pre-miRNAs. We predicted a number of miRNA target genes involved in various biological pathways. miR-24 binding to kisspeptin 1 receptor-2 (kiss1-r2 was confirmed using luciferase assay. CONCLUSION: This study demonstrates that identification of conserved and novel pre-miRNAs in a non-model vertebrate lacking substantial genomic resources can be performed by combining different next generation sequencing technologies. Our results indicate a wide conservation of miRNA precursors and involvement of miRNA in multiple regulatory pathways, and provide resources for further research on miRNA

  9. RNA interference in designing transgenic crops.

    Science.gov (United States)

    Ali, Nusrat; Datta, Swapan K; Datta, Karabi

    2010-01-01

    RNA interference (RNAi) is a sequence specific gene silencing mechanism, triggered by the introduction of dsRNA leading to mRNA degradation. It helps in switching on and off the targeted gene, which might have significant impact in developmental biology. Discovery of RNAi represents one of the most promising and rapidly advancing frontiers in plant functional genomics and in crop improvement by plant metabolic engineering and also plays an important role in reduction of allergenicity by silencing specific plant allergens. In plants the RNAi technology has been employed successfully in improvement of several plant species- by increasing their nutritional value, overall quality and by conferring resistance against pathogens and diseases. The review gives an insight to the perspective use of the technology in designing crops with innovation, to bring improvement to crop productivity and quality.

  10. Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome.

    Science.gov (United States)

    Anda, Mizue; Ohtsubo, Yoshiyuki; Okubo, Takashi; Sugawara, Masayuki; Nagata, Yuji; Tsuda, Masataka; Minamisawa, Kiwamu; Mitsui, Hisayuki

    2015-11-17

    rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the "main" chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general.

  11. Construction of RNA nanocages by re-engineering the packaging RNA of Phi29 bacteriophage

    Science.gov (United States)

    Hao, Chenhui; Li, Xiang; Tian, Cheng; Jiang, Wen; Wang, Guansong; Mao, Chengde

    2014-05-01

    RNA nanotechnology promises rational design of RNA nanostructures with wide array of structural diversities and functionalities. Such nanostructures could be used in applications such as small interfering RNA delivery and organization of in vivo chemical reactions. Though having impressive development in recent years, RNA nanotechnology is still quite limited and its programmability and complexity could not rival the degree of its closely related cousin: DNA nanotechnology. Novel strategies are needed for programmed RNA self-assembly. Here, we have assembled RNA nanocages by re-engineering a natural, biological RNA motif: the packaging RNA of phi29 bacteriophage. The resulting RNA nanostructures have been thoroughly characterized by gel electrophoresis, cryogenic electron microscopy imaging and dynamic light scattering.

  12. Biomarker discovery for colon cancer using a 761 gene RT-PCR assay

    Directory of Open Access Journals (Sweden)

    Hackett James R

    2007-08-01

    Full Text Available Abstract Background Reverse transcription PCR (RT-PCR is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan® RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX™ assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis and the likelihood of tumor response to standard chemotherapy regimens (prediction. We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. Results RNA was extracted from formalin fixed paraffin embedded (FPE tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan® reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. Conclusion We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of

  13. Small RNA-Seq analysis reveals microRNA-regulation of the Imd pathway during Escherichia coli infection in Drosophila.

    Science.gov (United States)

    Li, Shengjie; Shen, Li; Sun, Lianjie; Xu, Jiao; Jin, Ping; Chen, Liming; Ma, Fei

    2017-05-01

    Drosophila have served as a model for research on innate immunity for decades. However, knowledge of the post-transcriptional regulation of immune gene expression by microRNAs (miRNAs) remains rudimentary. In the present study, using small RNA-seq and bioinformatics analysis, we identified 67 differentially expressed miRNAs in Drosophila infected with Escherichia coli compared to injured flies at three time-points. Furthermore, we found that 21 of these miRNAs were potentially involved in the regulation of Imd pathway-related genes. Strikingly, based on UAS-miRNAs line screening and Dual-luciferase assay, we identified that miR-9a and miR-981 could both negatively regulate Drosophila antibacterial defenses and decrease the level of the antibacterial peptide, Diptericin. Taken together, these data support the involvement of miRNAs in the regulation of the Drosophila Imd pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

    DEFF Research Database (Denmark)

    Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine

    2015-01-01

    ) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer......Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA......-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer...

  15. Early days of tRNA research: Discovery, function, purification and ...

    Indian Academy of Sciences (India)

    Madhu

    2006-10-04

    Oct 4, 2006 ... function in protein synthesis and methods for its purification ... intermediate carrier of the amino acid in protein synthesis. (table 1). .... 14C-leucine were incubated with GTP, PEP, and pyruvate kinase as indicated (adapted from: Hoagland et al 1958). .... Purification of N. crassa mitochondrial initiator tRNA.

  16. 13 CFR 142.25 - Can a party or witness object to discovery?

    Science.gov (United States)

    2010-01-01

    ... discovery? 142.25 Section 142.25 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION PROGRAM FRAUD CIVIL REMEDIES ACT REGULATIONS Hearing Provisions § 142.25 Can a party or witness object to discovery? Any party or prospective witness may file a motion to quash a subpoena or to limit discovery or the...

  17. MicroRNAs in Amoebozoa: deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs.

    Science.gov (United States)

    Avesson, Lotta; Reimegård, Johan; Wagner, E Gerhart H; Söderbom, Fredrik

    2012-10-01

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.

  18. lncRNA Structure: Message to the Heart.

    Science.gov (United States)

    Fazal, Furqan M; Chang, Howard Y

    2016-10-06

    In this issue, Xue et al. (2016) describe the secondary structure of the heart-specific long non-coding RNA Braveheart, leading to the discovery of a short, asymmetric G-rich loop that controls cardiac lineage commitment by interacting with the transcription factor CNBP. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Misinterpreting the therapeutic effects of small interfering RNA caused by immune stimulation.

    Science.gov (United States)

    Robbins, Marjorie; Judge, Adam; Ambegia, Ellen; Choi, Catherine; Yaworski, Ed; Palmer, Lorne; McClintock, Kevin; MacLachlan, Ian

    2008-10-01

    Activation of innate immunity has direct effects in modulating viral replication, tumor growth, angiogenesis, and inflammatory and other immunological processes. It is now established that unmodified siRNA can activate this innate immune response and therefore there is real potential for siRNA to elicit nonspecific therapeutic effects in a wide range of disease models. Here we demonstrate that in a murine model of influenza infection, the antiviral activity of siRNA is due primarily to immune stimulation elicited by the active siRNA duplexes and is not the result of therapeutic RNA interference (RNAi) as previously reported. We show that the misinterpretation stems from the use of a particular control green fluorescent protein (GFP) siRNA that we identify as having unusually low immunostimulatory activity compared with the active anti-influenza siRNA. Curiously, this GFP siRNA has served as a negative control for a surprising number of groups reporting therapeutic effects of siRNA. The inert immunologic profile of the GFP sequence was unique among a broad panel of published siRNAs, all of which could elicit significant interferon induction from primary immune cells. This panel included eight active siRNAs against viral, angiogenic, and oncologic targets, the reported therapeutic efficacy of which was based on comparison with the nonimmunostimulatory GFP siRNA. These results emphasize the need for researchers to anticipate, monitor, and adequately control for siRNA-mediated immune stimulation and calls into question the interpretation of numerous published reports of therapeutic RNAi in vivo. The use of chemically modified siRNA with minimal immunostimulatory capacity will help to delineate more accurately the mechanism of action underlying such studies.

  20. Single nucleotide polymorphism discovery in bovine liver using RNA-seq technology.

    Directory of Open Access Journals (Sweden)

    Chandra Shekhar Pareek

    Full Text Available RNA-seq is a useful next-generation sequencing (NGS technology that has been widely used to understand mammalian transcriptome architecture and function. In this study, a breed-specific RNA-seq experiment was utilized to detect putative single nucleotide polymorphisms (SNPs in liver tissue of young bulls of the Polish Red, Polish Holstein-Friesian (HF and Hereford breeds, and to understand the genomic variation in the three cattle breeds that may reflect differences in production traits.The RNA-seq experiment on bovine liver produced 107,114,4072 raw paired-end reads, with an average of approximately 60 million paired-end reads per library. Breed-wise, a total of 345.06, 290.04 and 436.03 million paired-end reads were obtained from the Polish Red, Polish HF, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA read alignments showed that 81.35%, 82.81% and 84.21% of the mapped sequencing reads were properly paired to the Polish Red, Polish HF, and Hereford breeds, respectively. This study identified 5,641,401 SNPs and insertion and deletion (indel positions expressed in the bovine liver with an average of 313,411 SNPs and indel per young bull. Following the removal of the indel mutations, a total of 195,3804, 152,7120 and 205,3184 raw SNPs expressed in bovine liver were identified for the Polish Red, Polish HF, and Hereford breeds, respectively. Breed-wise, three highly reliable breed-specific SNP-databases (SNP-dbs with 31,562, 24,945 and 28,194 SNP records were constructed for the Polish Red, Polish HF, and Hereford breeds, respectively. Using a combination of stringent parameters of a minimum depth of ≥10 mapping reads that support the polymorphic nucleotide base and 100% SNP ratio, 4,368, 3,780 and 3,800 SNP records were detected in the Polish Red, Polish HF, and Hereford breeds, respectively. The SNP detections using RNA-seq data were successfully validated by kompetitive allele-specific PCR (KASPTM SNP genotyping assay. The

  1. Single nucleotide polymorphism discovery in bovine liver using RNA-seq technology.

    Science.gov (United States)

    Pareek, Chandra Shekhar; Błaszczyk, Paweł; Dziuba, Piotr; Czarnik, Urszula; Fraser, Leyland; Sobiech, Przemysław; Pierzchała, Mariusz; Feng, Yaping; Kadarmideen, Haja N; Kumar, Dibyendu

    2017-01-01

    RNA-seq is a useful next-generation sequencing (NGS) technology that has been widely used to understand mammalian transcriptome architecture and function. In this study, a breed-specific RNA-seq experiment was utilized to detect putative single nucleotide polymorphisms (SNPs) in liver tissue of young bulls of the Polish Red, Polish Holstein-Friesian (HF) and Hereford breeds, and to understand the genomic variation in the three cattle breeds that may reflect differences in production traits. The RNA-seq experiment on bovine liver produced 107,114,4072 raw paired-end reads, with an average of approximately 60 million paired-end reads per library. Breed-wise, a total of 345.06, 290.04 and 436.03 million paired-end reads were obtained from the Polish Red, Polish HF, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed that 81.35%, 82.81% and 84.21% of the mapped sequencing reads were properly paired to the Polish Red, Polish HF, and Hereford breeds, respectively. This study identified 5,641,401 SNPs and insertion and deletion (indel) positions expressed in the bovine liver with an average of 313,411 SNPs and indel per young bull. Following the removal of the indel mutations, a total of 195,3804, 152,7120 and 205,3184 raw SNPs expressed in bovine liver were identified for the Polish Red, Polish HF, and Hereford breeds, respectively. Breed-wise, three highly reliable breed-specific SNP-databases (SNP-dbs) with 31,562, 24,945 and 28,194 SNP records were constructed for the Polish Red, Polish HF, and Hereford breeds, respectively. Using a combination of stringent parameters of a minimum depth of ≥10 mapping reads that support the polymorphic nucleotide base and 100% SNP ratio, 4,368, 3,780 and 3,800 SNP records were detected in the Polish Red, Polish HF, and Hereford breeds, respectively. The SNP detections using RNA-seq data were successfully validated by kompetitive allele-specific PCR (KASPTM) SNP genotyping assay. The comprehensive

  2. Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis.

    Science.gov (United States)

    Nath, B Surendra; Gupta, S K; Bajpai, A K

    2012-12-01

    The life cycle, spore morphology, pathogenicity, tissue specificity, mode of transmission and small subunit rRNA (SSU-rRNA) gene sequence analysis of the five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworm, Bombyx mori have been studied along with type species, NIK-1s_mys. The life cycle of the microsporidians identified exhibited the sequential developmental cycles that are similar to the general developmental cycle of the genus, Nosema. The spores showed considerable variations in their shape, length and width. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIWB-15mb was found to be more virulent than other isolates. All of the microsporidians were found to infect most of the tissues examined and showed gonadal infection and transovarial transmission in the infected silkworms. SSU-rRNA sequence based phylogenetic tree placed NIWB-14b, NIWB-12n and NIWB-11bp in a separate branch along with other Nosema species and Nosema bombycis; while NIWB-15mb and NIWB-13md together formed another cluster along with other Nosema species. NIK-1s_mys revealed a signature sequence similar to standard type species, N. bombycis, indicating that NIK-1s_mys is similar to N. bombycis. Based on phylogenetic relationships, branch length information based on genetic distance and nucleotide differences, we conclude that the microsporidian isolates identified are distinctly different from the other known species and belonging to the genus, Nosema. This SSU-rRNA gene sequence analysis method is found to be more useful approach in detecting different and closely related microsporidians of this economically important domestic insect.

  3. Discovery of the "RNA continent" through a contrarian's research strategy.

    Science.gov (United States)

    Hayashizaki, Yoshihide

    2011-01-01

    The International Human Genome Sequencing Consortium completed the decoding of the human genome sequence in 2003. Readers will be aware of the paradigm shift which has occurred since then in the field of life science research. At last, mankind has been able to focus on a complete picture of the full extent of the genome, on which is recorded the basic information that controls all life. Meanwhile, another genome project, centered on Japan and known as the mouse genome encyclopedia project, was progressing with participation from around the world. Led by our research group at RIKEN, it was a full-length cDNA project which aimed to decode the whole RNA (transcriptome) using the mouse as a model. The basic information that controls all life is recorded on the genome, but in order to obtain a complete picture of this extensive information, the decoding of the genome alone is far from sufficient. These two genome projects established that the number of letters in the genome, which is the blueprint of life, is finite, that the number of RNA molecules derived from it is also finite, and that the number of protein molecules derived from the RNA is probably finite too. A massive number of combinations is still involved, but we are now able to understand one section of the network formed by these data. Once an object of study has been understood to be finite, establishing an image of the whole is certain to lead us to an understanding of the whole. Omics is an approach that views the information controlling life as finite and seeks to assemble and analyze it as a whole. Here, I would like to present our transcriptome research while making reference to our unique research strategy.

  4. Small RNA Transcriptome of Hibiscus Syriacus Provides Insights into the Potential Influence of microRNAs in Flower Development and Terpene Synthesis.

    Science.gov (United States)

    Kim, Taewook; Park, June Hyun; Lee, Sang-Gil; Kim, Soyoung; Kim, Jihyun; Lee, Jungho; Shin, Chanseok

    2017-08-01

    MicroRNAs (miRNAs) are essential small RNA molecules that regulate the expression of target mRNAs in plants and animals. Here, we aimed to identify miRNAs and their putative targets in Hibiscus syriacus , the national flower of South Korea. We employed high-throughput sequencing of small RNAs obtained from four different tissues ( i.e. , leaf, root, flower, and ovary) and identified 33 conserved and 30 novel miRNA families, many of which showed differential tissue-specific expressions. In addition, we computationally predicted novel targets of miRNAs and validated some of them using 5' rapid amplification of cDNA ends analysis. One of the validated novel targets of miR477 was a terpene synthase, the primary gene involved in the formation of disease-resistant terpene metabolites such as sterols and phytoalexins. In addition, a predicted target of conserved miRNAs, miR396, is SHORT VEGETATIVE PHASE , which is involved in flower initiation and is duplicated in H. syriacus . Collectively, this study provides the first reliable draft of the H. syriacus miRNA transcriptome that should constitute a basis for understanding the biological roles of miRNAs in H. syriacus.

  5. Potent host-directed small-molecule inhibitors of myxovirus RNA-dependent RNA-polymerases.

    Directory of Open Access Journals (Sweden)

    Stefanie A Krumm

    Full Text Available Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid

  6. Small molecule probes finely differentiate between various ds- and ss-DNA and RNA by fluorescence, CD and NMR response

    Energy Technology Data Exchange (ETDEWEB)

    Crnolatac, Ivo; Rogan, Iva; Majić, Boris; Tomić, Sanja [Division of Organic Chemistry and Biochemistry, Division of Physical Chemistry, Ruđer Bošković Institute, P.O. Box 180, 10002 Zagreb (Croatia); Deligeorgiev, Todor [Faculty of Chemistry and Pharmacy, University of Sofia (Bulgaria); Horvat, Gordan [Department of Physical Chemistry, Faculty of Science/Chemistry, Horvatovac 102A, HR-10000 Zagreb (Croatia); Makuc, Damjan; Plavec, Janez [Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, Ljubljana (Slovenia); EN-FIST Centre of Excellence, Trg Osvobodilne Fronte 13, Ljubljana (Slovenia); Pescitelli, Gennaro [Department of Chemistry, University of Pisa, Via Moruzzi 13, Pisa (Italy); Piantanida, Ivo, E-mail: pianta@irb.hr [Division of Organic Chemistry and Biochemistry, Division of Physical Chemistry, Ruđer Bošković Institute, P.O. Box 180, 10002 Zagreb (Croatia)

    2016-10-12

    Two small molecules showed intriguing properties of analytical multipurpose probes, whereby one chromophore gives different signal for many different DNA/RNA by application of several highly sensitive spectroscopic methods. Dyes revealed pronounced fluorescence ratiomeric differentiation between ds-AU-RNA, AT-DNA and GC-DNA in approximate order 10:8:1. Particularly interesting, dyes showed specific fluorimetric response for poly rA even at 10-fold excess of any other ss-RNA, and moreover such emission selectivity is preserved in multicomponent ss-RNA mixtures. The dyes also showed specific chiral recognition of poly rU in respect to the other ss-RNA by induced CD (ICD) pattern in visible range (400–500 nm), which was attributed to the dye-side-chain contribution to binding (confirmed by absence of any ICD band for reference compound lacking side-chain). Most intriguingly, minor difference in the side-chain attached to dye chromophore resulted in opposite sign of dye-ICD pattern, whereby differences in NMR NOESY contacts and proton chemical shifts between two dye/oligo rU complexes combined with MD simulations and CD calculations attributed observed bisignate ICD to the dimeric dye aggregate within oligo rU. - Highlights: • Novel dyes emit fluorescence only for poly rA even at high excess of all other ss-RNA. • Fluorescence response for AT-DNA is 8 times stronger than for GC-DNA. • Florescence induced by ds-RNA is 20% stronger that emission induced by ds-DNA. • Intrinsically non-chiral, dyes show strong and characteristic ICD response for poly rU.

  7. Discovery of the Higgs boson and beyond

    International Nuclear Information System (INIS)

    Godbole, Rohini

    2014-01-01

    This talk is about the Higgs mechanism, the theoretical discovery of which, was awarded the 2013 Nobel Prize. It also discusses the discovery of the Higgs boson at the large hadron collider which provided the experimental proof that made the Nobel prize possible. It covers the implications of these for the quest of unravelling the fundamental laws of nature which seem to govern both, the behavior of the ultra small (subatomic particles) and the ultra large (the cosmos)

  8. Molecular phylogenetic studies on an unnamed bovine Babesia sp. based on small subunit ribosomal RNA gene sequences.

    Science.gov (United States)

    Luo, Jianxun; Yin, Hong; Liu, Zhijie; Yang, Dongying; Guan, Guiquan; Liu, Aihong; Ma, Miling; Dang, Shengzhi; Lu, Bingyi; Sun, Caiqin; Bai, Qi; Lu, Wenshun; Chen, Puyan

    2005-10-10

    The 18S small subunit ribosomal RNA (18S rRNA) gene of an unnamed Babesia species (designated B. U sp.) was sequenced and analyzed in an attempt to distinguish it from other Babesia species in China. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was ligated to the pGEM-T Easy vector for sequencing. It was found that the length of the 18S rRNA gene of all B. U sp. Kashi 1 and B. U sp. Kashi 2 was 1699 bp and 1689 bp. Two phylogenetic trees were, respectively, inferred based on 18S rRNA sequence of the Chinese bovine Babesia isolates and all of Babesia species available in GenBank. The first tree showed that B. U sp. was situated in the branch between B. major Yili and B. bovis Shannxian, and the second tree revealed that B. U sp. was confined to the same group as B. caballi. The percent identity of B. U sp. with other Chinese Babesia species was between 74.2 and 91.8, while the percent identity between two B. U sp. isolates was 99.7. These results demonstrated that this B. U sp. is different from other Babesia species, but that two B. U sp. isolates obtained with nymphal and adultal Hyalomma anatolicum anatolicum tick belong to the same species.

  9. Small regulatory RNAs of the RNA interference (RNAi) pathway as a prophylactic treatment against fish pathogenic viruses

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Hajiabadi, Seyed Amir Hossein Jalali; Kristensen, Lasse Bøgelund Juel

    2011-01-01

    Small RNAs acting in the recently discovered gene regulatory mechanism called RNA interference has a potential as diagnostic signatures of disease and immunological state and when produced synthetically as prophylactic treatment of such diseases. In the RNAi mechanism the cell produces different....... The mechanism can be programmed with several types of small double stranded RNAs - the type of which defines the destiny of the target. One such class of regulatory RNAs called microRNAs are upregulated due to various physiological responses of the cell and they suppress many genes simultaneously believed...... small RNAs which inhibit gene expression through more or less specific interaction with messenger RNAs resulting in repression of translation to protein. In this way cells can turn of genes of specific pathways thereby leading to altered physiological stages of tissues and possibly of whole organisms...

  10. Small RNA-Sequencing Links Physiological Changes and RdDM Process to Vegetative-to-Floral Transition in Apple

    Directory of Open Access Journals (Sweden)

    Xinwei Guo

    2017-05-01

    Full Text Available Transition from vegetative to floral buds is a critical physiological change during flower induction that determines fruit productivity. Small non-coding RNAs (sRNAs including microRNAs (miRNAs and small interfering RNAs (siRNAs are pivotal regulators of plant growth and development. Although the key role of sRNAs in flowering regulation has been well-described in Arabidopsis and some other annual plants, their relevance to vegetative-to-floral transition (hereafter, referred to floral transition in perennial woody trees remains under defined. Here, we performed Illumina sequencing of sRNA libraries prepared from vegetative and floral bud during flower induction of the apple trees. A large number of sRNAs exemplified by 33 previously annotated miRNAs and six novel members display significant differential expression (DE patterns. Notably, most of these DE-miRNAs in floral transition displayed opposite expression changes in reported phase transition in apple trees. Bioinformatics analysis suggests most of the DE-miRNAs targeted transcripts involved in SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL gene regulation, stress responses, and auxin and gibberellin (GA pathways, with further suggestion that there is an inherent link between physiological stress response and metabolism reprogramming during floral transition. We also observed significant changes in 24 nucleotide (nt sRNAs that are hallmarks for RNA-dependent DNA methylation (RdDM pathway, suggestive of the correlation between epigenetic modifications and the floral transition. The study not only provides new insight into our understanding of fundamental mechanism of poorly studied floral transition in apple and other woody plants, but also presents important sRNA resource for future in-depth research in the apple flowering physiology.

  11. The discovery of Halictivirus resolves the Sinaivirus phylogeny.

    Science.gov (United States)

    Bigot, Diane; Dalmon, Anne; Roy, Bronwen; Hou, Chunsheng; Germain, Michèle; Romary, Manon; Deng, Shuai; Diao, Qingyun; Weinert, Lucy A; Cook, James M; Herniou, Elisabeth A; Gayral, Philippe

    2017-11-01

    By providing pollination services, bees are among the most important insects, both in ecological and economical terms. Combined next-generation and classical sequencing approaches were applied to discover and study new insect viruses potentially harmful to bees. A bioinformatics virus discovery pipeline was used on individual Illumina transcriptomes of 13 wild bees from three species from the genus Halictus and 30 ants from six species of the genera Messor and Aphaenogaster. This allowed the discovery and description of three sequences of a new virus termed Halictus scabiosae Adlikon virus (HsAV). Phylogenetic analyses of ORF1, RNA-dependent RNA-polymerase (RdRp) and capsid genes showed that HsAV is closely related to (+)ssRNA viruses of the unassigned Sinaivirus genus but distant enough to belong to a different new genus we called Halictivirus. In addition, our study of ant transcriptomes revealed the first four sinaivirus sequences from ants (Messor barbarus, M. capitatus and M. concolor). Maximum likelihood phylogenetic analyses were performed on a 594 nt fragment of the ORF1/RdRp region from 84 sinaivirus sequences, including 31 new Lake Sinai viruses (LSVs) from honey bees collected in five countries across the globe and the four ant viral sequences. The phylogeny revealed four main clades potentially representing different viral species infecting honey bees. Moreover, the ant viruses belonged to the LSV4 clade, suggesting a possible cross-species transmission between bees and ants. Lastly, wide honey bee screening showed that all four LSV clades have worldwide distributions with no obvious geographical segregation.

  12. miRBase: integrating microRNA annotation and deep-sequencing data.

    Science.gov (United States)

    Kozomara, Ana; Griffiths-Jones, Sam

    2011-01-01

    miRBase is the primary online repository for all microRNA sequences and annotation. The current release (miRBase 16) contains over 15,000 microRNA gene loci in over 140 species, and over 17,000 distinct mature microRNA sequences. Deep-sequencing technologies have delivered a sharp rise in the rate of novel microRNA discovery. We have mapped reads from short RNA deep-sequencing experiments to microRNAs in miRBase and developed web interfaces to view these mappings. The user can view all read data associated with a given microRNA annotation, filter reads by experiment and count, and search for microRNAs by tissue- and stage-specific expression. These data can be used as a proxy for relative expression levels of microRNA sequences, provide detailed evidence for microRNA annotations and alternative isoforms of mature microRNAs, and allow us to revisit previous annotations. miRBase is available online at: http://www.mirbase.org/.

  13. Small RNA and transcriptome deep sequencing proffers insight into floral gene regulation in Rosa cultivars

    Directory of Open Access Journals (Sweden)

    Kim Jungeun

    2012-11-01

    Full Text Available Abstract Background Roses (Rosa sp., which belong to the family Rosaceae, are the most economically important ornamental plants—making up 30% of the floriculture market. However, given high demand for roses, rose breeding programs are limited in molecular resources which can greatly enhance and speed breeding efforts. A better understanding of important genes that contribute to important floral development and desired phenotypes will lead to improved rose cultivars. For this study, we analyzed rose miRNAs and the rose flower transcriptome in order to generate a database to expound upon current knowledge regarding regulation of important floral characteristics. A rose genetic database will enable comprehensive analysis of gene expression and regulation via miRNA among different Rosa cultivars. Results We produced more than 0.5 million reads from expressed sequences, totalling more than 110 million bp. From these, we generated 35,657, 31,434, 34,725, and 39,722 flower unigenes from Rosa hybrid: ‘Vital’, ‘Maroussia’, and ‘Sympathy’ and Rosa rugosa Thunb. , respectively. The unigenes were assigned functional annotations, domains, metabolic pathways, Gene Ontology (GO terms, Plant Ontology (PO terms, and MIPS Functional Catalogue (FunCat terms. Rose flower transcripts were compared with genes from whole genome sequences of Rosaceae members (apple, strawberry, and peach and grape. We also produced approximately 40 million small RNA reads from flower tissue for Rosa, representing 267 unique miRNA tags. Among identified miRNAs, 25 of them were novel and 242 of them were conserved miRNAs. Statistical analyses of miRNA profiles revealed both shared and species-specific miRNAs, which presumably effect flower development and phenotypes. Conclusions In this study, we constructed a Rose miRNA and transcriptome database, and we analyzed the miRNAs and transcriptome generated from the flower tissues of four Rosa cultivars. The database provides a

  14. Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

    Science.gov (United States)

    Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

    2014-11-01

    MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  15. Small interfering RNA targeted to stem-loop II of the 5' untranslated region effectively inhibits expression of six HCV genotypes

    Directory of Open Access Journals (Sweden)

    Dash Srikanta

    2006-11-01

    Full Text Available Abstract Background The antiviral action of interferon alpha targets the 5' untranslated region (UTR used by hepatitis C virus (HCV to translate protein by an internal ribosome entry site (IRES mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance. Results Four different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains. Conclusion These in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon.

  16. Exploring complex miRNA-mRNA interactions with Bayesian networks by splitting-averaging strategy

    Directory of Open Access Journals (Sweden)

    Liu Lin

    2009-12-01

    Full Text Available Abstract Background microRNAs (miRNAs regulate target gene expression by controlling their mRNAs post-transcriptionally. Increasing evidence demonstrates that miRNAs play important roles in various biological processes. However, the functions and precise regulatory mechanisms of most miRNAs remain elusive. Current research suggests that miRNA regulatory modules are complicated, including up-, down-, and mix-regulation for different physiological conditions. Previous computational approaches for discovering miRNA-mRNA interactions focus only on down-regulatory modules. In this work, we present a method to capture complex miRNA-mRNA interactions including all regulatory types between miRNAs and mRNAs. Results We present a method to capture complex miRNA-mRNA interactions using Bayesian network structure learning with splitting-averaging strategy. It is designed to explore all possible miRNA-mRNA interactions by integrating miRNA-targeting information, expression profiles of miRNAs and mRNAs, and sample categories. We also present an analysis of data sets for epithelial and mesenchymal transition (EMT. Our results show that the proposed method identified all possible types of miRNA-mRNA interactions from the data. Many interactions are of tremendous biological significance. Some discoveries have been validated by previous research, for example, the miR-200 family negatively regulates ZEB1 and ZEB2 for EMT. Some are consistent with the literature, such as LOX has wide interactions with the miR-200 family members for EMT. Furthermore, many novel interactions are statistically significant and worthy of validation in the near future. Conclusions This paper presents a new method to explore the complex miRNA-mRNA interactions for different physiological conditions using Bayesian network structure learning with splitting-averaging strategy. The method makes use of heterogeneous data including miRNA-targeting information, expression profiles of miRNAs and

  17. MicroRNA and tasiRNA diversity in mature pollen of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Hafidh Said

    2009-12-01

    Full Text Available Abstract Background New generation sequencing technology has allowed investigation of the small RNA populations of flowering plants at great depth. However, little is known about small RNAs in their reproductive cells, especially in post-meiotic cells of the gametophyte generation. Pollen - the male gametophyte - is the specialised haploid structure that generates and delivers the sperm cells to the female gametes at fertilisation. Whether development and differentiation of the male gametophyte depends on the action of microRNAs and trans-acting siRNAs guiding changes in gene expression is largely unknown. Here we have used 454 sequencing to survey the various small RNA populations present in mature pollen of Arabidopsis thaliana. Results In this study we detected the presence of 33 different microRNA families in mature pollen and validated the expression levels of 17 selected miRNAs by Q-RT-PCR. The majority of the selected miRNAs showed pollen-enriched expression compared with leaves. Furthermore, we report for the first time the presence of trans-acting siRNAs in pollen. In addition to describing new patterns of expression for known small RNAs in each of these classes, we identified 7 putative novel microRNAs. One of these, ath-MIR2939, targets a pollen-specific F-box transcript and we demonstrate cleavage of its target mRNA in mature pollen. Conclusions Despite the apparent simplicity of the male gametophyte, comprising just two different cell types, pollen not only utilises many miRNAs and trans-acting siRNAs expressed in the somatic tissues but also expresses novel miRNAs.

  18. Dendrimers for siRNA Delivery

    Directory of Open Access Journals (Sweden)

    Swati Biswas

    2013-02-01

    Full Text Available Since the discovery of the “starburst polymer”, later renamed as dendrimer, this class of polymers has gained considerable attention for numerous biomedical applications, due mainly to the unique characteristics of this macromolecule, including its monodispersity, uniformity, and the presence of numerous functionalizable terminal groups. In recent years, dendrimers have been studied extensively for their potential application as carriers for nucleic acid therapeutics, which utilize the cationic charge of the dendrimers for effective dendrimer-nucleic acid condensation. siRNA is considered a promising, versatile tool among various RNAi-based therapeutics, which can effectively regulate gene expression if delivered successfully inside the cells. This review reports on the advancements in the development of dendrimers as siRNA carriers.

  19. Drug Repositioning Discovery for Early- and Late-Stage Non-Small-Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Chien-Hung Huang

    2014-01-01

    Full Text Available Drug repositioning is a popular approach in the pharmaceutical industry for identifying potential new uses for existing drugs and accelerating the development time. Non-small-cell lung cancer (NSCLC is one of the leading causes of death worldwide. To reduce the biological heterogeneity effects among different individuals, both normal and cancer tissues were taken from the same patient, hence allowing pairwise testing. By comparing early- and late-stage cancer patients, we can identify stage-specific NSCLC genes. Differentially expressed genes are clustered separately to form up- and downregulated communities that are used as queries to perform enrichment analysis. The results suggest that pathways for early- and late-stage cancers are different. Sets of up- and downregulated genes were submitted to the cMap web resource to identify potential drugs. To achieve high confidence drug prediction, multiple microarray experimental results were merged by performing meta-analysis. The results of a few drug findings are supported by MTT assay or clonogenic assay data. In conclusion, we have been able to assess the potential existing drugs to identify novel anticancer drugs, which may be helpful in drug repositioning discovery for NSCLC.

  20. Protection and systemic translocation of siRNA following oral administration of chitosan/siRNA nanoparticles

    DEFF Research Database (Denmark)

    Gonzalez, Borja Ballarin; Dagnæs-Hansen, Frederik; Fenton, Robert A.

    2013-01-01

    , gastrointestinal (GI) deposition, and translocation into peripheral tissue of nonmodified siRNA after oral gavage of chitosan/siRNA nanoparticles in mice. In contrast to naked siRNA, retained structural integrity and deposition in the stomach, proximal and distal small intestine, and colon was observed at 1 and 5...... hours for siRNA within nanoparticles. Furthermore, histological detection of fluorescent siRNA at the apical regions of the intestinal epithelium suggests mucoadhesion provided by chitosan. Detection of intact siRNA in the liver, spleen, and kidney was observed 1 hour after oral gavage, with an organ...

  1. Dicer uses distinct modules for recognizing dsRNA termini.

    Science.gov (United States)

    Sinha, Niladri K; Iwasa, Janet; Shen, Peter S; Bass, Brenda L

    2018-01-19

    Invertebrates rely on Dicer to cleave viral double-stranded RNA (dsRNA), and Drosophila Dicer-2 distinguishes dsRNA substrates by their termini. Blunt termini promote processive cleavage, while 3' overhanging termini are cleaved distributively. To understand this discrimination, we used cryo-electron microscopy to solve structures of Drosophila Dicer-2 alone and in complex with blunt dsRNA. Whereas the Platform-PAZ domains have been considered the only Dicer domains that bind dsRNA termini, unexpectedly, we found that the helicase domain is required for binding blunt, but not 3' overhanging, termini. We further showed that blunt dsRNA is locally unwound and threaded through the helicase domain in an adenosine triphosphate-dependent manner. Our studies reveal a previously unrecognized mechanism for optimizing antiviral defense and set the stage for the discovery of helicase-dependent functions in other Dicers. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  2. Applied metabolomics in drug discovery.

    Science.gov (United States)

    Cuperlovic-Culf, M; Culf, A S

    2016-08-01

    The metabolic profile is a direct signature of phenotype and biochemical activity following any perturbation. Metabolites are small molecules present in a biological system including natural products as well as drugs and their metabolism by-products depending on the biological system studied. Metabolomics can provide activity information about possible novel drugs and drug scaffolds, indicate interesting targets for drug development and suggest binding partners of compounds. Furthermore, metabolomics can be used for the discovery of novel natural products and in drug development. Metabolomics can enhance the discovery and testing of new drugs and provide insight into the on- and off-target effects of drugs. This review focuses primarily on the application of metabolomics in the discovery of active drugs from natural products and the analysis of chemical libraries and the computational analysis of metabolic networks. Metabolomics methodology, both experimental and analytical is fast developing. At the same time, databases of compounds are ever growing with the inclusion of more molecular and spectral information. An increasing number of systems are being represented by very detailed metabolic network models. Combining these experimental and computational tools with high throughput drug testing and drug discovery techniques can provide new promising compounds and leads.

  3. Know Your Enemy: Successful Bioinformatic Approaches to Predict Functional RNA Structures in Viral RNAs

    Science.gov (United States)

    Lim, Chun Shen; Brown, Chris M.

    2018-01-01

    Structured RNA elements may control virus replication, transcription and translation, and their distinct features are being exploited by novel antiviral strategies. Viral RNA elements continue to be discovered using combinations of experimental and computational analyses. However, the wealth of sequence data, notably from deep viral RNA sequencing, viromes, and metagenomes, necessitates computational approaches being used as an essential discovery tool. In this review, we describe practical approaches being used to discover functional RNA elements in viral genomes. In addition to success stories in new and emerging viruses, these approaches have revealed some surprising new features of well-studied viruses e.g., human immunodeficiency virus, hepatitis C virus, influenza, and dengue viruses. Some notable discoveries were facilitated by new comparative analyses of diverse viral genome alignments. Importantly, comparative approaches for finding RNA elements embedded in coding and non-coding regions differ. With the exponential growth of computer power we have progressed from stem-loop prediction on single sequences to cutting edge 3D prediction, and from command line to user friendly web interfaces. Despite these advances, many powerful, user friendly prediction tools and resources are underutilized by the virology community. PMID:29354101

  4. MicroRNA Array Normalization: An Evaluation Using a Randomized Dataset as the Benchmark

    Science.gov (United States)

    Qin, Li-Xuan; Zhou, Qin

    2014-01-01

    MicroRNA arrays possess a number of unique data features that challenge the assumption key to many normalization methods. We assessed the performance of existing normalization methods using two microRNA array datasets derived from the same set of tumor samples: one dataset was generated using a blocked randomization design when assigning arrays to samples and hence was free of confounding array effects; the second dataset was generated without blocking or randomization and exhibited array effects. The randomized dataset was assessed for differential expression between two tumor groups and treated as the benchmark. The non-randomized dataset was assessed for differential expression after normalization and compared against the benchmark. Normalization improved the true positive rate significantly in the non-randomized data but still possessed a false discovery rate as high as 50%. Adding a batch adjustment step before normalization further reduced the number of false positive markers while maintaining a similar number of true positive markers, which resulted in a false discovery rate of 32% to 48%, depending on the specific normalization method. We concluded the paper with some insights on possible causes of false discoveries to shed light on how to improve normalization for microRNA arrays. PMID:24905456

  5. [Satellite RNA (RNA3) of tomato black ring virus is found with one of the 2 major RNAs (RNA2) in a new capsid nucleoprotein].

    Science.gov (United States)

    Doz, B; Dunez, J; Bove, J M

    1977-12-19

    Tomato Black Ring Virus (TBRV) like other NEPOviruses posseses two nucleoproteins M and B and two major RNAs, RNA1 and RNA2 respectively distributed in B and M. A new nucleoprotein has just been discovered and comprises one molecule of RNA2 associated with one molecule of RNA3. RNA3 is a small RNA of molecular weight 500,000 d considered to be a satellite RNA. Its level appears to depend on the infection stage, local or systemic. RNA3 is able to modify the relative proportions of nucleoproteins M and B and their respective RNAs. The satellite RNA, might be part of the genome and represent a monocistronic mRNA for protein capsid synthesis. However it seems perhaps more tempting to correlate TBRV-RNA3 with satellite RNA5 of certain strains of Cucumber mosaic virus.

  6. Re-editing the paradigm of Cytidine (C) to Uridine (U) RNA editing.

    Science.gov (United States)

    Fossat, Nicolas; Tam, Patrick P L

    2014-01-01

    Cytidine (C) to Uridine (U) RNA editing is a post-trancriptional modification that until recently was known to only affect Apolipoprotein b (Apob) RNA and minimally require 2 components of the C to U editosome, the deaminase APOBEC1 and the RNA-binding protein A1CF. Our latest work has identified a novel RNA-binding protein, RBM47, as a core component of the editosome, which can substitute A1CF for the editing of ApoB mRNA. In addition, new RNA species that are subjected to C to U editing have been identified. Here, we highlight these recent discoveries and discuss how they change our view of the composition of the C to U editing machinery and expand our knowledge of the functional attributes of C to U RNA editing.

  7. Noise processing by microRNA-mediated circuits: The Incoherent Feed-Forward Loop, revisited

    Directory of Open Access Journals (Sweden)

    Silvia Grigolon

    2016-04-01

    Full Text Available The intrinsic stochasticity of gene expression is usually mitigated in higher eukaryotes by post-transcriptional regulation channels that stabilise the output layer, most notably protein levels. The discovery of small non-coding RNAs (miRNAs in specific motifs of the genetic regulatory network has led to identifying noise buffering as the possible key function they exert in regulation. Recent in vitro and in silico studies have corroborated this hypothesis. It is however also known that miRNA-mediated noise reduction is hampered by transcriptional bursting in simple topologies. Here, using stochastic simulations validated by analytical calculations based on van Kampen's expansion, we revisit the noise-buffering capacity of the miRNA-mediated Incoherent Feed Forward Loop (IFFL, a small module that is widespread in the gene regulatory networks of higher eukaryotes, in order to account for the effects of intermittency in the transcriptional activity of the modulator gene. We show that bursting considerably alters the circuit's ability to control static protein noise. By comparing with other regulatory architectures, we find that direct transcriptional regulation significantly outperforms the IFFL in a broad range of kinetic parameters. This suggests that, under pulsatile inputs, static noise reduction may be less important than dynamical aspects of noise and information processing in characterising the performance of regulatory elements.

  8. Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Wang Beibei; Lu Rui; Wang Weicheng; Jin Ying

    2006-01-01

    The tetracycline (Tc)-inducible small interference RNA (siRNA) is a powerful tool for studying gene function in mammalian cells. However, the system is infrequently utilized in embryonic stem (ES) cells. Here, we present First application of the Tc-inducible, stably integrated plasmid-based siRNA system in mouse ES cells to down-regulate expression of Npm1, an essential gene for embryonic development. The physiological role of Npm1 in ES cells has not been defined. Our data show that the knock-down of Npm1 expression by this siRNA system was not only highly efficient, but also Tc- dose- and induction time-dependent. Particularly, the down-regulation of Npm1 expression was reversible. Importantly, suppression of Npm1 expression in ES cells resulted in reduced cell proliferation. Taken together, this system allows for studying gene function in a highly controlled manner, otherwise difficult to achieve in ES cells. Moreover, our results demonstrate that Npm1 is essential for ES cell proliferation

  9. Establishment of a continuous culture system for Entamoeba muris and analysis of the small subunit rRNA gene

    Directory of Open Access Journals (Sweden)

    Kobayashi S.

    2009-06-01

    Full Text Available We established a culture system for Entamoeba muris (MG-EM-01 strain isolated from a Mongolian gerbil using a modified Balamuth’s egg yolk infusion medium supplemented with 4% adult bovine serum and Bacteroides fragilis cocultured with Escherichia coli. Further, encystation was observed in the culture medium. The morphological characteristics of E. muris are similar to those of Entamoeba coli (E. coli; moreover, the malic isoenzyme electrophoretic band, which shows species-specific electrophoretic mobility, of E. muris had almost the same mobility as that observed with the malic isoenzyme electrophorectic band of E. coli (UZG-EC-01 strain isolated from a gorilla. We determined the small subunit rRNA (SSU-rRNA gene sequence of the MG-EM-01 strain, and this sequence was observed to show 82.7% homology with that of the UZG-EC-01 strain. Further, the resultant phylogenetic tree for molecular taxonomy based on the SSU-rRNA genes of the 21 strains of the intestinal parasitic amoeba species indicated that the MG-EM-01 strain was most closely related to E. coli.

  10. Peripheral blood microRNA and VEGFA mRNA changes following electroconvulsive therapy: implications for psychotic depression.

    Science.gov (United States)

    Kolshus, E; Ryan, K M; Blackshields, G; Smyth, P; Sheils, O; McLoughlin, D M

    2017-12-01

    MicroRNAs are short, non-coding molecules that regulate gene expression. Here, we investigate the role of microRNAs in depression and electroconvulsive therapy (ECT). We performed three studies: a deep sequencing discovery-phase study of miRNA changes in whole blood following ECT (n = 16), followed by a validation study in a separate cohort of patients pre-/post-ECT (n = 37) and matched healthy controls (n = 34). Changes in an experimentally validated gene target (VEGFA) were then analysed in patients pre-/post-ECT (n = 97) and in matched healthy controls (n = 53). In the discovery-phase study, we found no statistically significant differences in miRNA expression from baseline to end of treatment in the group as a whole, but post hoc analysis indicated a difference in patients with psychotic depression (n = 3). In a follow-up validation study, patients with psychotic depression (n = 7) had elevated baseline levels of miR-126-3p (t = 3.015, P = 0.006) and miR-106a-5p (t = 2.598, P = 0.025) compared to healthy controls. Following ECT, these differences disappeared. Baseline VEGFA levels were significantly higher in depressed patients compared to healthy controls (F(1,144) = 27.688, P = <0.001). Following ECT, there was a significant change in VEGFA levels in the psychotic group only (t = 2.915, P = 0.010). Molecular differences (miRNA and VEGFA) may exist between psychotic and non-psychotic depression treated with ECT. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. MAJOR MERGERS WITH SMALL GALAXIES: THE DISCOVERY OF A MAGELLANIC-TYPE GALAXY AT z = 0.12

    International Nuclear Information System (INIS)

    Koch, Andreas; Frank, Matthias J.; Pasquali, Anna; Rich, R. Michael; Rabitz, Andreas

    2015-01-01

    We report on the serendipitous discovery of a star-forming galaxy at redshift z = 0.116 with morphological features that indicate an ongoing merger. This object exhibits two clearly separated components with significantly different colors, plus a possible tidal stream. Follow-up spectroscopy of the bluer component revealed a low star-forming activity of 0.09 M ⊙ yr −1 and a high metallicity of 12 + log(O/H) = 8.6. Based on comparison with mass–star formation-rate and mass–metallicity relations, and on fitting of spectral energy distributions, we obtain a stellar mass of 3 × 10 9 M ⊙ , which renders this object comparable to the Large Magellanic Cloud. Thus our finding provides a further piece of evidence of a major merger already acting on small, dwarf-galaxy-like scales

  12. MAJOR MERGERS WITH SMALL GALAXIES: THE DISCOVERY OF A MAGELLANIC-TYPE GALAXY AT z = 0.12

    Energy Technology Data Exchange (ETDEWEB)

    Koch, Andreas; Frank, Matthias J. [Zentrum für Astronomie der Universität Heidelberg, Landessternwarte, Königstuhl 12, D-69117 Heidelberg (Germany); Pasquali, Anna [Zentrum für Astronomie der Universität Heidelberg, Astronomisches Rechen-Institut, Mönchhofstrasse 12, D-69117 Heidelberg (Germany); Rich, R. Michael [University of California Los Angeles, Department of Physics and Astronomy, Los Angeles, CA (United States); Rabitz, Andreas, E-mail: akoch@lsw.uni-heidelberg.de [Leibniz-Institut für Astrophysik Potsdam, An der Sternwarte 16, D-14482 Potsdam (Germany)

    2015-12-20

    We report on the serendipitous discovery of a star-forming galaxy at redshift z = 0.116 with morphological features that indicate an ongoing merger. This object exhibits two clearly separated components with significantly different colors, plus a possible tidal stream. Follow-up spectroscopy of the bluer component revealed a low star-forming activity of 0.09 M{sub ⊙} yr{sup −1} and a high metallicity of 12 + log(O/H) = 8.6. Based on comparison with mass–star formation-rate and mass–metallicity relations, and on fitting of spectral energy distributions, we obtain a stellar mass of 3 × 10{sup 9} M{sub ⊙}, which renders this object comparable to the Large Magellanic Cloud. Thus our finding provides a further piece of evidence of a major merger already acting on small, dwarf-galaxy-like scales.

  13. RNA-Targeted Therapies and Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Stéphane Mathis

    2018-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal motor disease in adults. Its pathophysiology remains mysterious, but tremendous advances have been made with the discovery of the most frequent mutations of its more common familial form linked to the C9ORF72 gene. Although most cases are still considered sporadic, these genetic mutations have revealed the role of RNA production, processing and transport in ALS, and may be important players in all ALS forms. There are no disease-modifying treatments for adult human neurodegenerative diseases, including ALS. As in spinal muscular atrophy, RNA-targeted therapies have been proposed as potential strategies for treating this neurodegenerative disorder. Successes achieved in various animal models of ALS have proven that RNA therapies are both safe and effective. With careful consideration of the applicability of such therapies in humans, it is possible to anticipate ongoing in vivo research and clinical trial development of RNA therapies for treating ALS.

  14. RNA-Targeted Therapies and Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Mathis, Stéphane; Le Masson, Gwendal

    2018-01-15

    Amyotrophic lateral sclerosis (ALS) is a fatal motor disease in adults. Its pathophysiology remains mysterious, but tremendous advances have been made with the discovery of the most frequent mutations of its more common familial form linked to the C9ORF72 gene. Although most cases are still considered sporadic, these genetic mutations have revealed the role of RNA production, processing and transport in ALS, and may be important players in all ALS forms. There are no disease-modifying treatments for adult human neurodegenerative diseases, including ALS. As in spinal muscular atrophy, RNA-targeted therapies have been proposed as potential strategies for treating this neurodegenerative disorder. Successes achieved in various animal models of ALS have proven that RNA therapies are both safe and effective. With careful consideration of the applicability of such therapies in humans, it is possible to anticipate ongoing in vivo research and clinical trial development of RNA therapies for treating ALS.

  15. Design of small-molecule epigenetic modulators

    Science.gov (United States)

    Pachaiyappan, Boobalan

    2013-01-01

    The field of epigenetics has expanded rapidly to reveal multiple new targets for drug discovery. The functional elements of the epigenomic machinery can be catagorized as writers, erasers and readers, and together these elements control cellular gene expression and homeostasis. It is increasingly clear that aberrations in the epigenome can underly a variety of diseases, and thus discovery of small molecules that modulate the epigenome in a specific manner is a viable approach to the discovery of new therapeutic agents. In this Digest, the components of epigenetic control of gene expression will be briefly summarized, and efforts to identify small molecules that modulate epigenetic processes will be described. PMID:24300735

  16. MysiRNA-designer: a workflow for efficient siRNA design.

    Directory of Open Access Journals (Sweden)

    Mohamed Mysara

    Full Text Available The design of small interfering RNA (siRNA is a multi factorial problem that has gained the attention of many researchers in the area of therapeutic and functional genomics. MysiRNA score was previously introduced that improves the correlation of siRNA activity prediction considering state of the art algorithms. In this paper, a new program, MysiRNA-Designer, is described which integrates several factors in an automated work-flow considering mRNA transcripts variations, siRNA and mRNA target accessibility, and both near-perfect and partial off-target matches. It also features the MysiRNA score, a highly ranked correlated siRNA efficacy prediction score for ranking the designed siRNAs, in addition to top scoring models Biopredsi, DISR, Thermocomposition21 and i-Score, and integrates them in a unique siRNA score-filtration technique. This multi-score filtration layer filters siRNA that passes the 90% thresholds calculated from experimental dataset features. MysiRNA-Designer takes an accession, finds conserved regions among its transcript space, finds accessible regions within the mRNA, designs all possible siRNAs for these regions, filters them based on multi-scores thresholds, and then performs SNP and off-target filtration. These strict selection criteria were tested against human genes in which at least one active siRNA was designed from 95.7% of total genes. In addition, when tested against an experimental dataset, MysiRNA-Designer was found capable of rejecting 98% of the false positive siRNAs, showing superiority over three state of the art siRNA design programs. MysiRNA is a freely accessible (Microsoft Windows based desktop application that can be used to design siRNA with a high accuracy and specificity. We believe that MysiRNA-Designer has the potential to play an important role in this area.

  17. RNA interference-mediated intrinsic antiviral immunity in invertebrates.

    Science.gov (United States)

    Nayak, Arabinda; Tassetto, Michel; Kunitomi, Mark; Andino, Raul

    2013-01-01

    In invertebrates such as insects and nematodes, RNA interference (RNAi) provides RNA-based protection against viruses. This form of immunity restricts viral replication and dissemination from infected cells and viruses, in turn, have evolved evasion mechanisms or RNAi suppressors to counteract host defenses. Recent advances indicate that, in addition to RNAi, other related small RNA pathways contribute to antiviral functions in invertebrates. This has led to a deeper understanding of fundamental aspects of small RNA-based antiviral immunity in invertebrates and its contribution to viral spread and pathogenesis.

  18. The Piwi pathway: from piRNA methylation to histone methylation

    NARCIS (Netherlands)

    Luteijn, M.J.

    2013-01-01

    Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defense and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. These small RNA molecules play

  19. ISEV position paper: extracellular vesicle RNA analysis and bioinformatics

    Directory of Open Access Journals (Sweden)

    Andrew F. Hill

    2013-12-01

    Full Text Available Extracellular vesicles (EVs are the collective term for the various vesicles that are released by cells into the extracellular space. Such vesicles include exosomes and microvesicles, which vary by their size and/or protein and genetic cargo. With the discovery that EVs contain genetic material in the form of RNA (evRNA has come the increased interest in these vesicles for their potential use as sources of disease biomarkers and potential therapeutic agents. Rapid developments in the availability of deep sequencing technologies have enabled the study of EV-related RNA in detail. In October 2012, the International Society for Extracellular Vesicles (ISEV held a workshop on “evRNA analysis and bioinformatics.” Here, we report the conclusions of one of the roundtable discussions where we discussed evRNA analysis technologies and provide some guidelines to researchers in the field to consider when performing such analysis.

  20. Divergent homologs of the predicted small RNA BpCand697 in Burkholderia spp.

    Science.gov (United States)

    Damiri, Nadzirah; Mohd-Padil, Hirzahida; Firdaus-Raih, Mohd

    2015-09-01

    The small RNA (sRNA) gene candidate, BpCand697 was previously reported to be unique to Burkholderia spp. and is encoded at 3' non-coding region of a putative AraC family transcription regulator gene. This study demonstrates the conservation of BpCand697 sequence across 32 Burkholderia spp. including B. pseudomallei, B. mallei, B. thailandensis and Burkholderia sp. by integrating both sequence homology and secondary structural analyses of BpCand697 within the dataset. The divergent sequence of BpCand697 was also used as a discriminatory power in clustering the dataset according to the potential virulence of Burkholderia spp., showing that B. thailandensis was clearly secluded from the virulent cluster of B. pseudomallei and B. mallei. Finally, the differential co-transcript expression of BpCand697 and its flanking gene, bpsl2391 was detected in Burkholderia pseudomallei D286 after grown under two different culture conditions using nutrient-rich and minimal media. It is hypothesized that the differential expression of BpCand697-bpsl2391 co-transcript between the two standard prepared media might correlate with nutrient availability in the culture media, suggesting that the physical co-localization of BpCand697 in B. pseudomallei D286 might be directly or indirectly involved with the transcript regulation of bpsl2391 under the selected in vitro culture conditions.

  1. MicroRNA-429 induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes

    Energy Technology Data Exchange (ETDEWEB)

    Lang, Yaoguo; Xu, Shidong; Ma, Jianqun; Wu, Jun [Department of Thoracic Surgery, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China); Jin, Shi; Cao, Shoubo [Department of Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China); Yu, Yan, E-mail: yuyan@hrbmu.edu.cn [Department of Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China)

    2014-07-18

    Highlights: • MiR-429 expression is upregulated in non-small cell lung cancer (NSCLC). • MiR-429 inhibits PTEN, RASSF8 and TIMP2 expression. • MiR-429 promotes metastasis and proliferation. • We report important regulatory mechanisms involved in NSCLC progression. • MiR-429 is a potential therapeutic target and diagnostic marker. - Abstract: Lung cancer is the major cause of cancer death globally. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression. Aberrant expression of microRNA (miRNA) has been implicated in cancer initiation and progression. In this study, we demonstrated that the expression of miR-429 are often upregulated in non-small cell lung cancer (NSCLC) compared with normal lung tissues, and its expression level is also increased in NSCLC cell lines compared with normal lung cells. Overexpression of miR-429 in A549 NSCLC cells significantly promoted cell proliferation, migration and invasion, whereas inhibition of miR-429 inhibits these effects. Furthermore, we demonstrated that miR-429 down-regulates PTEN, RASSF8 and TIMP2 expression by directly targeting the 3′-untranslated region of these target genes. Taken together, our results suggest that miR-429 plays an important role in promoting the proliferation and metastasis of NSCLC cells and is a potential target for NSCLC therapy.

  2. Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

    Science.gov (United States)

    Kangaspeska, Sara; Hultsch, Susanne; Edgren, Henrik; Nicorici, Daniel; Murumägi, Astrid; Kallioniemi, Olli

    2012-01-01

    RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60%) of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

  3. Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

    Directory of Open Access Journals (Sweden)

    Sara Kangaspeska

    Full Text Available RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60% of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

  4. Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization.

    Science.gov (United States)

    Locati, Mauro D; Terpstra, Inez; de Leeuw, Wim C; Kuzak, Mateusz; Rauwerda, Han; Ensink, Wim A; van Leeuwen, Selina; Nehrdich, Ulrike; Spaink, Herman P; Jonker, Martijs J; Breit, Timo M; Dekker, Rob J

    2015-08-18

    There is an increasing interest in complementing RNA-seq experiments with small-RNA (sRNA) expression data to obtain a comprehensive view of a transcriptome. Currently, two main experimental challenges concerning sRNA-seq exist: how to check the size distribution of isolated sRNAs, given the sensitive size-selection steps in the protocol; and how to normalize data between samples, given the low complexity of sRNA types. We here present two separate sets of synthetic RNA spike-ins for monitoring size-selection and for performing data normalization in sRNA-seq. The size-range quality control (SRQC) spike-in set, consisting of 11 oligoribonucleotides (10-70 nucleotides), was tested by intentionally altering the size-selection protocol and verified via several comparative experiments. We demonstrate that the SRQC set is useful to reproducibly track down biases in the size-selection in sRNA-seq. The external reference for data-normalization (ERDN) spike-in set, consisting of 19 oligoribonucleotides, was developed for sample-to-sample normalization in differential-expression analysis of sRNA-seq data. Testing and applying the ERDN set showed that it can reproducibly detect differential expression over a dynamic range of 2(18). Hence, biological variation in sRNA composition and content between samples is preserved while technical variation is effectively minimized. Together, both spike-in sets can significantly improve the technical reproducibility of sRNA-seq. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Diet and lifestyle factors associated with miRNA expression in colorectal tissue

    Directory of Open Access Journals (Sweden)

    Slattery ML

    2016-12-01

    Full Text Available Martha L Slattery,1 Jennifer S Herrick,1 Lila E Mullany,1 John R Stevens,2 Roger K Wolff1 1Department of Internal Medicine, The University of Utah, Salt Lake City, 2Department of Mathematics and Statistics, Utah State University, Logan, UT, USA Abstract: MicroRNAs (miRNAs are small non-protein-coding RNA molecules that regulate gene expression. Diet and lifestyle factors have been hypothesized to be involved in the regulation of miRNA expression. In this study it was hypothesized that diet and lifestyle factors are associated with miRNA expression. Data from 1,447 cases of colorectal cancer to evaluate 34 diet and lifestyle variables using miRNA expression in normal colorectal mucosa as well as for differential expression between paired carcinoma and normal tissue were used. miRNA data were obtained using an Agilent platform. Multiple comparisons were adjusted for using the false discovery rate q-value. There were 250 miRNAs differentially expressed between carcinoma and normal colonic tissue by level of carbohydrate intake and 198 miRNAs differentially expressed by the level of sucrose intake. Of these miRNAs, 166 miRNAs were differentially expressed for both carbohydrate intake and sucrose intake. Ninety-nine miRNAs were differentially expressed by the level of whole grain intake in normal colonic mucosa. Level of oxidative balance score was associated with 137 differentially expressed miRNAs between carcinoma and paired normal rectal mucosa. Additionally, 135 miRNAs were differentially expressed in colon tissue based on recent NSAID use. Other dietary factors, body mass index, waist and hip circumference, and long-term physical activity levels did not alter miRNA expression after adjustment for multiple comparisons. These results suggest that diet and lifestyle factors regulate miRNA level. They provide additional support for the influence of carbohydrate, sucrose, whole grains, NSAIDs, and oxidative balance score on colorectal cancer risk

  6. Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9).

    Science.gov (United States)

    Fu, Qinqin; Yuan, Y Adam

    2013-03-01

    Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA.

  7. mRNA processing in yeast

    International Nuclear Information System (INIS)

    Stevens, A.

    1982-01-01

    Investigations in this laboratory center on basic enzymatic reactions of RNA. Still undefined are reactions involved in the conversion of precursors of mRA (pre-mRNA) to mRNA in eukaryotes. The pre-mRNA is called heterogeneous nuclear RNA and is 2 to 6 times larger than mRNA. The conversion, called splicing, involves a removal of internal sequences called introns by endoribonuclease action followed by a rejoining of the 3'- and 5'-end fragments, called exons, by ligating activity. It has not been possible yet to study the enzymes involved in vitro. Also undefined are reactions involved in the turnover or discarding of certain of the pre-mRNA molecules. Yeast is a simple eukaryote and may be expected to have the same, but perhaps simpler, processing reactions as the higher eukaryotes. Two enzymes involved in the processing of pre-mRNA and mRNA in yeast are under investigation. Both enzymes have been partially purified from ribonucleoprotein particles of yeast. The first is a unique decapping enzyme which cleaves [ 3 H]m 7 Gppp [ 14 C]RNA-poly (A) of yeast, yielding [ 3 H]m 7 GDP and is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed. The second enzyme is an endoribonuclease which converts both the [ 3 H] and [ 14 C] labels of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from an oligo(dT)-cellulose bound form to an unbound, acid-insoluble form. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA. The inhibition of the enzyme by ethidium bromide and its stimulation by small nuclear RNA suggest that it may be a processing ribonuclease, requiring specific double-stranded features in its substrate. The characterization of the unique decapping enzyme and endoribonuclease may help to understand reactions involved in the processing of pre-mRNA and mRNA in eukaryotes

  8. DeAnnIso: a tool for online detection and annotation of isomiRs from small RNA sequencing data.

    Science.gov (United States)

    Zhang, Yuanwei; Zang, Qiguang; Zhang, Huan; Ban, Rongjun; Yang, Yifan; Iqbal, Furhan; Li, Ao; Shi, Qinghua

    2016-07-08

    Small RNA (sRNA) Sequencing technology has revealed that microRNAs (miRNAs) are capable of exhibiting frequent variations from their canonical sequences, generating multiple variants: the isoforms of miRNAs (isomiRs). However, integrated tool to precisely detect and systematically annotate isomiRs from sRNA sequencing data is still in great demand. Here, we present an online tool, DeAnnIso (Detection and Annotation of IsomiRs from sRNA sequencing data). DeAnnIso can detect all the isomiRs in an uploaded sample, and can extract the differentially expressing isomiRs from paired or multiple samples. Once the isomiRs detection is accomplished, detailed annotation information, including isomiRs expression, isomiRs classification, SNPs in miRNAs and tissue specific isomiR expression are provided to users. Furthermore, DeAnnIso provides a comprehensive module of target analysis and enrichment analysis for the selected isomiRs. Taken together, DeAnnIso is convenient for users to screen for isomiRs of their interest and useful for further functional studies. The server is implemented in PHP + Perl + R and available to all users for free at: http://mcg.ustc.edu.cn/bsc/deanniso/ and http://mcg2.ustc.edu.cn/bsc/deanniso/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Therapeutic miRNA and siRNA: Moving from Bench to Clinic as Next Generation Medicine

    Directory of Open Access Journals (Sweden)

    Chiranjib Chakraborty

    2017-09-01

    Full Text Available In the past few years, therapeutic microRNA (miRNA and small interfering RNA (siRNA are some of the most important biopharmaceuticals that are in commercial space as future medicines. This review summarizes the patents of miRNA- and siRNA-based new drugs, and also provides a snapshot about significant biopharmaceutical companies that are investing for the therapeutic development of miRNA and siRNA molecules. An insightful view about individual siRNA and miRNA drugs has been depicted with their present status, which is gaining attention in the therapeutic landscape. The efforts of the biopharmaceuticals are discussed with the status of their preclinical and/or clinical trials. Here, some of the setbacks have been highlighted during the biopharmaceutical development of miRNA and siRNA as individual therapeutics. Finally, a snapshot is illustrated about pharmacokinetics, pharmacodynamics with absorption, distribution, metabolism, and excretion (ADME, which is the fundamental development process of these therapeutics, as well as the delivery system for miRNA- and siRNA-based drugs. Keywords: miRNA, siRNA, drug development

  10. RIP-seq of BmAgo2-associated small RNAs reveal various types of small non-coding RNAs in the silkworm, Bombyx mori

    Science.gov (United States)

    2013-01-01

    Background Small non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm. Results Here, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and TψC loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8 s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm. Conclusions Using an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect

  11. Fatgraph models of RNA structure

    Directory of Open Access Journals (Sweden)

    Huang Fenix

    2017-01-01

    Full Text Available In this review paper we discuss fatgraphs as a conceptual framework for RNA structures. We discuss various notions of coarse-grained RNA structures and relate them to fatgraphs.We motivate and discuss the main intuition behind the fatgraph model and showcase its applicability to canonical as well as noncanonical base pairs. Recent discoveries regarding novel recursions of pseudoknotted (pk configurations as well as their translation into context-free grammars for pk-structures are discussed. This is shown to allow for extending the concept of partition functions of sequences w.r.t. a fixed structure having non-crossing arcs to pk-structures. We discuss minimum free energy folding of pk-structures and combine these above results outlining how to obtain an inverse folding algorithm for PK structures.

  12. Using Information Theory to Assess the Communicative Capacity of Circulating MicroRNA

    OpenAIRE

    Finn, Nnenna A.; Searles, Charles D.

    2013-01-01

    The discovery of extracellular microRNAs (miRNAs) and their transport modalities (i.e. microparticles, exosomes, proteins and lipoproteins) has sparked theories regarding their role in intercellular communication. Here, we assessed the information transfer capacity of different miRNA transport modalities in human serum by utilizing basic principles of information theory. Zipf Statistics were calculated for each of the miRNA transport modalities identified in human serum. Our analyses revealed...

  13. The Mechanisms of Virulence Regulation by Small Noncoding RNAs in Low GC Gram-Positive Pathogens

    Directory of Open Access Journals (Sweden)

    Stephanie Pitman

    2015-12-01

    Full Text Available The discovery of small noncoding regulatory RNAs (sRNAs in bacteria has grown tremendously recently, giving new insights into gene regulation. The implementation of computational analysis and RNA sequencing has provided new tools to discover and analyze potential sRNAs. Small regulatory RNAs that act by base-pairing to target mRNAs have been found to be ubiquitous and are the most abundant class of post-transcriptional regulators in bacteria. The majority of sRNA studies has been limited to E. coli and other gram-negative bacteria. However, examples of sRNAs in gram-positive bacteria are still plentiful although the detailed gene regulation mechanisms behind them are not as well understood. Strict virulence control is critical for a pathogen’s survival and many sRNAs have been found to be involved in that process. This review outlines the targets and currently known mechanisms of trans-acting sRNAs involved in virulence regulation in various gram-positive pathogens. In addition, their shared characteristics such as CU interaction motifs, the role of Hfq, and involvement in two-component regulators, riboswitches, quorum sensing, or toxin/antitoxin systems are described.

  14. Small interfering RNA targeting HIF-1{alpha} reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Staab, Adrian [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Paul Scherrer Institute (PSI), Villigen (Switzerland); Fleischer, Markus [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Wuerzburg Univ. (Germany). Medical Clinic II; Loeffler, Juergen; Einsele, Herrmann [Wuerzburg Univ. (Germany). Medical Clinic II; Said, Harun M.; Katzer, Astrid; Flentje, Michael [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Plathow, Christian [Freiburg Univ. (Germany). Dept. of Nuclear Medicine; Vordermark, Dirk [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Halle-Wittenberg Univ. (Germany). Dept. of Radiation Oncology

    2011-04-15

    Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1{alpha} expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1{alpha} siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1{alpha}. HIF-1{alpha} protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O{sub 2} (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1{alpha}-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O{sub 2} as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1{alpha}-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1{alpha}-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

  15. Small interfering RNA targeting HIF-1α reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

    International Nuclear Information System (INIS)

    Staab, Adrian; Fleischer, Markus; Wuerzburg Univ.; Loeffler, Juergen; Einsele, Herrmann; Said, Harun M.; Katzer, Astrid; Flentje, Michael; Plathow, Christian; Vordermark, Dirk; Halle-Wittenberg Univ.

    2011-01-01

    Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1α expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1α siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1α. HIF-1α protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O 2 (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1α-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O 2 as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1α-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1α-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1α-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1α-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

  16. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.

    Directory of Open Access Journals (Sweden)

    Jae-Su Moon

    Full Text Available The hepatitis C virus (HCV internal ribosome entry site (IRES that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343 where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.

  17. Regulatory RNAs derived from transfer RNA?

    Science.gov (United States)

    Pederson, Thoru

    2010-10-01

    Four recent studies suggest that cleavages of transfer RNAs generate products with microRNA-like features, with some evidence of function. If their regulatory functions were to be confirmed, these newly revealed RNAs would add to the expanding repertoire of small noncoding RNAs and would also provide new perspectives on the coevolution of transfer RNA and messenger RNA.

  18. A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

    Science.gov (United States)

    Tsai, Hsin-Yue; Chen, Chun-Chieh G; Conte, Darryl; Moresco, James J; Chaves, Daniel A; Mitani, Shohei; Yates, John R; Tsai, Ming-Daw; Mello, Craig C

    2015-01-29

    Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA.

    Science.gov (United States)

    Parker, Greg S; Eckert, Debra M; Bass, Brenda L

    2006-05-01

    In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not required in subsequent steps, and how RDE-4 distinguishes between long dsRNA and short siRNA is unclear. We report the first detailed analysis of RDE-4 binding, using purified recombinant RDE-4 and various truncated proteins. We find that, similar to other dsRBPs, RDE-4 is not sequence-specific. However, consistent with its in vivo roles, RDE-4 binds with higher affinity to long dsRNA. We also observe that RDE-4 is a homodimer in solution, and that the C-terminal domain of the protein is required for dimerization. Using extracts from wild-type and rde-4 mutant C. elegans, we show that the C-terminal dimerization domain is required for the production of siRNA. Our findings suggest a model for RDE-4 function during the initiation of RNAi.

  20. Tapping the RNA world for therapeutics.

    Science.gov (United States)

    Lieberman, Judy

    2018-04-16

    A recent revolution in RNA biology has led to the identification of new RNA classes with unanticipated functions, new types of RNA modifications, an unexpected multiplicity of alternative transcripts and widespread transcription of extragenic regions. This development in basic RNA biology has spawned a corresponding revolution in RNA-based strategies to generate new types of therapeutics. Here, I review RNA-based drug design and discuss barriers to broader applications and possible ways to overcome them. Because they target nucleic acids rather than proteins, RNA-based drugs promise to greatly extend the domain of 'druggable' targets beyond what can be achieved with small molecules and biologics.

  1. Emerging Concepts and Methodologies in Cancer Biomarker Discovery.

    Science.gov (United States)

    Lu, Meixia; Zhang, Jinxiang; Zhang, Lanjing

    2017-01-01

    Cancer biomarker discovery is a critical part of cancer prevention and treatment. Despite the decades of effort, only a small number of cancer biomarkers have been identified for and validated in clinical settings. Conceptual and methodological breakthroughs may help accelerate the discovery of additional cancer biomarkers, particularly their use for diagnostics. In this review, we have attempted to review the emerging concepts in cancer biomarker discovery, including real-world evidence, open access data, and data paucity in rare or uncommon cancers. We have also summarized the recent methodological progress in cancer biomarker discovery, such as high-throughput sequencing, liquid biopsy, big data, artificial intelligence (AI), and deep learning and neural networks. Much attention has been given to the methodological details and comparison of the methodologies. Notably, these concepts and methodologies interact with each other and will likely lead to synergistic effects when carefully combined. Newer, more innovative concepts and methodologies are emerging as the current emerging ones became mainstream and widely applied to the field. Some future challenges are also discussed. This review contributes to the development of future theoretical frameworks and technologies in cancer biomarker discovery and will contribute to the discovery of more useful cancer biomarkers.

  2. Alterations in Bronchial Airway miRNA Expression for Lung Cancer Detection.

    Science.gov (United States)

    Pavel, Ana B; Campbell, Joshua D; Liu, Gang; Elashoff, David; Dubinett, Steven; Smith, Kate; Whitney, Duncan; Lenburg, Marc E; Spira, Avrum

    2017-11-01

    We have previously shown that gene expression alterations in normal-appearing bronchial epithelial cells can serve as a lung cancer detection biomarker in smokers. Given that miRNAs regulate airway gene expression responses to smoking, we evaluated whether miRNA expression is also altered in the bronchial epithelium of smokers with lung cancer. Using epithelial brushings from the mainstem bronchus of patients undergoing bronchoscopy for suspected lung cancer (as part of the AEGIS-1/2 clinical trials), we profiled miRNA expression via small-RNA sequencing from 347 current and former smokers for which gene expression data were also available. Patients were followed for one year postbronchoscopy until a final diagnosis of lung cancer ( n = 194) or benign disease ( n = 153) was made. Following removal of 6 low-quality samples, we used 138 patients (AEGIS-1) as a discovery set to identify four miRNAs (miR-146a-5p, miR-324-5p, miR-223-3p, and miR-223-5p) that were downregulated in the bronchial airway of lung cancer patients (ANOVA P cancer patients (GSEA FDR lung cancer significantly improves its performance (AUC) in the 203 samples (AEGIS-1/2) serving an independent test set (DeLong P lung cancer, and that they may regulate cancer-associated gene expression differences. Cancer Prev Res; 10(11); 651-9. ©2017 AACR . ©2017 American Association for Cancer Research.

  3. The Battle of RNA Synthesis: Virus versus Host.

    Science.gov (United States)

    Harwig, Alex; Landick, Robert; Berkhout, Ben

    2017-10-21

    Transcription control is the foundation of gene regulation. Whereas a cell is fully equipped for this task, viruses often depend on the host to supply tools for their transcription program. Over the course of evolution and adaptation, viruses have found diverse ways to optimally exploit cellular host processes such as transcription to their own benefit. Just as cells are increasingly understood to employ nascent RNAs in transcription regulation, recent discoveries are revealing how viruses use nascent RNAs to benefit their own gene expression. In this review, we first outline the two different transcription programs used by viruses, i.e., transcription (DNA-dependent) and RNA-dependent RNA synthesis. Subsequently, we use the distinct stages (initiation, elongation, termination) to describe the latest insights into nascent RNA-mediated regulation in the context of each relevant stage.

  4. Role of Melt Curve Analysis in Interpretation of Nutrigenomics' MicroRNA Expression Data.

    Science.gov (United States)

    Ahmed, Farid E; Gouda, Mostafa M; Hussein, Laila A; Ahmed, Nancy C; Vos, Paul W; Mohammad, Mahmoud A

    2017-01-01

    This article illustrates the importance of melt curve analysis (MCA) in interpretation of mild nutrogenomic micro(mi)RNA expression data, by measuring the magnitude of the expression of key miRNA molecules in stool of healthy human adults as molecular markers, following the intake of Pomegranate juice (PGJ), functional fermented sobya (FS), rich in potential probiotic lactobacilli, or their combination. Total small RNA was isolated from stool of 25 volunteers before and following a three-week dietary intervention trial. Expression of 88 miRNA genes was evaluated using Qiagen's 96 well plate RT 2 miRNA qPCR arrays. Employing parallel coordinates plots, there was no observed significant separation for the gene expression (Cq) values, using Roche 480® PCR LightCycler instrument used in this study, and none of the miRNAs showed significant statistical expression after controlling for the false discovery rate. On the other hand, melting temperature profiles produced during PCR amplification run, found seven significant genes (miR-184, miR-203, miR-373, miR-124, miR-96, miR-373 and miR-301a), which separated candidate miRNAs that could function as novel molecular markers of relevance to oxidative stress and immunoglobulin function, for the intake of polyphenol (PP)-rich, functional fermented foods rich in lactobacilli (FS), or their combination. We elaborate on these data, and present a detailed review on use of melt curves for analyzing nutigenomic miRNA expression data, which initially appear to show no significant expressions, but are actually more subtle than this simplistic view, necessitating the understanding of the role of MCA for a comprehensive understanding of what the collective expression and MCA data collectively imply. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  5. Small Interference RNA Targeting TLR4 Gene Effectively Attenuates Pulmonary Inflammation in a Rat Model

    Directory of Open Access Journals (Sweden)

    Feixiang Wu

    2012-01-01

    Full Text Available Objective. The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA targeting Toll-like receptor 4 (TLR4 gene in ameliorating lipopolysaccharide- (LPS- induced acute lung injury (ALI. Methods. In vitro, alveolar macrophages (AMs were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group, 300 μL of Ad-EGFP (Ad-EGFP group, or 300 μL of Ad-siTLR4 (Ad-siTLR4 group and then were intravenously treated with LPS (50 mg/kg to induce ALI. Results. Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals. Conclusion. TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

  6. [Small interfering RNA-mediated COX-2 gene silencing enhances chemosensitivity of KB/VCR cells by suppressing MDR-1 gene expression and P-glycoprotein activity].

    Science.gov (United States)

    Mo, Xianchao; Li, Weizhong

    2014-05-01

    To investigate the effect of small interfering RNA (siRNA)-mediated COX-2 gene silencing in enhancing the chemosensitivity of KB/VCR cell lines. KB/VCR cells were trasnfected with COX-2 siRNA were examined for expressions of COX-2 and MDR-1 mRNAs with RT-PCR and for Rho-123 accumulation using flow cytometry. MTT assay was used to analyze the proliferation of the transfected KB/VCR cells. Compared with the negative and blank control groups, COX-2 siRNA transfection resulted in significant growth inhibition of KB/VCR cells exposed to vincristine (PKB/VCR cells. COX-2 gene silencing can enhance the chemosensitivity of KB/VCR cells to vincristine, the mechanism of which may involve down-regulated MDR-1 gene expression and inhibition of P-glycoprotein activity.

  7. Silencing of cytosolic NADP(+)-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine.

    Science.gov (United States)

    Lee, Su-Min; Park, Sin Young; Shin, Seoung Woo; Kil, In Sup; Yang, Eun Sun; Park, Jeen-Woo

    2009-02-01

    Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.

  8. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

    Science.gov (United States)

    van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

    2016-01-01

    RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

  9. Improving functional modules discovery by enriching interaction networks with gene profiles

    KAUST Repository

    Salem, Saeed

    2013-05-01

    Recent advances in proteomic and transcriptomic technologies resulted in the accumulation of vast amount of high-throughput data that span multiple biological processes and characteristics in different organisms. Much of the data come in the form of interaction networks and mRNA expression arrays. An important task in systems biology is functional modules discovery where the goal is to uncover well-connected sub-networks (modules). These discovered modules help to unravel the underlying mechanisms of the observed biological processes. While most of the existing module discovery methods use only the interaction data, in this work we propose, CLARM, which discovers biological modules by incorporating gene profiles data with protein-protein interaction networks. We demonstrate the effectiveness of CLARM on Yeast and Human interaction datasets, and gene expression and molecular function profiles. Experiments on these real datasets show that the CLARM approach is competitive to well established functional module discovery methods.

  10. Discovery of a dsRNA virus infecting the marine photosynthetic protist Micromonas pusilla

    International Nuclear Information System (INIS)

    Brussaard, C.P.D.; Noordeloos, A.A.M.; Sandaa, R.-A.; Heldal, M.; Bratbak, G.

    2004-01-01

    We report the isolation of the first double-stranded (ds) RNA virus in the family Reoviridae that infects a protist (microalga Micromonas pusilla, Prasinophyceae). The dsRNA genome was composed of 11 segments ranging between 0.8 and 5.8 kb, with a total size of approximately 25.5 kb. The virus (MpRNAV-01B) could not be assigned to the genus level because host type, genome size, and number of segments smaller than 2 kb did not correspond to either of the two existing 11-segmented dsRNA genera Rotavirus and Aquareovirus. MpRNAV-01B has a particle size of 65-80 nm, a narrow host range, a latent period of 36 h, and contains five major proteins (120, 95, 67, 53, and 32 kDa). MpRNAV-01B was stable to freeze-thawing, resistant to chloroform, ether, nonionic detergents, chelating and reducing agents. The virus was inactivated at temperatures above 35 deg. C and by ionic detergent, ethanol, acetone, and acidic conditions (pH 2-5)

  11. Reconstruction of ribosomal RNA genes from metagenomic data.

    Directory of Open Access Journals (Sweden)

    Lu Fan

    Full Text Available Direct sequencing of environmental DNA (metagenomics has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes.

  12. Modulation of microRNA activity by semi-microRNAs (smiRNAs

    Directory of Open Access Journals (Sweden)

    Isabelle ePlante

    2012-06-01

    Full Text Available The ribonuclease Dicer plays a central role in the microRNA pathway by catalyzing the formation of 19 to 24-nucleotide (nt long microRNAs. Subsequently incorporated into Ago2 effector complexes, microRNAs are known to regulate messenger RNA (mRNA translation. Whether shorter RNA species derived from microRNAs exist and play a role in mRNA regulation remains unknown. Here, we report the serendipitous discovery of a 12-nt long RNA species corresponding to the 5’ region of the microRNA let-7, and tentatively termed semi-microRNA, or smiRNA. Using a smiRNA derived from the precursor of miR-223 as a model, we show that 12-nt long smiRNA species are devoid of any direct mRNA regulatory activity, as assessed in a reporter gene activity assay in transfected cultured human cells. However, smiR-223 was found to modulate the ability of the microRNA from which it derives to mediate translational repression or cleavage of reporter mRNAs. Our findings suggest that smiRNAs may be generated along the microRNA pathway and participate to the control of gene expression by regulating the activity of the related full-length mature microRNA in vivo.

  13. RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins

    International Nuclear Information System (INIS)

    Hoffman, D.W.; Query, C.C.; Golden, B.L.; White, S.W.; Keene, J.D.

    1991-01-01

    An RNA recognition motif (RRM) of ∼80 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing. The U1 RNA-binding domain of the A protein component of the human U1 small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, was analyzed by using NMR spectroscopy. The domain of the A protein is a highly stable monomer in solution consisting of four antiparallel β-strands and two α-helices. The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent β-strands. Conserved aromatic side chains that are critical for RNA binding are clustered on the surface to the molecule adjacent to a variable loop that influences recognition of specific RNA sequences. The secondary structure and topology of the RRM are similar to those of ribosomal proteins L12 and L30, suggesting a distant evolutionary relationship between these two types of RNA-associated proteins

  14. Drawbacks of the ancient RNA-based life-like system under primitive earth conditions.

    Science.gov (United States)

    Kawamura, Kunio

    2012-07-01

    Following the discovery of ribozymes, the "RNA world" hypothesis has become the most accepted hypothesis concerning the origin of life and genetic information. However, this hypothesis has several drawbacks. Verification of the hypothesis from different viewpoints led us to proposals from the viewpoint of the hydrothermal origin of life, solubility of RNA and related biopolymers, and the possibility of creating an evolutionary system comparable to the in vitro selection technique for functional RNA molecules based on molecular biology. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  15. Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

    Directory of Open Access Journals (Sweden)

    Alexey Berezhnoy

    2012-01-01

    Full Text Available Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

  16. Quantification of RNA in bacteriophage MS2-like viruses in solution by small-angle X-ray scattering

    International Nuclear Information System (INIS)

    Kuzmanovic, Deborah A.; Elashvili, Ilya; Wick, Charles; O'Connell, Catherine; Krueger, Susan

    2006-01-01

    Recombinant forms of bacteriophage MS2 virus particles, wild-type MS2 and MS2 capsids have been examined in solution using small-angle X-ray scattering (SAXS). SAXS was used to determine the overall size of the virus particles and to quantify the amount of encapsulated viral RNA. These studies show that analysis of natural and recombinant forms of MS2 virus by SAXS can be used as both a quantitative measure of nucleic acid content in situ and diagnostic indicator of sample integrity

  17. Identification of RNA species in the RNA-toxin complex and structure of the complex in Clostridium botulinum type E.

    Science.gov (United States)

    Kitamura, Masaru

    2002-02-15

    Clostridium botulinum type E toxin was isolated in the form of a complex with RNA(s) from bacterial cells. Characterization of the complexed RNA remains to be elucidated. The RNA is identified here as ribosomal RNA (rRNA) having 23S and 16S components. The RNA-toxin complexes were found to be made up of three types with different molecular sizes. The three types of RNA-toxin complex are toxin bound to both the 23S and 16S rRNA, toxin bound to the 16S rRNA and a small amount of 23S rRNA, and toxin bound only to the 16S rRNA. ©2002 Elsevier Science (USA).

  18. Design of small molecule epigenetic modulators.

    Science.gov (United States)

    Pachaiyappan, Boobalan; Woster, Patrick M

    2014-01-01

    The field of epigenetics has expanded rapidly to reveal multiple new targets for drug discovery. The functional elements of the epigenomic machinery can be categorized as writers, erasers and readers, and together these elements control cellular gene expression and homeostasis. It is increasingly clear that aberrations in the epigenome can underly a variety of diseases, and thus discovery of small molecules that modulate the epigenome in a specific manner is a viable approach to the discovery of new therapeutic agents. In this Digest, the components of epigenetic control of gene expression will be briefly summarized, and efforts to identify small molecules that modulate epigenetic processes will be described. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. RNA Relics and Origin of Life

    Directory of Open Access Journals (Sweden)

    Laurent Vial

    2009-07-01

    Full Text Available A number of small RNA sequences, located in different non-coding sequences and highly preserved across the tree of life, have been suggested to be molecular fossils, of ancient (and possibly primordial origin. On the other hand, recent years have revealed the existence of ubiquitous roles for small RNA sequences in modern organisms, in functions ranging from cell regulation to antiviral activity. We propose that a single thread can be followed from the beginning of life in RNA structures selected only for stability reasons through the RNA relics and up to the current coevolution of RNA sequences; such an understanding would shed light both on the history and on the present development of the RNA machinery and interactions. After presenting the evidence (by comparing their sequences that points toward a common thread, we discuss a scenario of genome coevolution (with emphasis on viral infectious processes and finally propose a plan for the reevaluation of the stereochemical theory of the genetic code; we claim that it may still be relevant, and not only for understanding the origin of life, but also for a comprehensive picture of regulation in present-day cells.

  20. RNA Relics and Origin of Life

    Science.gov (United States)

    Demongeot, Jacques; Glade, Nicolas; Moreira, Andrés; Vial, Laurent

    2009-01-01

    A number of small RNA sequences, located in different non-coding sequences and highly preserved across the tree of life, have been suggested to be molecular fossils, of ancient (and possibly primordial) origin. On the other hand, recent years have revealed the existence of ubiquitous roles for small RNA sequences in modern organisms, in functions ranging from cell regulation to antiviral activity. We propose that a single thread can be followed from the beginning of life in RNA structures selected only for stability reasons through the RNA relics and up to the current coevolution of RNA sequences; such an understanding would shed light both on the history and on the present development of the RNA machinery and interactions. After presenting the evidence (by comparing their sequences) that points toward a common thread, we discuss a scenario of genome coevolution (with emphasis on viral infectious processes) and finally propose a plan for the reevaluation of the stereochemical theory of the genetic code; we claim that it may still be relevant, and not only for understanding the origin of life, but also for a comprehensive picture of regulation in present-day cells. PMID:20111682