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Sample records for human small rnas

  1. 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...

  2. A survey of small RNAs in human sperm

    Science.gov (United States)

    Krawetz, Stephen A.; Kruger, Adele; Lalancette, Claudia; Tagett, Rebecca; Anton, Ester; Draghici, Sorin; Diamond, Michael P.

    2011-01-01

    BACKGROUND There has been substantial interest in assessing whether RNAs (mRNAs and sncRNAs, i.e. small non-coding) delivered from mammalian spermatozoa play a functional role in early embryo development. While the cadre of spermatozoal mRNAs has been characterized, comparatively little is known about the distribution or function of the estimated 24 000 sncRNAs within each normal human spermatozoon. METHODS RNAs of libraries for Next Generation Sequencing. Known sncRNAs that uniquely mapped to a single location in the human genome were identified. RESULTS Bioinformatic analysis revealed the presence of multiple classes of small RNAs in human spermatozoa. The primary classes resolved included microRNA (miRNAs) (≈7%), Piwi-interacting piRNAs (≈17%), repeat-associated small RNAs (≈65%). A minor subset of short RNAs within the transcription start site/promoter fraction (≈11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. These have been termed quiescent RNAs. CONCLUSIONS A complex population of male derived sncRNAs that are available for delivery upon fertilization was revealed. Sperm miRNA-targeted enrichment in the human oocyte is consistent with their role as modifiers of early post-fertilization. The relative abundance of piRNAs and repeat-associated RNAs suggests that they may assume a role in confrontation and consolidation. This may ensure the compatibility of the genomes at fertilization. PMID:21989093

  3. Regulatory Role of Small Nucleolar RNAs in Human Diseases

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    Grigory A. Stepanov

    2015-01-01

    Full Text Available Small nucleolar RNAs (snoRNAs are appreciable players in gene expression regulation in human cells. The canonical function of box C/D and box H/ACA snoRNAs is posttranscriptional modification of ribosomal RNAs (rRNAs, namely, 2′-O-methylation and pseudouridylation, respectively. A series of independent studies demonstrated that snoRNAs, as well as other noncoding RNAs, serve as the source of various short regulatory RNAs. Some snoRNAs and their fragments can also participate in the regulation of alternative splicing and posttranscriptional modification of mRNA. Alterations in snoRNA expression in human cells can affect numerous vital cellular processes. SnoRNA level in human cells, blood serum, and plasma presents a promising target for diagnostics and treatment of human pathologies. Here we discuss the relation between snoRNAs and oncological, neurodegenerative, and viral diseases and also describe changes in snoRNA level in response to artificial stress and some drugs.

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

    DEFF Research Database (Denmark)

    Pantano, Lorena; Jodar, Meritxell; Bak, Mads

    2015-01-01

    -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....

  5. 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

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

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    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. Nanoparticle (MPG)-mediated delivery of small RNAs into human ...

    African Journals Online (AJOL)

    The cellular membrane constitutes an effective barrier that protects the complex, yet highly ordered, intracellular compartment of the cell. Passage of molecules across this barrier is highly regulated and highly restricted. Cell penetrating peptides (CPPs) are a class of small cationic peptides that are able to defy the rules of ...

  8. Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin

    Directory of Open Access Journals (Sweden)

    Jakobsen Maria

    2011-02-01

    Full Text Available Abstract Background Psoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα, interleukin-12 (IL-12, and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs. Methods Effective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels. Results Potent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimick the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by

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

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    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.

  10. An Algorithm for Generating Small RNAs Capable of Epigenetically Modulating Transcriptional Gene Silencing and Activation in Human Cells

    Directory of Open Access Journals (Sweden)

    Amanda Ackley

    2013-01-01

    Full Text Available Small noncoding antisense RNAs (sasRNAs guide epigenetic silencing complexes to target loci in human cells and modulate gene transcription. When these targeted loci are situated within a promoter, long-term, stable epigenetic silencing of transcription can occur. Recent studies suggest that there exists an endogenous form of such epigenetic regulation in human cells involving long noncoding RNAs. In this article, we present and validate an algorithm for the generation of highly effective sasRNAs that can mimic the endogenous noncoding RNAs involved in the epigenetic regulation of gene expression. We validate this algorithm by targeting several oncogenes including AKT-1, c-MYC, K-RAS, and H-RAS. We also target a long antisense RNA that mediates the epigenetic repression of the tumor suppressor gene DUSP6, silenced in pancreatic cancer. An algorithm that can efficiently design small noncoding RNAs for the epigenetic transcriptional silencing or activation of specific genes has potential therapeutic and experimental applications.

  11. Random small interfering RNA library screen identifies siRNAs that induce human erythroleukemia cell differentiation.

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    Fan, Cuiqing; Xiong, Yuan; Zhu, Ning; Lu, Yabin; Zhang, Jiewen; Wang, Song; Liang, Zicai; Shen, Yan; Chen, Meihong

    2011-03-01

    Cancers are characterized by poor differentiation. Differentiation therapy is a strategy to alleviate malignant phenotypes by inducing cancer cell differentiation. Here we carried out a combinatorial high-throughput screen with a random siRNA library on human erythroleukemia K-562 cell differentiation. Two siRNAs screened from the library were validated to be able to induce erythroid differentiation to varying degrees, determined by CD235 and globin up-regulation, GATA-2 down-regulation, and cell growth inhibition. The screen we performed here is the first trial of screening cancer differentiation-inducing agents from a random siRNA library, demonstrating that a random siRNA library can be considered as a new resource in efforts to seek new therapeutic agents for cancers. As a random siRNA library has a broad coverage for the entire genome, including known/unknown genes and protein coding/non-coding sequences, screening using a random siRNA library can be expected to greatly augment the repertoire of therapeutic siRNAs for cancers.

  12. 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.

  13. The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells

    International Nuclear Information System (INIS)

    Xu, Y.; Mirmalek-Sani, S.-H.; Yang, X.; Zhang, J.; Oreffo, R.O.C.

    2006-01-01

    RNA interference (RNAi) has been used in functional genomics and offers innovative approaches in the development of novel therapeutics. Human mesenchymal stem cells offer a unique cell source for tissue engineering/regeneration strategies. The current study examined the potential of small interfering RNAs (siRNA) against human peroxisome proliferator activated receptor gamma (PPARγ) to suppress adipocyte differentiation (adipogenesis) in human preadipocytes and fetal-femur-derived mesenchymal cells. Adipogenesis was investigated using cellular and biochemical analysis. Transient transfection with PPARγ-siRNA using a liposomal-based strategy resulted in a significant inhibition of adipogenesis in human preadipocytes and fetal-femur-derived mesenchymal cells, compared to controls (cell, liposomal and negative siRNA). The inhibitory effect of PPARγ-siRNA was supported by testing human PPARγ mRNA and adipogenic associated genes using reverse transcription polymerase chain reaction (RT-PCR) to adiponectin receptor 1 and 2 as well as examination of fatty acid binding protein 3 (FABP 3 ) expression, an adipocyte-specific marker. The current studies indicate that PPARγ-siRNA is a useful tool to study adipogenesis in human cells, with potential applications both therapeutic and in the elucidation of mesenchymal cell differentiation in the modulation of cell differentiation in human mesenchymal cells

  14. Role of Small RNAs in Trypanosomatid Infections

    Science.gov (United States)

    Linhares-Lacerda, Leandra; Morrot, Alexandre

    2016-01-01

    Trypanosomatid parasites survive and replicate in the host by using mechanisms that aim to establish a successful infection and ensure parasite survival. Evidence points to microRNAs as new players in the host-parasite interplay. MicroRNAs are small non-coding RNAs that control proteins levels via post-transcriptional gene down-regulation, either within the cells where they were produced or in other cells via intercellular transfer. These microRNAs can be modulated in host cells during infection and are among the growing group of small regulatory RNAs, for which many classes have been described, including the transfer RNA-derived small RNAs. Parasites can either manipulate microRNAs to evade host-driven damage and/or transfer small RNAs to host cells. In this mini-review, we present evidence for the involvement of small RNAs, such as microRNAs, in trypanosomatid infections which lack RNA interference. We highlight both microRNA profile alterations in host cells during those infections and the horizontal transfer of small RNAs and proteins from parasites to the host by membrane-derived extracellular vesicles in a cell communication mechanism. PMID:27065454

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

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    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.

  16. Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs

    DEFF Research Database (Denmark)

    Khan, Aly A; Betel, Doron; Miller, Martin L

    2009-01-01

    Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competiti...

  17. Mammalian small nucleolar RNAs are mobile genetic elements.

    Directory of Open Access Journals (Sweden)

    Michel J Weber

    2006-12-01

    Full Text Available Small nucleolar RNAs (snoRNAs of the H/ACA box and C/D box categories guide the pseudouridylation and the 2'-O-ribose methylation of ribosomal RNAs by forming short duplexes with their target. Similarly, small Cajal body-specific RNAs (scaRNAs guide modifications of spliceosomal RNAs. The vast majority of vertebrate sno/scaRNAs are located in introns of genes transcribed by RNA polymerase II and processed by exonucleolytic trimming after splicing. A bioinformatic search for orthologues of human sno/scaRNAs in sequenced mammalian genomes reveals the presence of species- or lineage-specific sno/scaRNA retroposons (sno/scaRTs characterized by an A-rich tail and an approximately 14-bp target site duplication that corresponds to their insertion site, as determined by interspecific genomic alignments. Three classes of snoRTs are defined based on the extent of intron and exon sequences from the snoRNA parental host gene they contain. SnoRTs frequently insert in gene introns in the sense orientation at genomic hot spots shared with other genetic mobile elements. Previously characterized human snoRNAs are encoded in retroposons whose parental copies can be identified by phylogenic analysis, showing that snoRTs can be faithfully processed. These results identify snoRNAs as a new family of mobile genetic elements. The insertion of new snoRNA copies might constitute a safeguard mechanism by which the biological activity of snoRNAs is maintained in spite of the risk of mutations in the parental copy. I furthermore propose that retroposition followed by genetic drift is a mechanism that increased snoRNA diversity during vertebrate evolution to eventually acquire new RNA-modification functions.

  18. Small RNAs controlling outer membrane porins

    DEFF Research Database (Denmark)

    Valentin-Hansen, Poul; Johansen, Jesper; Rasmussen, Anders A

    2007-01-01

    are key regulators of environmental stress. Recent work has revealed an intimate interplay between small RNA regulation of outer membrane proteins and the stress-induced sigmaE-signalling system, which has an essential role in the maintenance of the integrity of the outer membrane.......Gene regulation by small non-coding RNAs has been recognized as an important post-transcriptional regulatory mechanism for several years. In Gram-negative bacteria such as Escherichia coli and Salmonella, these RNAs control stress response and translation of outer membrane proteins and therefore...

  19. Extracellular small RNAs: what, where, why?

    Science.gov (United States)

    Hoy, Anna M.; Buck, Amy H.

    2012-01-01

    miRNAs (microRNAs) are a class of small RNA that regulate gene expression by binding to mRNAs and modulating the precise amount of proteins that get expressed in a cell at a given time. This form of gene regulation plays an important role in developmental systems and is critical for the proper function of numerous biological pathways. Although miRNAs exert their functions inside the cell, these and other classes of RNA are found in body fluids in a cell-free form that is resistant to degradation by RNases. A broad range of cell types have also been shown to secrete miRNAs in association with components of the RISC (RNA-induced silencing complex) and/or encapsulation within vesicles, which can be taken up by other cells. In the present paper, we provide an overview of the properties of extracellular miRNAs in relation to their capacity as biomarkers, stability against degradation and mediators of cell–cell communication. PMID:22817753

  20. deepBase v2.0: identification, expression, evolution and function of small RNAs, LncRNAs and circular RNAs from deep-sequencing data.

    Science.gov (United States)

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

    2016-01-04

    Small non-coding RNAs (e.g. miRNAs) and long non-coding RNAs (e.g. lincRNAs and circRNAs) are emerging as key regulators of various cellular processes. However, only a very small fraction of these enigmatic RNAs have been well functionally characterized. In this study, we describe deepBase v2.0 (http://biocenter.sysu.edu.cn/deepBase/), an updated platform, to decode evolution, expression patterns and functions of diverse ncRNAs across 19 species. deepBase v2.0 has been updated to provide the most comprehensive collection of ncRNA-derived small RNAs generated from 588 sRNA-Seq datasets. Moreover, we developed a pipeline named lncSeeker to identify 176 680 high-confidence lncRNAs from 14 species. Temporal and spatial expression patterns of various ncRNAs were profiled. We identified approximately 24 280 primate-specific, 5193 rodent-specific lncRNAs, and 55 highly conserved lncRNA orthologs between human and zebrafish. We annotated 14 867 human circRNAs, 1260 of which are orthologous to mouse circRNAs. By combining expression profiles and functional genomic annotations, we developed lncFunction web-server to predict the function of lncRNAs based on protein-lncRNA co-expression networks. This study is expected to provide considerable resources to facilitate future experimental studies and to uncover ncRNA functions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Small non coding RNAs in adipocyte biology and obesity.

    Science.gov (United States)

    Amri, Ez-Zoubir; Scheideler, Marcel

    2017-11-15

    Obesity has reached epidemic proportions world-wide and constitutes a substantial risk factor for hypertension, type 2 diabetes, cardiovascular diseases and certain cancers. So far, regulation of energy intake by dietary and pharmacological treatments has met limited success. The main interest of current research is focused on understanding the role of different pathways involved in adipose tissue function and modulation of its mass. Whole-genome sequencing studies revealed that the majority of the human genome is transcribed, with thousands of non-protein-coding RNAs (ncRNA), which comprise small and long ncRNAs. ncRNAs regulate gene expression at the transcriptional and post-transcriptional level. Numerous studies described the involvement of ncRNAs in the pathogenesis of many diseases including obesity and associated metabolic disorders. ncRNAs represent potential diagnostic biomarkers and promising therapeutic targets. In this review, we focused on small ncRNAs involved in the formation and function of adipocytes and obesity. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Horizontal Transfer of Small RNAs To and From Plants

    Directory of Open Access Journals (Sweden)

    Lu eHan

    2015-12-01

    Full Text Available Genetic information is traditionally thought to be transferred from parents to offspring. However, there is evidence indicating that gene transfer can also occur from microbes to higher species, such as plants, invertebrates and vertebrates. This horizontal transfer can be carried out by small RNAs (sRNAs. sRNAs have been recently reported to move across kingdoms as mobile signals, spreading silencing information toward targeted genes. sRNAs, especially microRNAs (miRNAs and small interfering RNAs (siRNAs, are non-coding molecules that control gene expression at the transcriptional or post-transcriptional level. Some sRNAs act in a cross-kingdom manner between animals and their parasites, but little is known about such sRNAs associated with plants. In this report, we provide a brief introduction to miRNAs that are transferred from plants to mammals/viruses and siRNAs that are transferred from microbes to plants. Both miRNAs and siRNAs can exert corresponding functions in the target organisms. Additionally, we provide information concerning a host-induced gene silencing (HIGS system as a potential application that utilizes the transgenic trafficking of RNA molecules to silence the genes of interacting organisms. Moreover, we lay out the controversial views regarding cross-kingdom miRNAs and call for better methodology and experimental design to confirm this unique function of miRNAs.

  3. Endogenous small RNAs and antibacterial immunity in plants.

    Science.gov (United States)

    Jin, Hailing

    2008-08-06

    Small RNAs are non-coding regulatory RNA molecules that control gene expression by mediating mRNA degradation, translational inhibition, or chromatin modification. Virus-derived small RNAs induce silencing of viral RNAs and are essential for antiviral defense in both animal and plant systems. The role of host endogenous small RNAs on antibacterial immunity has only recently been recognized. Host disease resistance and defense responses are achieved by activation and repression of a large array of genes. Certain endogenous small RNAs in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are induced or repressed in response to pathogen attack and subsequently regulate the expression of genes involved in disease resistance and defense responses by mediating transcriptional or post-transcriptional gene silencing. Thus, these small RNAs play an important role in gene expression reprogramming in plant disease resistance and defense responses. This review focuses on the recent findings of plant endogenous small RNAs in antibacterial immunity.

  4. Small Engine, Big Power: MicroRNAs as Regulators of Cardiac Diseases and Regeneration

    Directory of Open Access Journals (Sweden)

    Darukeshwara Joladarashi

    2014-09-01

    Full Text Available Cardiac diseases are the predominant cause of human mortality in the United States and around the world. MicroRNAs (miRNAs are small non-coding RNAs that have been shown to modulate a wide range of biological functions under various pathophysiological conditions. miRNAs alter target expression by post-transcriptional regulation of gene expression. Numerous studies have implicated specific miRNAs in cardiovascular development, pathology, regeneration and repair. These observations suggest that miRNAs are potential therapeutic targets to prevent or treat cardiovascular diseases. This review focuses on the emerging role of miRNAs in cardiac development, pathogenesis of cardiovascular diseases, cardiac regeneration and stem cell-mediated cardiac repair. We also discuss the novel diagnostic and therapeutic potential of these miRNAs and their targets in patients with cardiac diseases.

  5. Deep sequencing of small RNA libraries from human prostate epithelial and stromal cells reveal distinct pattern of microRNAs primarily predicted to target growth factors.

    Science.gov (United States)

    Singh, Savita; Zheng, Yun; Jagadeeswaran, Guru; Ebron, Jey Sabith; Sikand, Kavleen; Gupta, Sanjay; Sunker, Ramanjulu; Shukla, Girish C

    2016-02-28

    Complex epithelial and stromal cell interactions are required during the development and progression of prostate cancer. Regulatory small non-coding microRNAs (miRNAs) participate in the spatiotemporal regulation of messenger RNA (mRNA) and regulation of translation affecting a large number of genes involved in prostate carcinogenesis. In this study, through deep-sequencing of size fractionated small RNA libraries we profiled the miRNAs of prostate epithelial (PrEC) and stromal (PrSC) cells. Over 50 million reads were obtained for PrEC in which 860,468 were unique sequences. Similarly, nearly 76 million reads for PrSC were obtained in which over 1 million were unique reads. Expression of many miRNAs of broadly conserved and poorly conserved miRNA families were identified. Sixteen highly expressed miRNAs with significant change in expression in PrSC than PrEC were further analyzed in silico. ConsensusPathDB showed the target genes of these miRNAs were significantly involved in adherence junction, cell adhesion, EGRF, TGF-β and androgen signaling. Let-7 family of tumor-suppressor miRNAs expression was highly pervasive in both, PrEC and PrSC cells. In addition, we have also identified several miRNAs that are unique to PrEC or PrSC cells and their predicted putative targets are a group of transcription factors. This study provides perspective on the miRNA expression in PrEC and PrSC, and reveals a global trend in miRNA interactome. We conclude that the most abundant miRNAs are potential regulators of development and differentiation of the prostate gland by targeting a set of growth factors. Additionally, high level expression of the most members of let-7 family miRNAs suggests their role in the fine tuning of the growth and proliferation of prostate epithelial and stromal cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Guardian small RNAs and sex determination.

    Science.gov (United States)

    Katsuma, Susumu; Kawamoto, Munetaka; Kiuchi, Takashi

    2014-01-01

    The W chromosome of the silkworm Bombyx mori has been known to determine femaleness for more than 80 years. However, the feminizing gene has not been molecularly identified, because the B. mori W chromosome is almost fully occupied by a large number of transposable elements. The W chromosome-derived feminizing factor of B. mori was recently shown to be a female-specific PIWI-interacting RNA (piRNA). piRNAs are small RNAs that potentially repress invading "non-self" elements (e.g., transposons and virus-like elements) by associating with PIWI proteins. Our results revealed that female-specific piRNA precursors, which we named Fem, are transcribed from the sex-determining region of the W chromosome at the early embryonic stage and are processed into a single mature piRNA (Fem piRNA). Fem piRNA forms a complex with Siwi (silkworm Piwi), which cleaves a protein-coding mRNA transcribed from the Z chromosome. RNA interference of this Z-linked gene, which we named Masc, revealed that this gene encodes a protein required for masculinization and dosage compensation. Fem and Masc both participate in the ping-pong cycle of the piRNA amplification loop by associating with the 2 B. mori PIWI proteins Siwi and BmAgo3 (silkworm Ago3), respectively, indicating that the piRNA-mediated interaction between the 2 sex chromosomes is the primary signal for the B. mori sex determination cascade. Fem is a non-transposable repetitive sequence on the W chromosome, whereas Masc is a single-copy protein-coding gene. It is of great interest how the piRNA system recognizes "self "Masc mRNA as "non-self" RNA.

  7. A role for small RNAs in DNA double-strand break repair

    DEFF Research Database (Denmark)

    Wei, W.; Ba, Z.; Wu, Y.

    2012-01-01

    Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ∼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells....... We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, di...

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

    NARCIS (Netherlands)

    Miesen, P.; Ivens, A.; Buck, A.H.; Rij, R.P. van

    2016-01-01

    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

  9. Dynamic evolution and biogenesis of small RNAs during sex reversal

    OpenAIRE

    Liu, Jie; Luo, Majing; Sheng, Yue; Hong, Qiang; Cheng, Hanhua; Zhou, Rongjia

    2015-01-01

    Understanding origin, evolution and functions of small RNA (sRNA) genes has been a great challenge in the past decade. Molecular mechanisms underlying sexual reversal in vertebrates, particularly sRNAs involved in this process, are largely unknown. By deep-sequencing of small RNA transcriptomes in combination with genomic analysis, we identified a large amount of piRNAs and miRNAs including over 1,000 novel miRNAs, which were differentially expressed during gonad reversal from ovary to testis...

  10. Dynamic evolution and biogenesis of small RNAs during sex reversal.

    Science.gov (United States)

    Liu, Jie; Luo, Majing; Sheng, Yue; Hong, Qiang; Cheng, Hanhua; Zhou, Rongjia

    2015-05-06

    Understanding origin, evolution and functions of small RNA (sRNA) genes has been a great challenge in the past decade. Molecular mechanisms underlying sexual reversal in vertebrates, particularly sRNAs involved in this process, are largely unknown. By deep-sequencing of small RNA transcriptomes in combination with genomic analysis, we identified a large amount of piRNAs and miRNAs including over 1,000 novel miRNAs, which were differentially expressed during gonad reversal from ovary to testis via ovotesis. Biogenesis and expressions of miRNAs were dynamically changed during the reversal. Notably, phylogenetic analysis revealed dynamic expansions of miRNAs in vertebrates and an evolutionary trajectory of conserved miR-17-92 cluster in the Eukarya. We showed that the miR-17-92 cluster in vertebrates was generated through multiple duplications from ancestor miR-92 in invertebrates Tetranychus urticae and Daphnia pulex from the Chelicerata around 580 Mya. Moreover, we identified the sexual regulator Dmrt1 as a direct target of the members miR-19a and -19b in the cluster. These data suggested dynamic biogenesis and expressions of small RNAs during sex reversal and revealed multiple expansions and evolutionary trajectory of miRNAs from invertebrates to vertebrates, which implicate small RNAs in sexual reversal and provide new insight into evolutionary and molecular mechanisms underlying sexual reversal.

  11. Wheat hybridization and polyploidization results in deregulation of small RNAs.

    Science.gov (United States)

    Kenan-Eichler, Michal; Leshkowitz, Dena; Tal, Lior; Noor, Elad; Melamed-Bessudo, Cathy; Feldman, Moshe; Levy, Avraham A

    2011-06-01

    Speciation via interspecific or intergeneric hybridization and polyploidization triggers genomic responses involving genetic and epigenetic alterations. Such modifications may be induced by small RNAs, which affect key cellular processes, including gene expression, chromatin structure, cytosine methylation and transposable element (TE) activity. To date, the role of small RNAs in the context of wide hybridization and polyploidization has received little attention. In this work, we performed high-throughput sequencing of small RNAs of parental, intergeneric hybrid, and allopolyploid plants that mimic the genomic changes occurring during bread wheat speciation. We found that the percentage of small RNAs corresponding to miRNAs increased with ploidy level, while the percentage of siRNAs corresponding to TEs decreased. The abundance of most miRNA species was similar to midparent values in the hybrid, with some deviations, as seen in overrepresentation of miR168, in the allopolyploid. In contrast, the number of siRNAs corresponding to TEs strongly decreased upon allopolyploidization, but not upon hybridization. The reduction in corresponding siRNAs, together with decreased CpG methylation, as shown here for the Veju element, represent hallmarks of TE activation. TE-siRNA downregulation in the allopolyploid may contribute to genome destabilization at the initial stages of speciation. This phenomenon is reminiscent of hybrid dysgenesis in Drosophila.

  12. 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....

  13. Integration of Bacterial Small RNAs in Regulatory Networks.

    Science.gov (United States)

    Nitzan, Mor; Rehani, Rotem; Margalit, Hanah

    2017-05-22

    Small RNAs (sRNAs) are central regulators of gene expression in bacteria, controlling target genes posttranscriptionally by base pairing with their mRNAs. sRNAs are involved in many cellular processes and have unique regulatory characteristics. In this review, we discuss the properties of regulation by sRNAs and how it differs from and combines with transcriptional regulation. We describe the global characteristics of the sRNA-target networks in bacteria using graph-theoretic approaches and review the local integration of sRNAs in mixed regulatory circuits, including feed-forward loops and their combinations, feedback loops, and circuits made of an sRNA and another regulator, both derived from the same transcript. Finally, we discuss the competition effects in posttranscriptional regulatory networks that may arise over shared targets, shared regulators, and shared resources and how they may lead to signal propagation across the network.

  14. 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.

  15. Small regulatory RNAs may sharpen spatial expression patterns.

    Directory of Open Access Journals (Sweden)

    Erel Levine

    2007-11-01

    Full Text Available The precise establishment of gene expression patterns is a crucial step in development. Formation of a sharp boundary between high and low spatial expression domains requires a genetic mechanism that exhibits sensitivity, yet is robust to fluctuations, a demand that may not be easily achieved by morphogens alone. Recently, it has been demonstrated that small RNAs (and, in particular, microRNAs play many roles in embryonic development. Whereas some RNAs are essential for embryogenesis, others are limited to fine-tuning a predetermined gene expression pattern. Here, we explore the possibility that small RNAs participate in sharpening a gene expression profile that was crudely established by a morphogen. To this end, we study a model in which small RNAs interact with a target gene and diffusively move from cell to cell. Though diffusion generally smoothens spatial expression patterns, we find that intercellular mobility of small RNAs is actually critical in sharpening the interface between target expression domains in a robust manner. This sharpening occurs as small RNAs diffuse into regions of low mRNA expression and eliminate target molecules therein, but cannot affect regions of high mRNA levels. We discuss the applicability of our results, as examples, to the case of leaf polarity establishment in maize and Hox patterning in the early Drosophila embryo. Our findings point out the functional significance of some mechanistic properties, such as mobility of small RNAs and the irreversibility of their interactions. These properties are yet to be established directly for most classes of small RNAs. An indirect yet simple experimental test of the proposed mechanism is suggested in some detail.

  16. Identification and validation of human papillomavirus encoded microRNAs.

    Directory of Open Access Journals (Sweden)

    Kui Qian

    Full Text Available We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.

  17. miRNAs: Small but deadly

    African Journals Online (AJOL)

    Jane

    2011-08-24

    Aug 24, 2011 ... Levels of some miRNAs are found altered in cancers, so we might expect these regulatory ..... males is the prostate cancer (PCa) (Jemal et al., 2008). ..... 1 growth factor receptor family members HER-1, HER-2, and HER-3.

  18. Plant Responses to Pathogen Attack: Small RNAs in Focus.

    Science.gov (United States)

    Islam, Waqar; Noman, Ali; Qasim, Muhammad; Wang, Liande

    2018-02-08

    Small RNAs (sRNA) are a significant group of gene expression regulators for multiple biological processes in eukaryotes. In plants, many sRNA silencing pathways produce extensive array of sRNAs with specialized roles. The evidence on record advocates for the functions of sRNAs during plant microbe interactions. Host sRNAs are reckoned as mandatory elements of plant defense. sRNAs involved in plant defense processes via different pathways include both short interfering RNA (siRNA) and microRNA (miRNA) that actively regulate immunity in response to pathogenic attack via tackling pathogen-associated molecular patterns (PAMPs) and other effectors. In response to pathogen attack, plants protect themselves with the help of sRNA-dependent immune systems. That sRNA-mediated plant defense responses play a role during infections is an established fact. However, the regulations of several sRNAs still need extensive research. In this review, we discussed the topical advancements and findings relevant to pathogen attack and plant defense mediated by sRNAs. We attempted to point out diverse sRNAs as key defenders in plant systems. It is hoped that sRNAs would be exploited as a mainstream player to achieve food security by tackling different plant diseases.

  19. Diversity of small RNAs expressed in Pseudomonas species

    DEFF Research Database (Denmark)

    Gomez-Lozano, Mara; Marvig, Rasmus Lykke; Molina-Santiago, Carlos

    2015-01-01

    RNA sequencing (RNA-seq) has revealed several hundreds of previously undetected small RNAs (sRNAs) in all bacterial species investigated, including strains of Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas syringae. Nonetheless, only little is known about the extent of conservation...... of expressed sRNAs across strains and species. In this study, we have used RNA-seq to identify sRNAs in P.putidaDOT-T1E and Pseudomonas extremaustralis 14-3b. This is the first strain of P.extremaustralis and the second strain of P.putida to have their transcriptomes analysed for sRNAs, and we identify...... the presence of around 150 novel sRNAs in each strain. Furthermore, we provide a comparison based on sequence conservation of all the sRNAs detected by RNA-seq in the Pseudomonas species investigated so far. Our results show that the extent of sRNA conservation across different species is very limited...

  20. Molecular Basis for the Immunostimulatory Potency of Small Interfering RNAs

    Directory of Open Access Journals (Sweden)

    Mouldy Sioud

    2006-01-01

    Full Text Available Small interfering RNAs (siRNAs represent a new class of antigene agents, which has emerged as a powerful tool for functional genomics and might serve as a potent therapeutic approach. However, several studies have showed that they could trigger several bystander effects, including immune activation and inhibition of unintended target genes. Although activation of innate immunity by siRNAs might be beneficial for therapy in some instances, uncontrolled activation can be toxic, and is therefore a major challenging problem. Interestingly, replacement of uridines in siRNA sequences with their 2′-modified counterparts abrogated siRNA bystander effects. Here we highlight these important findings that are expected to facilitate the rational design of siRNAs that avoid the induction of bystander effects.

  1. RISC assembly: Coordination between small RNAs and Argonaute proteins.

    Science.gov (United States)

    Kobayashi, Hotaka; Tomari, Yukihide

    2016-01-01

    Non-coding RNAs generally form ribonucleoprotein (RNP) complexes with their partner proteins to exert their functions. Small RNAs, including microRNAs, small interfering RNAs, and PIWI-interacting RNAs, assemble with Argonaute (Ago) family proteins into the effector complex called RNA-induced silencing complex (RISC), which mediates sequence-specific target gene silencing. RISC assembly is not a simple binding between a small RNA and Ago; rather, it follows an ordered multi-step pathway that requires specific accessory factors. Some steps of RISC assembly and RISC-mediated gene silencing are dependent on or facilitated by particular intracellular platforms, suggesting their spatial regulation. In this review, we summarize the currently known mechanisms for RISC assembly of each small RNA class and propose a revised model for the role of the chaperone machinery in the duplex-initiated RISC assembly pathway. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. A comparative study of sequence- and structure-based features of small RNAs and other RNAs of bacteria.

    Science.gov (United States)

    Barik, Amita; Das, Santasabuj

    2018-01-02

    Small RNAs (sRNAs) in bacteria have emerged as key players in transcriptional and post-transcriptional regulation of gene expression. Here, we present a statistical analysis of different sequence- and structure-related features of bacterial sRNAs to identify the descriptors that could discriminate sRNAs from other bacterial RNAs. We investigated a comprehensive and heterogeneous collection of 816 sRNAs, identified by northern blotting across 33 bacterial species and compared their various features with other classes of bacterial RNAs, such as tRNAs, rRNAs and mRNAs. We observed that sRNAs differed significantly from the rest with respect to G+C composition, normalized minimum free energy of folding, motif frequency and several RNA-folding parameters like base-pairing propensity, Shannon entropy and base-pair distance. Based on the selected features, we developed a predictive model using Random Forests (RF) method to classify the above four classes of RNAs. Our model displayed an overall predictive accuracy of 89.5%. These findings would help to differentiate bacterial sRNAs from other RNAs and further promote prediction of novel sRNAs in different bacterial species.

  3. 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

  4. A comparative study of small RNAs in Toxoplasma gondii of distinct genotypes

    Directory of Open Access Journals (Sweden)

    Wang Jielin

    2012-09-01

    Full Text Available Abstract Background Toxoplasma gondii is an intracellular parasite with a significant impact on human health. Inside the mammalian and avian hosts, the parasite can undergo rapid development or remain inactive in the cysts. The mechanism that regulates parasite proliferation has not been fully understood. Small noncoding RNAs (sncRNA such as microRNAs (miRNAs are endogenous regulatory factors that can modulate cell differentiation and development. It is anticipated that hundreds of miRNAs regulate the expression of thousands of genes in a single organism. SncRNAs have been identified in T. gondii, however the profiles of sncRNAs expression and their potential regulatory function in parasites of distinct genotypes has largely been unknown. Methods The transcription profiles of miRNAs in the two genetically distinct strains, RH and ME49, of T. gondii were investigated and compared by a high-through-put RNA sequencing technique and systematic bioinformatics analysis. The expression of some of the miRNAs was confirmed by Northern blot analysis. Results 1,083,320 unique sequences were obtained. Of which, 17 conserved miRNAs related to 2 metazoan miRNA families and 339 novel miRNAs were identified. A total of 175 miRNAs showed strain-specific expression, of which 155 miRNAs were up-regulated in RH strain and 20 miRNAs were up-regulated in ME49 strain. Strain-specific expression of miRNAs in T. gondii could be due to activation of specific genes at different genomic loci or due to arm-switching of the same pre-miRNA duplex. Conclusions Evidence for the differential expression of miRNAs in the two genetically distinct strains of T. gondii has been identified and defined. MiRNAs of T. gondii are more species-specific as compared to other organisms, which can be developed as diagnostic biomarkers for toxoplasmosis. The data also provide a framework for future studies on RNAi-dependent regulatory mechanisms in the zoonotic parasite.

  5. Deep sequencing of Brachypodium small RNAs at the global genome level identifies microRNAs involved in cold stress response

    Directory of Open Access Journals (Sweden)

    Chong Kang

    2009-09-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are endogenous small RNAs having large-scale regulatory effects on plant development and stress responses. Extensive studies of miRNAs have only been performed in a few model plants. Although miRNAs are proved to be involved in plant cold stress responses, little is known for winter-habit monocots. Brachypodium distachyon, with close evolutionary relationship to cool-season cereals, has recently emerged as a novel model plant. There are few reports of Brachypodium miRNAs. Results High-throughput sequencing and whole-genome-wide data mining led to the identification of 27 conserved miRNAs, as well as 129 predicted miRNAs in Brachypodium. For multiple-member conserved miRNA families, their sizes in Brachypodium were much smaller than those in rice and Populus. The genome organization of miR395 family in Brachypodium was quite different from that in rice. The expression of 3 conserved miRNAs and 25 predicted miRNAs showed significant changes in response to cold stress. Among these miRNAs, some were cold-induced and some were cold-suppressed, but all the conserved miRNAs were up-regulated under cold stress condition. Conclusion Our results suggest that Brachypodium miRNAs are composed of a set of conserved miRNAs and a large proportion of non-conserved miRNAs with low expression levels. Both kinds of miRNAs were involved in cold stress response, but all the conserved miRNAs were up-regulated, implying an important role for cold-induced miRNAs. The different size and genome organization of miRNA families in Brachypodium and rice suggest that the frequency of duplication events or the selection pressure on duplicated miRNAs are different between these two closely related plant species.

  6. Highly Complementary Target RNAs Promote Release of Guide RNAs from Human Argonaute2

    Science.gov (United States)

    De, Nabanita; Young, Lisa; Lau, Pick-Wei; Meisner, Nicole-Claudia; Morrissey, David V.; MacRae, Ian J.

    2013-01-01

    SUMMARY Argonaute proteins use small RNAs to guide the silencing of complementary target RNAs in many eukaryotes. Although small RNA biogenesis pathways are well studied, mechanisms for removal of guide RNAs from Argonaute are poorly understood. Here we show that the Argonaute2 (Ago2) guide RNA complex is extremely stable, with a half-life on the order of days. However, highly complementary target RNAs destabilize the complex and significantly accelerate release of the guide RNA from Ago2. This “unloading” activity can be enhanced by mismatches between the target and the guide 5′ end and attenuated by mismatches to the guide 3′ end. The introduction of 3′ mismatches leads to more potent silencing of abundant mRNAs in mammalian cells. These findings help to explain why the 3′ ends of mammalian microRNAs (miRNAs) rarely match their targets, suggest a mechanism for sequence-specific small RNA turnover, and offer insights for controlling small RNAs in mammalian cells. PMID:23664376

  7. MicroRNAs in Human Placental Development and Pregnancy Complications

    Directory of Open Access Journals (Sweden)

    Chun Peng

    2013-03-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs, which function as critical posttranscriptional regulators of gene expression by promoting mRNA degradation and translational inhibition. Placenta expresses many ubiquitous as well as specific miRNAs. These miRNAs regulate trophoblast cell differentiation, proliferation, apoptosis, invasion/migration, and angiogenesis, suggesting that miRNAs play important roles during placental development. Aberrant miRNAs expression has been linked to pregnancy complications, such as preeclampsia. Recent research of placental miRNAs focuses on identifying placental miRNA species, examining differential expression of miRNAs between placentas from normal and compromised pregnancies, and uncovering the function of miRNAs in the placenta. More studies are required to further understand the functional significance of miRNAs in placental development and to explore the possibility of using miRNAs as biomarkers and therapeutic targets for pregnancy-related disorders. In this paper, we reviewed the current knowledge about the expression and function of miRNAs in placental development, and propose future directions for miRNA studies.

  8. Identifying and annotating human bifunctional RNAs reveals their versatile functions.

    Science.gov (United States)

    Chen, Geng; Yang, Juan; Chen, Jiwei; Song, Yunjie; Cao, Ruifang; Shi, Tieliu; Shi, Leming

    2016-10-01

    Bifunctional RNAs that possess both protein-coding and noncoding functional properties were less explored and poorly understood. Here we systematically explored the characteristics and functions of such human bifunctional RNAs by integrating tandem mass spectrometry and RNA-seq data. We first constructed a pipeline to identify and annotate bifunctional RNAs, leading to the characterization of 132 high-confidence bifunctional RNAs. Our analyses indicate that bifunctional RNAs may be involved in human embryonic development and can be functional in diverse tissues. Moreover, bifunctional RNAs could interact with multiple miRNAs and RNA-binding proteins to exert their corresponding roles. Bifunctional RNAs may also function as competing endogenous RNAs to regulate the expression of many genes by competing for common targeting miRNAs. Finally, somatic mutations of diverse carcinomas may generate harmful effect on corresponding bifunctional RNAs. Collectively, our study not only provides the pipeline for identifying and annotating bifunctional RNAs but also reveals their important gene-regulatory functions.

  9. Characterizing ncRNAs in Human Pathogenic Protists Using High-Throughput Sequencing Technology

    Science.gov (United States)

    Collins, Lesley Joan

    2011-01-01

    ncRNAs are key genes in many human diseases including cancer and viral infection, as well as providing critical functions in pathogenic organisms such as fungi, bacteria, viruses, and protists. Until now the identification and characterization of ncRNAs associated with disease has been slow or inaccurate requiring many years of testing to understand complicated RNA and protein gene relationships. High-throughput sequencing now offers the opportunity to characterize miRNAs, siRNAs, small nucleolar RNAs (snoRNAs), and long ncRNAs on a genomic scale, making it faster and easier to clarify how these ncRNAs contribute to the disease state. However, this technology is still relatively new, and ncRNA discovery is not an application of high priority for streamlined bioinformatics. Here we summarize background concepts and practical approaches for ncRNA analysis using high-throughput sequencing, and how it relates to understanding human disease. As a case study, we focus on the parasitic protists Giardia lamblia and Trichomonas vaginalis, where large evolutionary distance has meant difficulties in comparing ncRNAs with those from model eukaryotes. A combination of biological, computational, and sequencing approaches has enabled easier classification of ncRNA classes such as snoRNAs, but has also aided the identification of novel classes. It is hoped that a higher level of understanding of ncRNA expression and interaction may aid in the development of less harsh treatment for protist-based diseases. PMID:22303390

  10. Phloem small RNAs, nutrient stress responses, and systemic mobility

    Directory of Open Access Journals (Sweden)

    Kehr Julia

    2010-04-01

    Full Text Available Abstract Background Nutrient availabilities and needs have to be tightly coordinated between organs to ensure a balance between uptake and consumption for metabolism, growth, and defense reactions. Since plants often have to grow in environments with sub-optimal nutrient availability, a fine tuning is vital. To achieve this, information has to flow cell-to-cell and over long-distance via xylem and phloem. Recently, specific miRNAs emerged as a new type of regulating molecules during stress and nutrient deficiency responses, and miR399 was suggested to be a phloem-mobile long-distance signal involved in the phosphate starvation response. Results We used miRNA microarrays containing all known plant miRNAs and a set of unknown small (s RNAs earlier cloned from Brassica phloem sap 1, to comprehensively analyze the phloem response to nutrient deficiency by removing sulfate, copper or iron, respectively, from the growth medium. We show that phloem sap contains a specific set of sRNAs that is distinct from leaves and roots, and that the phloem also responds specifically to stress. Upon S and Cu deficiencies phloem sap reacts with an increase of the same miRNAs that were earlier characterized in other tissues, while no clear positive response to -Fe was observed. However, -Fe led to a reduction of Cu- and P-responsive miRNAs. We further demonstrate that under nutrient starvation miR399 and miR395 can be translocated through graft unions from wild type scions to rootstocks of the miRNA processing hen1-1 mutant. In contrast, miR171 was not transported. Translocation of miR395 led to a down-regulation of one of its targets in rootstocks, suggesting that this transport is of functional relevance, and that miR395, in addition to the well characterized miR399, could potentially act as a long-distance information transmitter. Conclusions Phloem sap contains a specific set of sRNAs, of which some specifically accumulate in response to nutrient deprivation. From

  11. Psmir: a database of potential associations between small molecules and miRNAs.

    Science.gov (United States)

    Meng, Fanlin; Wang, Jing; Dai, Enyu; Yang, Feng; Chen, Xiaowen; Wang, Shuyuan; Yu, Xuexin; Liu, Dianming; Jiang, Wei

    2016-01-13

    miRNAs are key post-transcriptional regulators of many essential biological processes, and their dysregulation has been validated in almost all human cancers. Restoring aberrantly expressed miRNAs might be a novel therapeutics. Recently, many studies have demonstrated that small molecular compounds can affect miRNA expression. Thus, prediction of associations between small molecules and miRNAs is important for investigation of miRNA-targeted drugs. Here, we analyzed 39 miRNA-perturbed gene expression profiles, and then calculated the similarity of transcription responses between miRNA perturbation and drug treatment to predict drug-miRNA associations. At the significance level of 0.05, we obtained 6501 candidate associations between 1295 small molecules and 25 miRNAs, which included 624 FDA approved drugs. Finally, we constructed the Psmir database to store all potential associations and the related materials. In a word, Psmir served as a valuable resource for dissecting the biological significance in small molecules' effects on miRNA expression, which will facilitate developing novel potential therapeutic targets or treatments for human cancers. Psmir is supported by all major browsers, and is freely available at http://www.bio-bigdata.com/Psmir/.

  12. Identification of Small RNAs in Desulfovibrio vulgaris Hildenborough

    International Nuclear Information System (INIS)

    Burns, Andrew; Joachimiak, Marcin; Deutschbauer, Adam; Arkin, Adam; Bender, Kelly

    2010-01-01

    Desulfovibrio vulgaris is an anaerobic sulfate-reducing bacterium capable of facilitating the removal of toxic metals such as uranium from contaminated sites via reduction. As such, it is essential to understand the intricate regulatory cascades involved in how D. vulgaris and its relatives respond to stressors in such sites. One approach is the identification and analysis of small non-coding RNAs (sRNAs); molecules ranging in size from 20-200 nucleotides that predominantly affect gene regulation by binding to complementary mRNA in an anti-sense fashion and therefore provide an immediate regulatory response. To identify sRNAs in D. vulgaris, a bacterium that does not possess an annotated hfq gene, RNA was pooled from stationary and exponential phases, nitrate exposure, and biofilm conditions. The subsequent RNA was size fractionated, modified, and converted to cDNA for high throughput transcriptomic deep sequencing. A computational approach to identify sRNAs via the alignment of seven separate Desulfovibrio genomes was also performed. From the deep sequencing analysis, 2,296 reads between 20 and 250 nt were identified with expression above genome background. Analysis of those reads limited the number of candidates to ∼87 intergenic, while ∼140 appeared to be antisense to annotated open reading frames (ORFs). Further BLAST analysis of the intergenic candidates and other Desulfovibrio genomes indicated that eight candidates were likely portions of ORFs not previously annotated in the D. vulgaris genome. Comparison of the intergenic and antisense data sets to the bioinformatical predicted candidates, resulted in ∼54 common candidates. Current approaches using Northern analysis and qRT-PCR are being used toverify expression of the candidates and to further develop the role these sRNAs play in D. vulgaris regulation.

  13. Dynamic localisation of mature microRNAs in Human nucleoli is influenced by exogenous genetic materials.

    Science.gov (United States)

    Li, Zhou Fang; Liang, Yi Min; Lau, Pui Ngan; Shen, Wei; Wang, Dai Kui; Cheung, Wing Tai; Xue, Chun Jason; Poon, Lit Man; Lam, Yun Wah

    2013-01-01

    Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials.

  14. Dynamic localisation of mature microRNAs in Human nucleoli is influenced by exogenous genetic materials.

    Directory of Open Access Journals (Sweden)

    Zhou Fang Li

    Full Text Available Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials.

  15. Insights into the regulation of human CNV-miRNAs from the view of their target genes

    Directory of Open Access Journals (Sweden)

    Wu Xudong

    2012-12-01

    Full Text Available Abstract Background microRNAs (miRNAs represent a class of small (typically 22 nucleotides in length non-coding RNAs that can degrade their target mRNAs or block their translation. Recent research showed that copy number alterations of miRNAs and their target genes are highly prevalent in cancers; however, the evolutionary and biological functions of naturally existing copy number variable miRNAs (CNV-miRNAs among individuals have not been studied extensively throughout the genome. Results In this study, we comprehensively analyzed the properties of genes regulated by CNV-miRNAs, and found that CNV-miRNAs tend to target a higher average number of genes and prefer to synergistically regulate the same genes; further, the targets of CNV-miRNAs tend to have higher variability of expression within and between populations. Finally, we found the targets of CNV-miRNAs are more likely to be differentially expressed among tissues and developmental stages, and participate in a wide range of cellular responses. Conclusions Our analyses of CNV-miRNAs provide new insights into the impact of copy number variations on miRNA-mediated post-transcriptional networks. The deeper interpretation of patterns of gene expression variation and the functional characterization of CNV-miRNAs will help to broaden the current understanding of the molecular basis of human phenotypic diversity.

  16. Small RNAs in plants: Recent development and application for crop improvement

    Directory of Open Access Journals (Sweden)

    Ayushi eKamthan

    2015-04-01

    Full Text Available The phenomenon of RNA interference (RNAi which involves sequence specific gene regulation by small non-coding RNAs i.e small interfering RNA (siRNA and micro RNA (miRNA has emerged as one of most powerful approaches for crop improvement. RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species. This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits. RNAi has been used for enhancing the crop yield and productivity by manipulating the gene involved in biomass, grain yield and enhanced shelf life of fruits & vegetables. It has also been applied for developing resistance against various biotic (bacteria, fungi, viruses, nematodes, insects and abiotic stresses (drought, salinity, cold etc.. Nutritional improvements of crops have also been achieved by enriching the crops with essential amino acids, fatty acids, antioxidants and other nutrients beneficial for human health or by reducing allergens or anti-nutrients. Micro RNAs are key regulators of important plant processes like growth, development and response to various stresses. In spite of similarity in size (20-24nt, miRNA differ from siRNA in precursor structures, pathway of biogenesis, and modes of action. This review also highlights the miRNA based genetic modification technology where various miRNAs/artificial miRNAs and their targets can be utilized for improving several desirable plant traits. Micro RNA based strategies are much efficient than siRNA-based RNAi strategies due to its specificity and less undesirable off target effects. As per the FDA guidelines, small RNA based transgenics are much safer for consumption than those over expressing proteins. This review thereby summarizes the emerging advances and achievement in the field of small RNAs and its application for crop improvement.

  17. 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.

  18. 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.

  19. Emerging Roles of Small Epstein-Barr Virus Derived Non-Coding RNAs in Epithelial Malignancy

    Science.gov (United States)

    Lung, Raymond Wai-Ming; Tong, Joanna Hung-Man; To, Ka-Fai

    2013-01-01

    Latent Epstein-Barr virus (EBV) infection is an etiological factor in the progression of several human epithelial malignancies such as nasopharyngeal carcinoma (NPC) and a subset of gastric carcinoma. Reports have shown that EBV produces several viral oncoproteins, yet their pathological roles in carcinogenesis are not fully elucidated. Studies on the recently discovered of EBV-encoded microRNAs (ebv-miRNAs) showed that these small molecules function as post-transcriptional gene regulators and may play a role in the carcinogenesis process. In NPC and EBV positive gastric carcinoma (EBVaGC), 22 viral miRNAs which are located in the long alternative splicing EBV transcripts, named BamH1 A rightward transcripts (BARTs), are abundantly expressed. The importance of several miR-BARTs in carcinogenesis has recently been demonstrated. These novel findings enhance our understanding of the oncogenic properties of EBV and may lead to a more effective design of therapeutic regimens to combat EBV-associated malignancies. This article will review the pathological roles of miR-BARTs in modulating the expression of cancer-related genes in both host and viral genomes. The expression of other small non-coding RNAs in NPC and the expression pattern of miR-BARTs in rare EBV-associated epithelial cancers will also be discussed. PMID:23979421

  20. Small RNAs in plants: recent development and application for crop improvement.

    Science.gov (United States)

    Kamthan, Ayushi; Chaudhuri, Abira; Kamthan, Mohan; Datta, Asis

    2015-01-01

    The phenomenon of RNA interference (RNAi) which involves sequence-specific gene regulation by small non-coding RNAs, i.e., small interfering RNA (siRNA) and microRNA (miRNA) has emerged as one of most powerful approaches for crop improvement. RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species. This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits. RNAi has been used for enhancing the crop yield and productivity by manipulating the gene involved in biomass, grain yield and enhanced shelf life of fruits and vegetables. It has also been applied for developing resistance against various biotic (bacteria, fungi, viruses, nematodes, insects) and abiotic stresses (drought, salinity, cold, etc.). Nutritional improvements of crops have also been achieved by enriching the crops with essential amino acids, fatty acids, antioxidants and other nutrients beneficial for human health or by reducing allergens or anti-nutrients. microRNAs are key regulators of important plant processes like growth, development, and response to various stresses. In spite of similarity in size (20-24 nt), miRNA differ from siRNA in precursor structures, pathway of biogenesis, and modes of action. This review also highlights the miRNA based genetic modification technology where various miRNAs/artificial miRNAs and their targets can be utilized for improving several desirable plant traits. microRNA based strategies are much efficient than siRNA-based RNAi strategies due to its specificity and less undesirable off target effects. As per the FDA guidelines, small RNA (sRNA) based transgenics are much safer for consumption than those over-expressing proteins. This review thereby summarizes the emerging advances and achievement in the field of sRNAs and its application for crop improvement.

  1. Isolation and characterization of the genes for two small RNAs of herpesvirus papio and their comparison with Epstein-Barr virus-encoded EBER RNAs.

    Science.gov (United States)

    Howe, J G; Shu, M D

    1988-08-01

    Genes for the Epstein-Barr virus-encoded RNAs (EBERs), two low-molecular-weight RNAs encoded by the human gammaherpesvirus Epstein-Barr virus (EBV), hybridize to two small RNAs in a baboon cell line that contains a similar virus, herpesvirus papio (HVP). The genes for the HVP RNAs (HVP-1 and HVP-2) are located together in the small unique region at the left end of the viral genome and are transcribed by RNA polymerase III in a rightward direction, similar to the EBERs. There is significant similarity between EBER1 and HVP-1 RNA, except for an insert of 22 nucleotides which increases the length of HVP-1 RNA to 190 nucleotides. There is less similarity between the sequences of EBER2 and HVP-2 RNA, but both have a length of about 170 nucleotides. The predicted secondary structure of each HVP RNA is remarkably similar to that of the respective EBER, implying that the secondary structures are important for function. Upstream from the initiation sites of all four RNA genes are several highly conserved sequences which may function in the regulation of transcription. The HVP RNAs, together with the EBERs, are highly abundant in transformed cells and are efficiently bound by the cellular La protein.

  2. Detecting new microRNAs in human osteoarthritic chondrocytes identifies miR-3085 as a human, chondrocyte-selective, microRNA

    OpenAIRE

    Crowe, N.; Swingler, T.E.; Le, L.T.T.; Barter, M.J.; Wheeler, G.; Pais, H.; Donell, S.T.; Young, D.A.; Dalmay, T.; Clark, I.M.

    2016-01-01

    Summary Objective To use deep sequencing to identify novel microRNAs (miRNAs) in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function. Design A small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate miRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray...

  3. Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells

    International Nuclear Information System (INIS)

    Su, Ming-Wei; Yu, Sung-Liang; Lin, Wen-Chang; Tsai, Ching-Hui; Chen, Po-Hua; Lee, Yungling Leo

    2016-01-01

    Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressed genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke. - Highlights: • We conducted a smoke reduction trial program and investigated the causal relationship between smoke and gene regulation. • MicroRNA and mRNA expression changes were examined in human PBMC. • MicroRNAs are important in regulating disease-causal genes after tobacco smoke reduction.

  4. Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells

    Energy Technology Data Exchange (ETDEWEB)

    Su, Ming-Wei [Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan (China); Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (China); Yu, Sung-Liang [Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Lin, Wen-Chang [Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (China); Tsai, Ching-Hui [Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan (China); Chen, Po-Hua [School of Medicine, National Taiwan University, Taipei, Taiwan (China); Lee, Yungling Leo, E-mail: leolee@ntu.edu.tw [Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan (China); Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (China)

    2016-08-15

    Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressed genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke. - Highlights: • We conducted a smoke reduction trial program and investigated the causal relationship between smoke and gene regulation. • MicroRNA and mRNA expression changes were examined in human PBMC. • MicroRNAs are important in regulating disease-causal genes after tobacco smoke reduction.

  5. Network analysis of microRNAs and their regulation in human ovarian cancer

    KAUST Repository

    Schmeier, Sebastian

    2011-11-03

    Background: MicroRNAs (miRNAs) are small non-coding RNA molecules that repress the translation of messenger RNAs (mRNAs) or degrade mRNAs. These functions of miRNAs allow them to control key cellular processes such as development, differentiation and apoptosis, and they have also been implicated in several cancers such as leukaemia, lung, pancreatic and ovarian cancer (OC). Unfortunately, the specific machinery of miRNA regulation, involving transcription factors (TFs) and transcription co-factors (TcoFs), is not well understood. In the present study we focus on computationally deciphering the underlying network of miRNAs, their targets, and their control mechanisms that have an influence on OC development.Results: We analysed experimentally verified data from multiple sources that describe miRNA influence on diseases, miRNA targeting of mRNAs, and on protein-protein interactions, and combined this data with ab initio transcription factor binding site predictions within miRNA promoter regions. From these analyses, we derived a network that describes the influence of miRNAs and their regulation in human OC. We developed a methodology to analyse the network in order to find the nodes that have the largest potential of influencing the network\\'s behaviour (network hubs). We further show the potentially most influential miRNAs, TFs and TcoFs, showing subnetworks illustrating the involved mechanisms as well as regulatory miRNA network motifs in OC. We find an enrichment of miRNA targeted OC genes in the highly relevant pathways cell cycle regulation and apoptosis.Conclusions: We combined several sources of interaction and association data to analyse and place miRNAs within regulatory pathways that influence human OC. These results represent the first comprehensive miRNA regulatory network analysis for human OC. This suggests that miRNAs and their regulation may play a major role in OC and that further directed research in this area is of utmost importance to enhance

  6. Network analysis of microRNAs and their regulation in human ovarian cancer

    KAUST Repository

    Schmeier, Sebastian; Schaefer, Ulf; Essack, Magbubah; Bajic, Vladimir B.

    2011-01-01

    Background: MicroRNAs (miRNAs) are small non-coding RNA molecules that repress the translation of messenger RNAs (mRNAs) or degrade mRNAs. These functions of miRNAs allow them to control key cellular processes such as development, differentiation and apoptosis, and they have also been implicated in several cancers such as leukaemia, lung, pancreatic and ovarian cancer (OC). Unfortunately, the specific machinery of miRNA regulation, involving transcription factors (TFs) and transcription co-factors (TcoFs), is not well understood. In the present study we focus on computationally deciphering the underlying network of miRNAs, their targets, and their control mechanisms that have an influence on OC development.Results: We analysed experimentally verified data from multiple sources that describe miRNA influence on diseases, miRNA targeting of mRNAs, and on protein-protein interactions, and combined this data with ab initio transcription factor binding site predictions within miRNA promoter regions. From these analyses, we derived a network that describes the influence of miRNAs and their regulation in human OC. We developed a methodology to analyse the network in order to find the nodes that have the largest potential of influencing the network's behaviour (network hubs). We further show the potentially most influential miRNAs, TFs and TcoFs, showing subnetworks illustrating the involved mechanisms as well as regulatory miRNA network motifs in OC. We find an enrichment of miRNA targeted OC genes in the highly relevant pathways cell cycle regulation and apoptosis.Conclusions: We combined several sources of interaction and association data to analyse and place miRNAs within regulatory pathways that influence human OC. These results represent the first comprehensive miRNA regulatory network analysis for human OC. This suggests that miRNAs and their regulation may play a major role in OC and that further directed research in this area is of utmost importance to enhance our

  7. 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.

  8. The expression pattern of small nucleolar and small Cajal body-specific RNAs characterizes distinct molecular subtypes of multiple myeloma

    International Nuclear Information System (INIS)

    Ronchetti, D; Todoerti, K; Tuana, G; Agnelli, L; Mosca, L; Lionetti, M; Fabris, S; Colapietro, P; Miozzo, M; Ferrarini, M; Tassone, P; Neri, A

    2012-01-01

    Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs involved in the maturation of other RNA molecules and generally located in the introns of host genes. It is now emerging that altered sno/scaRNAs expression may have a pathological role in cancer. This study elucidates the patterns of sno/scaRNAs expression in multiple myeloma (MM) by profiling purified malignant plasma cells from 55 MMs, 8 secondary plasma cell leukemias (sPCLs) and 4 normal controls. Overall, a global sno/scaRNAs downregulation was found in MMs and, even more, in sPCLs compared with normal plasma cells. Whereas SCARNA22 resulted the only sno/scaRNA characterizing the translocation/cyclin D4 (TC4) MM, TC2 group displayed a distinct sno/scaRNA signature overexpressing members of SNORD115 and SNORD116 families located in a region finely regulated by an imprinting center at 15q11, which, however, resulted overall hypomethylated in MMs independently of the SNORD115 and SNORD116 expression levels. Finally, integrative analyses with available gene expression and genome-wide data revealed the occurrence of significant sno/scaRNAs/host genes co-expression and the putative influence of allelic imbalances on specific snoRNAs expression. Our data extend the current view of sno/scaRNAs deregulation in cancer and add novel information to the bio-molecular complexity of plasma cell dyscrasias

  9. New Insight into Inter-kingdom Communication: Horizontal Transfer of Mobile Small RNAs.

    Science.gov (United States)

    Zhou, Geyu; Zhou, Yu; Chen, Xi

    2017-01-01

    Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), are conventionally regarded as critical molecular regulators of various intracellular processes. However, recent accumulating evidence indicates that sRNAs can be transferred within cells and tissues and even across species. In plants, nematodes and microbes, these mobile sRNAs can mediate inter-kingdom communication, environmental sensing, gene expression regulation, host-parasite defense and many other biological functions. Strikingly, a recent study by our group suggested that ingested plant miRNAs are transferred to blood, accumulate in tissues and regulate transcripts in consuming animals. While our and other independent groups' subsequent studies further explored the emerging field of sRNA-mediated crosstalk between species, some groups reported negative results and questioned its general applicability. Thus, further studies carefully evaluating the horizontal transfer of exogenous sRNAs and its potential biological functions are urgently required. Here, we review the current state of knowledge in the field of the horizontal transfer of mobile sRNAs, suggest its future directions and key points for examination and discuss its potential mechanisms and application prospects in nutrition, agriculture and medicine.

  10. New Insight into Inter-kingdom Communication: Horizontal Transfer of Mobile Small RNAs

    Directory of Open Access Journals (Sweden)

    Xi Chen

    2017-05-01

    Full Text Available Small RNAs (sRNAs, including small interfering RNAs (siRNAs and microRNAs (miRNAs, are conventionally regarded as critical molecular regulators of various intracellular processes. However, recent accumulating evidence indicates that sRNAs can be transferred within cells and tissues and even across species. In plants, nematodes and microbes, these mobile sRNAs can mediate inter-kingdom communication, environmental sensing, gene expression regulation, host-parasite defense and many other biological functions. Strikingly, a recent study by our group suggested that ingested plant miRNAs are transferred to blood, accumulate in tissues and regulate transcripts in consuming animals. While our and other independent groups’ subsequent studies further explored the emerging field of sRNA-mediated crosstalk between species, some groups reported negative results and questioned its general applicability. Thus, further studies carefully evaluating the horizontal transfer of exogenous sRNAs and its potential biological functions are urgently required. Here, we review the current state of knowledge in the field of the horizontal transfer of mobile sRNAs, suggest its future directions and key points for examination and discuss its potential mechanisms and application prospects in nutrition, agriculture and medicine.

  11. Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus

    Directory of Open Access Journals (Sweden)

    Schrago Carlos EG

    2011-08-01

    Full Text Available Abstract Background In response to infection, viral genomes are processed by Dicer-like (DCL ribonuclease proteins into viral small RNAs (vsRNAs of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some groups of viruses. However, nothing is known about the vsRNAs produced during infections of members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, we report the characterization of a population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV, a member of the genus Polerovirus, family Luteoviridae. Results Deep sequencing of small RNAs (sRNAs from leaves of CLRDV-infected cotton plants revealed that the vsRNAs were 21- to 24-nucleotides (nt long and that their sequences matched the viral genome, with higher frequencies of matches in the 3- region. There were equivalent amounts of sense and antisense vsRNAs, and the 22-nt class of small RNAs was predominant. During infection, cotton Dcl transcripts appeared to be up-regulated, while Dcl2 appeared to be down-regulated. Conclusions This is the first report on the profile of sRNAs in a plant infected with a virus from the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAs. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpectedly high accumulation of 22-nt vsRNAs are discussed. CLRDV is the causal agent of Cotton blue disease, which occurs worldwide. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases.

  12. Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus.

    Science.gov (United States)

    Silva, Tatiane F; Romanel, Elisson A C; Andrade, Roberto R S; Farinelli, Laurent; Østerås, Magne; Deluen, Cécile; Corrêa, Régis L; Schrago, Carlos E G; Vaslin, Maite F S

    2011-08-24

    In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some groups of viruses. However, nothing is known about the vsRNAs produced during infections of members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, we report the characterization of a population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus Polerovirus, family Luteoviridae. Deep sequencing of small RNAs (sRNAs) from leaves of CLRDV-infected cotton plants revealed that the vsRNAs were 21- to 24-nucleotides (nt) long and that their sequences matched the viral genome, with higher frequencies of matches in the 3- region. There were equivalent amounts of sense and antisense vsRNAs, and the 22-nt class of small RNAs was predominant. During infection, cotton Dcl transcripts appeared to be up-regulated, while Dcl2 appeared to be down-regulated. This is the first report on the profile of sRNAs in a plant infected with a virus from the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAs. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpectedly high accumulation of 22-nt vsRNAs are discussed. CLRDV is the causal agent of Cotton blue disease, which occurs worldwide. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases.

  13. Clinical implications of microRNAs in human glioblastoma

    Directory of Open Access Journals (Sweden)

    Masahiro eMizoguchi

    2013-02-01

    Full Text Available Glioblastoma (GBM is one of the most common and dismal brain tumors in adults. Further elucidation of the molecular pathogenesis of GBM is mandatory to improve the overall survival of patients. A novel small non-coding RNA molecule, microRNA (miRNA, appears to represent one of the most attractive target molecules contributing to the pathogenesis of various types of tumors. Recent global analyses have revealed that several miRNAs are clinically implicated in GBM, with some reports indicating the association of miRNA dysregulation with acquired temozolomide (TMZ resistance. More recent studies have revealed that miRNAs could play a role in cancer stem cell (CSC properties, contributing to treatment resistance. In addition, greater impact might be expected from miRNA-targeted therapies based on tumor-derived exosomes that contain numerous functional miRNAs, which could be transferred between tumor cells and surrounding structures. Tumor-derived miRNAs are now considered to be a novel molecular mechanism promoting the progression of GBM. Establishment of miRNA-targeted therapies based on miRNA dysregulation of CSCs could provide effective therapeutic strategies for TMZ-resistant GBM. Recent progress has revealed that miRNAs are not only putative biological markers for diagnosis, but also one of the most promising targets for GBM treatment. Herein, we summarize the translational aspects of miRNAs in the diagnosis and treatment of GBM.

  14. Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection.

    Science.gov (United States)

    Romanel, Elisson; Silva, Tatiane F; Corrêa, Régis L; Farinelli, Laurent; Hawkins, Jennifer S; Schrago, Carlos E G; Vaslin, Maite F S

    2012-11-01

    Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new miRNA families that had not been previously described in cotton. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks.

  15. Differential and coherent processing patterns from small RNAs

    DEFF Research Database (Denmark)

    Pundhir, Sachin; Gorodkin, Jan

    2015-01-01

    Post-transcriptional processing events related to short RNAs are often reflected in their read profile patterns emerging from high-throughput sequencing data. MicroRNA arm switching across different tissues is a well-known example of what we define as differential processing. Here, short RNAs from...

  16. Targeting of microRNAs for therapeutics

    DEFF Research Database (Denmark)

    Stenvang, Jan; Lindow, Morten; Kauppinen, Sakari

    2008-01-01

    miRNAs (microRNAs) comprise a class of small endogenous non-coding RNAs that post-transcriptionally repress gene expression by base-pairing with their target mRNAs. Recent evidence has shown that miRNAs play important roles in a wide variety of human diseases, such as viral infections, cancer...

  17. 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

  18. Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles.

    Science.gov (United States)

    Caswell, Clayton C; Oglesby-Sherrouse, Amanda G; Murphy, Erin R

    2014-01-01

    Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators.

  19. Sibling rivalry: Related bacterial small RNAs and their redundant and non-redundant roles

    Directory of Open Access Journals (Sweden)

    Clayton eCaswell

    2014-10-01

    Full Text Available Small RNA molecules (sRNAs are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators.

  20. Roles of small RNAs in soybean defense against Phytophthora sojae infection.

    Science.gov (United States)

    Wong, James; Gao, Lei; Yang, Yang; Zhai, Jixian; Arikit, Siwaret; Yu, Yu; Duan, Shuyi; Chan, Vicky; Xiong, Qin; Yan, Jun; Li, Shengben; Liu, Renyi; Wang, Yuanchao; Tang, Guiliang; Meyers, Blake C; Chen, Xuemei; Ma, Wenbo

    2014-09-01

    The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat-inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding-leucine rich repeat proteins and genes encoding pentatricopeptide repeat-containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense-associated genes in soybean during Phytophthora infection. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  1. Computational analysis of human miRNAs phylogenetics

    African Journals Online (AJOL)

    User

    2011-05-02

    May 2, 2011 ... Human DNA. 71. 100.00. 1.94E-28. AL138714. Human DNA sequence from clone RP11-. 121J7 on chromosome 13q32.1-32.3. Contains the 3' end of a novel gene, the 5' end of the GPC5 gene for glypican 5, 5 ..... including human, chimpanzee, orangutan, and macaque, and find that miRNAs were ...

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. Discovery and Characterization of piRNAs in the Human Fetal Ovary

    Directory of Open Access Journals (Sweden)

    Zev Williams

    2015-10-01

    Full Text Available Piwi-interacting RNAs (piRNAs, a class of 26- to 32-nt non-coding RNAs (ncRNAs, function in germline development, transposon silencing, and epigenetic regulation. We performed deep sequencing and annotation of untreated and periodate-treated small RNA cDNA libraries from human fetal and adult germline and reference somatic tissues. This revealed abundant piRNAs originating from 150 piRNA-encoding genes, including some exhibiting gender-specific expression, in fetal ovary and adult testis—developmental periods coinciding with mitotic cell divisions expanding fetal germ cells prior to meiotic divisions. The absence of reads mapping uniquely to annotated piRNA genes demonstrated their paucity in fetal testis and adult ovary and absence in somatic tissues. We curated human piRNA-expressing regions and defined their precise borders and observed piRNA-guided cleavage of transcripts antisense to some piRNA-producing genes. This study provides insights into sex-specific mammalian piRNA expression and function and serves as a reference for human piRNA analysis and annotation.

  8. A simple and efficient method for isolating small RNAs from different plant species

    Directory of Open Access Journals (Sweden)

    de Folter Stefan

    2011-02-01

    Full Text Available Abstract Background Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species. Results We developed a simple and efficient method to isolate small RNAs from different plant species by first comparing different total RNA extraction protocols, followed by streamlining the best one, finally resulting in a small RNA extraction method that has no need of first total RNA extraction and is not based on the commercially available TRIzol® Reagent or columns. This small RNA extraction method not only works well for plant tissues with high polysaccharide content, like cactus, agave, banana, and tomato, but also for plant species like Arabidopsis or tobacco. Furthermore, the obtained small RNA samples were successfully used in northern blot assays. Conclusion Here we provide a simple and efficient method to isolate small RNAs from different plant species, such as cactus, agave, banana, tomato, Arabidopsis, and tobacco, and the small RNAs from this simplified and low cost method is suitable for downstream handling like northern blot assays.

  9. Evolutionary patterns of Escherichia coli small RNAs and their regulatory interactions.

    Science.gov (United States)

    Peer, Asaf; Margalit, Hanah

    2014-07-01

    Most bacterial small RNAs (sRNAs) are post-transcriptional regulators of gene expression, exerting their regulatory function by base-pairing with their target mRNAs. While it has become evident that sRNAs play central regulatory roles in the cell, little is known about their evolution and the evolution of their regulatory interactions. Here we used the prokaryotic phylogenetic tree to reconstruct the evolutionary history of Escherichia coli sRNAs and their binding sites on target mRNAs. We discovered that sRNAs currently present in E. coli mainly accumulated inside the Enterobacteriales order, succeeding the appearance of other types of noncoding RNAs and concurrently with the evolution of a variant of the Hfq protein exhibiting a longer C-terminal region. Our analysis of the evolutionary ages of sRNA-mRNA interactions revealed that while all sRNAs were evolutionarily older than most of their known binding sites on mRNA targets, for quite a few sRNAs there was at least one binding site that coappeared with or preceded them. It is conceivable that the establishment of these first interactions forced selective pressure on the sRNAs, after which additional targets were acquired by fitting a binding site to the active region of the sRNA. This conjecture is supported by the appearance of many binding sites on target mRNAs only after the sRNA gain, despite the prior presence of the target gene in ancestral genomes. Our results suggest a selective mechanism that maintained the sRNAs across the phylogenetic tree, and shed light on the evolution of E. coli post-transcriptional regulatory network. © 2014 Peer and Margalit; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  10. 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

  11. 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

  12. Expression of Mitochondrial Non-coding RNAs (ncRNAs) Is Modulated by High Risk Human Papillomavirus (HPV) Oncogenes*

    Science.gov (United States)

    Villota, Claudio; Campos, América; Vidaurre, Soledad; Oliveira-Cruz, Luciana; Boccardo, Enrique; Burzio, Verónica A.; Varas, Manuel; Villegas, Jaime; Villa, Luisa L.; Valenzuela, Pablo D. T.; Socías, Miguel; Roberts, Sally; Burzio, Luis O.

    2012-01-01

    The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis. PMID:22539350

  13. Expression of mitochondrial non-coding RNAs (ncRNAs) is modulated by high risk human papillomavirus (HPV) oncogenes.

    Science.gov (United States)

    Villota, Claudio; Campos, América; Vidaurre, Soledad; Oliveira-Cruz, Luciana; Boccardo, Enrique; Burzio, Verónica A; Varas, Manuel; Villegas, Jaime; Villa, Luisa L; Valenzuela, Pablo D T; Socías, Miguel; Roberts, Sally; Burzio, Luis O

    2012-06-15

    The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

  14. Let-7 microRNAs are developmentally regulated in circulating human erythroid cells

    Directory of Open Access Journals (Sweden)

    Reed Christopher

    2009-11-01

    Full Text Available Abstract Background MicroRNAs are ~22nt-long small non-coding RNAs that negatively regulate protein expression through mRNA degradation or translational repression in eukaryotic cells. Based upon their importance in regulating development and terminal differentiation in model systems, erythrocyte microRNA profiles were examined at birth and in adults to determine if changes in their abundance coincide with the developmental phenomenon of hemoglobin switching. Methods Expression profiling of microRNA was performed using total RNA from four adult peripheral blood samples compared to four cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNAs were hybridized to custom spotted arrays containing 474 human microRNA species (miRBase release 9.1. Total RNA from Epstein-Barr virus (EBV-transformed lymphoblastoid cell lines provided a hybridization reference for all samples to generate microRNA abundance profile for each sample. Results Among 206 detected miRNAs, 79% of the microRNAs were present at equivalent levels in both cord and adult cells. By comparison, 37 microRNAs were up-regulated and 4 microRNAs were down-regulated in adult erythroid cells (fold change > 2; p let-7 miRNA family consistently demonstrated increased abundance in the adult samples by array-based analyses that were confirmed by quantitative PCR (4.5 to 18.4 fold increases in 6 of 8 let-7 miRNA. Profiling studies of messenger RNA (mRNA in these cells additionally demonstrated down-regulation of ten let-7 target genes in the adult cells. Conclusion These data suggest that a consistent pattern of up-regulation among let-7 miRNA in circulating erythroid cells occurs in association with hemoglobin switching during the fetal-to-adult developmental transition in humans.

  15. Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

    Science.gov (United States)

    Gangras, Pooja; Dayeh, Daniel M; Mabin, Justin W; Nakanishi, Kotaro; Singh, Guramrit

    2018-01-01

    Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

  16. Evolutionary Dynamics of Small RNAs in 27 Escherichia coli and Shigella Genomes

    Science.gov (United States)

    Skippington, Elizabeth; Ragan, Mark A.

    2012-01-01

    Small RNAs (sRNAs) are widespread in bacteria and play critical roles in regulating physiological processes. They are best characterized in Escherichia coli K-12 MG1655, where 83 sRNAs constitute nearly 2% of the gene complement. Most sRNAs act by base pairing with a target mRNA, modulating its translation and/or stability; many of these RNAs share only limited complementarity to their mRNA target, and require the chaperone Hfq to facilitate base pairing. Little is known about the evolutionary dynamics of bacterial sRNAs. Here, we apply phylogenetic and network analyses to investigate the evolutionary processes and principles that govern sRNA gene distribution in 27 E. coli and Shigella genomes. We identify core (encoded in all 27 genomes) and variable sRNAs; more than two-thirds of the E. coli K-12 MG1655 sRNAs are core, whereas the others show patterns of presence and absence that are principally due to genetic loss, not duplication or lateral genetic transfer. We present evidence that variable sRNAs are less tightly integrated into cellular genetic regulatory networks than are the core sRNAs, and that Hfq facilitates posttranscriptional cross talk between the E. coli–Shigella core and variable genomes. Finally, we present evidence that more than 80% of genes targeted by Hfq-associated core sRNAs have been transferred within the E. coli–Shigella clade, and that most of these genes have been transferred intact. These results suggest that Hfq and sRNAs help integrate laterally acquired genes into established regulatory networks. PMID:22223756

  17. The Mechanisms of Virulence Regulation by Small Noncoding RNAs in Low GC Gram-Positive Pathogens

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    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.

  18. Identification and characterization of microRNAs in Clonorchis sinensis of human health significance

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    Yuan Zi-Guo

    2010-09-01

    Full Text Available Abstract Background Clonorchis sinensis is a zoonotic parasite causing clonorchiasis-associated human disease such as biliary calculi, cholecystitis, liver cirrhosis, and it is currently classified as carcinogenic to humans for cholangiocarcinoma. MicroRNAs (miRNAs are non-coding, regulating small RNA molecules which are essential for the complex life cycles of parasites and are involved in parasitic infections. To identify and characterize miRNAs expressed in adult C. sinensis residing chronically in the biliary tract, we developed an integrative approach combining deep sequencing and bioinformatic predictions with stem-loop real-time PCR analysis. Results Here we report the use of this approach to identify and clone 6 new and 62,512 conserved C. sinensis miRNAs which belonged to 284 families. There was strong bias on families, family members and sequence nucleotides in C. sinensis. Uracil was the dominant nucleotide, particularly at positions 1, 14 and 22, which were located approximately at the beginning, middle and end of conserved miRNAs. There was no significant "seed region" at the first and ninth positions which were commonly found in human, animals and plants. Categorization of conserved miRNAs indicated that miRNAs of C. sinensis were still innovated and concentrated along three branches of the phylogenetic tree leading to bilaterians, insects and coelomates. There were two miRNA strategies in C. sinensis for its parasitic life: keeping a large category of miRNA families of different animals and keeping stringent conserved seed regions with high active innovation in other places of miRNAs mainly in the middle and the end, which were perfect for the parasite to perform its complex life style and for host changes. Conclusions The present study represented the first large scale characterization of C. sinensis miRNAs, which have implications for understanding the complex biology of this zoonotic parasite, as well as miRNA studies of other

  19. Extracellular RNAs: development as biomarkers of human disease

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    Joseph F. Quinn

    2015-08-01

    Full Text Available Ten ongoing studies designed to test the possibility that extracellular RNAs may serve as biomarkers in human disease are described. These studies, funded by the NIH Common Fund Extracellular RNA Communication Program, examine diverse extracellular body fluids, including plasma, serum, urine and cerebrospinal fluid. The disorders studied include hepatic and gastric cancer, cardiovascular disease, chronic kidney disease, neurodegenerative disease, brain tumours, intracranial haemorrhage, multiple sclerosis and placental disorders. Progress to date and the plans for future studies are outlined.

  20. Regulation of cytokines by small RNAs during skin inflammation

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    Mikkelsen Jacob G

    2010-07-01

    Full Text Available Abstract Intercellular signaling by cytokines is a vital feature of the innate immune system. In skin, an inflammatory response is mediated by cytokines and an entwined network of cellular communication between T-cells and epidermal keratinocytes. Dysregulated cytokine production, orchestrated by activated T-cells homing to the skin, is believed to be the main cause of psoriasis, a common inflammatory skin disorder. Cytokines are heavily regulated at the transcriptional level, but emerging evidence suggests that regulatory mechanisms that operate after transcription play a key role in balancing the production of cytokines. Herein, we review the nature of cytokine signaling in psoriasis with particular emphasis on regulation by mRNA destabilizing elements and the potential targeting of cytokine-encoding mRNAs by miRNAs. The proposed linkage between mRNA decay mediated by AU-rich elements and miRNA association is described and discussed as a possible general feature of cytokine regulation in skin. Moreover, we describe the latest attempts to therapeutically target cytokines at the RNA level in psoriasis by exploiting the cellular RNA interference machinery. The applicability of cytokine-encoding mRNAs as future clinical drug targets is evaluated, and advances and obstacles related to topical administration of RNA-based drugs targeting the cytokine circuit in psoriasis are described.

  1. 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...

  2. Bistability and oscillations in gene regulation mediated by small noncoding RNAs.

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    Dengyu Liu

    Full Text Available The interplay of small noncoding RNAs (sRNAs, mRNAs, and proteins has been shown to play crucial roles in almost all cellular processes. As key post-transcriptional regulators of gene expression, the mechanisms and roles of sRNAs in various cellular processes still need to be fully understood. When participating in cellular processes, sRNAs mainly mediate mRNA degradation or translational repression. Here, we show how the dynamics of two minimal architectures is drastically affected by these two mechanisms. A comparison is also given to reveal the implication of the fundamental differences. This study may help us to analyze complex networks assembled by simple modules more easily. A better knowledge of the sRNA-mediated motifs is also of interest for bio-engineering and artificial control.

  3. 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.

  4. Identification of Conserved and Potentially Regulatory Small RNAs in Heterocystous Cyanobacteria

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    Manuel eBrenes-Álvarez

    2016-02-01

    Full Text Available Small RNAs (sRNAs are a growing class of non-protein-coding transcripts that participate in the regulation of virtually every aspect of bacterial physiology. Heterocystous cyanobacteria are a group of photosynthetic organisms that exhibit multicellular behaviour and developmental alternatives involving specific transcriptomes exclusive of a given physiological condition or even a cell type. In the context of our ongoing effort to understand developmental decisions in these organisms we have undertaken an approach to the global identification of sRNAs. Using differential RNA-Seq we have previously identified transcriptional start sites for the model heterocystous cyanobacterium Nostoc sp. PCC 7120. Here we combine this dataset with a prediction of Rho-independent transcriptional terminators and an analysis of phylogenetic conservation of potential sRNAs among 89 available cyanobacterial genomes. In contrast to predictive genome-wide approaches, the use of an experimental dataset comprising all active transcriptional start sites (differential RNA-Seq facilitates the identification of bona fide sRNAs. The output of our approach is a dataset of predicted potential sRNAs in Nostoc sp. PCC 7120, with different degrees of phylogenetic conservation across the 89 cyanobacterial genomes analyzed. Previously described sRNAs appear among the predicted sRNAs, demonstrating the performance of the algorithm. In addition, new predicted sRNAs are now identified that can be involved in regulation of different aspects of cyanobacterial physiology, including adaptation to nitrogen stress, the condition that triggers differentiation of heterocysts (specialized nitrogen-fixing cells. Transcription of several predicted sRNAs that appear exclusively in the genomes of heterocystous cyanobacteria is experimentally verified by Northern blot. Cell-specific transcription of one of these sRNAs, NsiR8 (nitrogen stress-induced RNA 8, in developing heterocysts is also

  5. Evidence for widespread exonic small RNAs in the glaucophyte alga Cyanophora paradoxa.

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    Jeferson Gross

    Full Text Available RNAi (RNA interference relies on the production of small RNAs (sRNAs from double-stranded RNA and comprises a major pathway in eukaryotes to restrict the propagation of selfish genetic elements. Amplification of the initial RNAi signal by generation of multiple secondary sRNAs from a targeted mRNA is catalyzed by RNA-dependent RNA polymerases (RdRPs. This phenomenon is known as transitivity and is particularly important in plants to limit the spread of viruses. Here we describe, using a genome-wide approach, the distribution of sRNAs in the glaucophyte alga Cyanophora paradoxa. C. paradoxa is a member of the supergroup Plantae (also known as Archaeplastida that includes red algae, green algae, and plants. The ancient (>1 billion years ago split of glaucophytes within Plantae suggests that C. paradoxa may be a useful model to learn about the early evolution of RNAi in the supergroup that ultimately gave rise to plants. Using next-generation sequencing and bioinformatic analyses we find that sRNAs in C. paradoxa are preferentially associated with mRNAs, including a large number of transcripts that encode proteins arising from different functional categories. This pattern of exonic sRNAs appears to be a general trend that affects a large fraction of mRNAs in the cell. In several cases we observe that sRNAs have a bias for a specific strand of the mRNA, including many instances of antisense predominance. The genome of C. paradoxa encodes four sequences that are homologous to RdRPs in Arabidopsis thaliana. We discuss the possibility that exonic sRNAs in the glaucophyte may be secondarily derived from mRNAs by the action of RdRPs. If this hypothesis is confirmed, then transitivity may have had an ancient origin in Plantae.

  6. MicroRNAs: From Female Fertility, Germ Cells, and Stem Cells to Cancer in Humans

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    Irma Virant-Klun

    2016-01-01

    Full Text Available MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human preimplantation development. They keep a balance between pluripotency and differentiation in the embryo and embryonic stem cells. Moreover, it became evident that dysregulation of microRNA expression may play a fundamental role in progression and dissemination of different cancers including ovarian cancer. The interest is still increased by the discovery of exosomes, that is, cell-derived vesicles, which can carry different proteins but also microRNAs between different cells and are involved in cell-to-cell communication. MicroRNAs, together with exosomes, have a great potential to be used for prognosis, therapy, and biomarkers of different diseases including infertility. The aim of this review paper is to summarize the existent knowledge on microRNAs related to female fertility and cancer: from primordial germ cells and ovarian function, germinal stem cells, oocytes, and embryos to embryonic stem cells.

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

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    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.

  8. Identification of differentially expressed microRNAs in human male breast cancer

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    Schipper Elisa

    2010-03-01

    Full Text Available Abstract Background The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases. Methods The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods. Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer. Results Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer. Conclusions Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.

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

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    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.

  10. Micropathogen Community Analysis in Hyalomma rufipes via High-Throughput Sequencing of Small RNAs

    Science.gov (United States)

    Luo, Jin; Liu, Min-Xuan; Ren, Qiao-Yun; Chen, Ze; Tian, Zhan-Cheng; Hao, Jia-Wei; Wu, Feng; Liu, Xiao-Cui; Luo, Jian-Xun; Yin, Hong; Wang, Hui; Liu, Guang-Yuan

    2017-01-01

    Ticks are important vectors in the transmission of a broad range of micropathogens to vertebrates, including humans. Because of the role of ticks in disease transmission, identifying and characterizing the micropathogen profiles of tick populations have become increasingly important. The objective of this study was to survey the micropathogens of Hyalomma rufipes ticks. Illumina HiSeq2000 technology was utilized to perform deep sequencing of small RNAs (sRNAs) extracted from field-collected H. rufipes ticks in Gansu Province, China. The resultant sRNA library data revealed that the surveyed tick populations produced reads that were homologous to St. Croix River Virus (SCRV) sequences. We also observed many reads that were homologous to microbial and/or pathogenic isolates, including bacteria, protozoa, and fungi. As part of this analysis, a phylogenetic tree was constructed to display the relationships among the homologous sequences that were identified. The study offered a unique opportunity to gain insight into the micropathogens of H. rufipes ticks. The effective control of arthropod vectors in the future will require knowledge of the micropathogen composition of vectors harboring infectious agents. Understanding the ecological factors that regulate vector propagation in association with the prevalence and persistence of micropathogen lineages is also imperative. These interactions may affect the evolution of micropathogen lineages, especially if the micropathogens rely on the vector or host for dispersal. The sRNA deep-sequencing approach used in this analysis provides an intuitive method to survey micropathogen prevalence in ticks and other vector species. PMID:28861401

  11. 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.

  12. Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs.

    LENUS (Irish Health Repository)

    Weissenmayer, Barbara A

    2011-01-01

    Second generation sequencing has prompted a number of groups to re-interrogate the transcriptomes of several bacterial and archaeal species. One of the central findings has been the identification of complex networks of small non-coding RNAs that play central roles in transcriptional regulation in all growth conditions and for the pathogen\\'s interaction with and survival within host cells. Legionella pneumophila is a gram-negative facultative intracellular human pathogen with a distinct biphasic lifestyle. One of its primary environmental hosts in the free-living amoeba Acanthamoeba castellanii and its infection by L. pneumophila mimics that seen in human macrophages. Here we present analysis of strand specific sequencing of the transcriptional response of L. pneumophila during exponential and post-exponential broth growth and during the replicative and transmissive phase of infection inside A. castellanii. We extend previous microarray based studies as well as uncovering evidence of a complex regulatory architecture underpinned by numerous non-coding RNAs. Over seventy new non-coding RNAs could be identified; many of them appear to be strain specific and in configurations not previously reported. We discover a family of non-coding RNAs preferentially expressed during infection conditions and identify a second copy of 6S RNA in L. pneumophila. We show that the newly discovered putative 6S RNA as well as a number of other non-coding RNAs show evidence for antisense transcription. The nature and extent of the non-coding RNAs and their expression patterns suggests that these may well play central roles in the regulation of Legionella spp. specific traits and offer clues as to how L. pneumophila adapts to its intracellular niche. The expression profiles outlined in the study have been deposited into Genbank\\'s Gene Expression Omnibus (GEO) database under the series accession GSE27232.

  13. Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis

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    Lonardi Stefano

    2008-01-01

    Full Text Available Abstract Background In spite of large intergenic spaces in plant and animal genomes, 7% to 30% of genes in the genomes encode overlapping cis-natural antisense transcripts (cis-NATs. The widespread occurrence of cis-NATs suggests an evolutionary advantage for this type of genomic arrangement. Experimental evidence for the regulation of two cis-NAT gene pairs by natural antisense transcripts-generated small interfering RNAs (nat-siRNAs via the RNA interference (RNAi pathway has been reported in Arabidopsis. However, the extent of siRNA-mediated regulation of cis-NAT genes is still unclear in any genome. Results The hallmarks of RNAi regulation of NATs are 1 inverse regulation of two genes in a cis-NAT pair by environmental and developmental cues and 2 generation of siRNAs by cis-NAT genes. We examined Arabidopsis transcript profiling data from public microarray databases to identify cis-NAT pairs whose sense and antisense transcripts show opposite expression changes. A subset of the cis-NAT genes displayed negatively correlated expression profiles as well as inverse differential expression changes under at least one of the examined developmental stages or treatment conditions. By searching the Arabidopsis Small RNA Project (ASRP and Massively Parallel Signature Sequencing (MPSS small RNA databases as well as our stress-treated small RNA dataset, we found small RNAs that matched at least one gene in 646 pairs out of 1008 (64% protein-coding cis-NAT pairs, which suggests that siRNAs may regulate the expression of many cis-NAT genes. 209 putative siRNAs have the potential to target more than one gene and half of these small RNAs could target multiple members of a gene family. Furthermore, the majority of the putative siRNAs within the overlapping regions tend to target only one transcript of a given NAT pair, which is consistent with our previous finding on salt- and bacteria-induced nat-siRNAs. In addition, we found that genes encoding plastid- or

  14. Monitoring the Spatiotemporal Activities of miRNAs in Small Animal Models Using Molecular Imaging Modalities

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    Patrick Baril

    2015-03-01

    Full Text Available MicroRNAs (miRNAs are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy.

  15. Monitoring the spatiotemporal activities of miRNAs in small animal models using molecular imaging modalities.

    Science.gov (United States)

    Baril, Patrick; Ezzine, Safia; Pichon, Chantal

    2015-03-04

    MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy.

  16. Ancestral vinclozolin exposure alters the epigenetic transgenerational inheritance of sperm small noncoding RNAs.

    Science.gov (United States)

    Schuster, Andrew; Skinner, Michael K; Yan, Wei

    Exposure to the agricultural fungicide vinclozolin during gestation promotes a higher incidence of various diseases in the subsequent unexposed F3 and F4 generations. This phenomenon is termed epigenetic transgenerational inheritance and has been shown to in part involve alterations in DNA methylation, but the role of other epigenetic mechanisms remains unknown. The current study investigated the alterations in small noncoding RNA (sncRNA) in the sperm from F3 generation control and vinclozolin lineage rats. Over 200 differentially expressed sncRNAs were identified and the tRNA-derived sncRNAs, namely 5' halves of mature tRNAs (5' halves), displayed the most dramatic changes. Gene targets of the altered miRNAs and tRNA 5' halves revealed associations between the altered sncRNAs and differentially DNA methylated regions. Dysregulated sncRNAs appear to correlate with mRNA profiles associated with the previously observed vinclozolin-induced disease phenotypes. Data suggest potential connections between sperm-borne RNAs and the vinclozolin-induced epigenetic transgenerational inheritance phenomenon.

  17. LncRNAWiki: harnessing community knowledge in collaborative curation of human long non-coding RNAs

    KAUST Repository

    Ma, L.

    2014-11-15

    Long non-coding RNAs (lncRNAs) perform a diversity of functions in numerous important biological processes and are implicated in many human diseases. In this report we present lncRNAWiki (http://lncrna.big.ac.cn), a wiki-based platform that is open-content and publicly editable and aimed at community-based curation and collection of information on human lncRNAs. Current related databases are dependent primarily on curation by experts, making it laborious to annotate the exponentially accumulated information on lncRNAs, which inevitably requires collective efforts in community-based curation of lncRNAs. Unlike existing databases, lncRNAWiki features comprehensive integration of information on human lncRNAs obtained from multiple different resources and allows not only existing lncRNAs to be edited, updated and curated by different users but also the addition of newly identified lncRNAs by any user. It harnesses community collective knowledge in collecting, editing and annotating human lncRNAs and rewards community-curated efforts by providing explicit authorship based on quantified contributions. LncRNAWiki relies on the underling knowledge of scientific community for collective and collaborative curation of human lncRNAs and thus has the potential to serve as an up-to-date and comprehensive knowledgebase for human lncRNAs.

  18. 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 amiRNAs

  19. 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

  20. Small RNA Sequencing Uncovers New miRNAs and moRNAs Differentially Expressed in Normal and Primary Myelofibrosis CD34+ Cells.

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    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. Identification and characterization of argonaute protein, Ago2 and its associated small RNAs in Schistosoma japonicum.

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    Pengfei Cai

    Full Text Available BACKGROUND: The complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC, have not yet been fully explored. METHODOLOGY/PRINCIPAL FINDINGS: Two monoclonal antibodies (mAbs specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2, but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of ≈50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs generated from transposable elements (TEs, especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons. CONCLUSIONS/SIGNIFICANCE: In this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived

  2. High Throughput Sequencing of Small RNAs in the Two Cucurbita Germplasm with Different Sodium Accumulation Patterns Identifies Novel MicroRNAs Involved in Salt Stress Response.

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    Xie, Junjun; Lei, Bo; Niu, Mengliang; Huang, Yuan; Kong, Qiusheng; Bie, Zhilong

    2015-01-01

    MicroRNAs (miRNAs), a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch.) and N15 (Cucurbita. moschata Duch.), with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs) were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small RNAs in the

  3. High Throughput Sequencing of Small RNAs in the Two Cucurbita Germplasm with Different Sodium Accumulation Patterns Identifies Novel MicroRNAs Involved in Salt Stress Response.

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    Junjun Xie

    Full Text Available MicroRNAs (miRNAs, a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch. and N15 (Cucurbita. moschata Duch., with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small

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

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    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.

  5. Identification of genes for small non-coding RNAs that belong to the regulon of the two-component regulatory system CiaRH in Streptococcus

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    Hakenbeck Regine

    2010-11-01

    Full Text Available Abstract Background Post-transcriptional regulation by small RNAs (sRNAs in bacteria is now recognized as a wide-spread regulatory mechanism modulating a variety of physiological responses including virulence. In Streptococcus pneumoniae, an important human pathogen, the first sRNAs to be described were found in the regulon of the CiaRH two-component regulatory system. Five of these sRNAs were detected and designated csRNAs for cia-dependent small RNAs. CiaRH pleiotropically affects β-lactam resistance, autolysis, virulence, and competence development by yet to be defined molecular mechanisms. Since CiaRH is highly conserved among streptococci, it is of interest to determine if csRNAs are also included in the CiaRH regulon in this group of organisms consisting of commensal as well as pathogenic species. Knowledge on the participation of csRNAs in CiaRH-dependent regulatory events will be the key to define the physiological role of this important control system. Results Genes for csRNAs were predicted in streptococcal genomes and data base entries other than S. pneumoniae by searching for CiaR-activated promoters located in intergenic regions that are followed by a transcriptional terminator. 61 different candidate genes were obtained specifying csRNAs ranging in size from 51 to 202 nt. Comparing these genes among each other revealed 40 different csRNA types. All streptococcal genomes harbored csRNA genes, their numbers varying between two and six. To validate these predictions, S. mitis, S. oralis, and S. sanguinis were subjected to csRNA-specific northern blot analysis. In addition, a csRNA gene from S. thermophilus plasmid pST0 introduced into S. pneumoniae was also tested. Each of the csRNAs was detected on these blots and showed the anticipated sizes. Thus, the method applied here is able to predict csRNAs with high precision. Conclusions The results of this study strongly suggest that genes for small non-coding RNAs, csRNAs, are part of

  6. In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce.

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    Yakovlev, Igor A; Fossdal, Carl G

    2017-01-01

    Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21-22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21-22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic

  7. In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce

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    Igor A. Yakovlev

    2017-09-01

    Full Text Available Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs and other small non-coding RNAs (sRNAs play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C. We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21–22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21–22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be

  8. Characterization and expression patterns of small RNAs in synthesized Brassica hexaploids.

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    Shen, Yanyue; Zhao, Qin; Zou, Jun; Wang, Wenliang; Gao, Yi; Meng, Jinling; Wang, Jianbo

    2014-06-01

    Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. We used high-throughput sequencing to compare miRNA expression profiles between Brassica hexaploid and its parents. A total of 613, 784 and 742 known miRNAs were identified in Brassica rapa, Brassica carinata, and Brassica hexaploid, respectively. We detected 618 miRNAs were differentially expressed (log(2)Ratio ≥ 1, P ≤ 0.05) between Brassica hexaploid and its parents, and 425 miRNAs were non-additively expressed in Brassica hexaploid, which suggest a trend of non-additive miRNA regulation following hybridization and polyploidization. Remarkably, majority of the non-additively expressed miRNAs in the Brassica hexaploid are repressed, and there was a bias toward repression of B. rapa miRNAs, which is consistent with the progenitor-biased gene repression in the synthetic allopolyploids. In addition, we identified 653 novel mature miRNAs in Brassica hexaploid and its parents. Finally, we found that almost all the non-additive accumulation of siRNA clusters exhibited a low-parent pattern in Brassica hexaploid. Non-additive small RNA regulation is involved in a range of biological pathways, probably providing a driving force for variation and adaptation in allopolyploids.

  9. Efficient and specific gene knockdown by small interfering RNAs produced in bacteria

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    Huang, Linfeng; Jin, Jingmin; Deighan, Padraig; Kiner, Evgeny; McReynolds, Larry; Lieberman, Judy

    2013-01-01

    Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells1,2 and may be used for therapeutic purposes to knockdown genes implicated in disease3. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in E. coli. This method relies on ectopic expression of p19, a siRNA-binding protein found in a plant RNA virus4, 5. When expressed in E. coli, p19 stabilizes ~21 nt siRNA-like species produced by bacterial RNase III. Transfection of mammalian cells with siRNAs, generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene, at low nanomolar concentrations reproducibly knocks down gene expression by ~90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes. PMID:23475073

  10. Revealing stable processing products from ribosome-associated small RNAs by deep-sequencing data analysis.

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    Zywicki, Marek; Bakowska-Zywicka, Kamilla; Polacek, Norbert

    2012-05-01

    The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.

  11. Deep sequencing-based identification of small regulatory RNAs in Synechocystis sp. PCC 6803.

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    Wen Xu

    Full Text Available Synechocystis sp. PCC 6803 is a genetically tractable model organism for photosynthesis research. The genome of Synechocystis sp. PCC 6803 consists of a circular chromosome and seven plasmids. The importance of small regulatory RNAs (sRNAs as mediators of a number of cellular processes in bacteria has begun to be recognized. However, little is known regarding sRNAs in Synechocystis sp. PCC 6803. To provide a comprehensive overview of sRNAs in this model organism, the sRNAs of Synechocystis sp. PCC 6803 were analyzed using deep sequencing, and 7,951,189 reads were obtained. High quality mapping reads (6,127,890 were mapped onto the genome and assembled into 16,192 transcribed regions (clusters based on read overlap. A total number of 5211 putative sRNAs were revealed from the genome and the 4 megaplasmids, and 27 of these molecules, including four from plasmids, were confirmed by RT-PCR. In addition, possible target genes regulated by all of the putative sRNAs identified in this study were predicted by IntaRNA and analyzed for functional categorization and biological pathways, which provided evidence that sRNAs are indeed involved in many different metabolic pathways, including basic metabolic pathways, such as glycolysis/gluconeogenesis, the citrate cycle, fatty acid metabolism and adaptations to environmentally stress-induced changes. The information from this study provides a valuable reservoir for understanding the sRNA-mediated regulation of the complex physiology and metabolic processes of cyanobacteria.

  12. 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.

  13. The role of Ctk1 kinase in termination of small non-coding RNAs.

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    Tineke L Lenstra

    Full Text Available Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is influenced by the phosphorylation status of the carboxy-terminal domain (CTD of RNA polymerase II (Pol II. Late termination of mRNAs is performed by the CPF/CF complex, the recruitment of which is dependent on CTD-Ser2 phosphorylation (Ser2P. Early termination of shorter cryptic unstable transcripts (CUTs and small nucleolar/nuclear RNAs (sno/snRNAs is performed by the Nrd1-Nab3-Sen1 (NNS complex that binds phosphorylated CTD-Ser5 (Ser5P via the CTD-interacting domain (CID of Nrd1p. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1p are more similar to those derived from alterations in the Ser5P-dependent NNS pathway, than from loss of CTD-Ser2P binding factors. Tiling array analysis of ctk1Δ cells reveals readthrough at snoRNAs, at many cryptic unstable transcripts (CUTs and stable uncharacterized transcripts (SUTs, but only at some mRNAs. Despite the suggested predominant role in termination of mRNAs, we observed that a CTK1 deletion or a Pol II CTD mutant lacking all Ser2 positions does not result in a global mRNA termination defect. Rather, termination defects in these strains are widely observed at NNS-dependent genes. These results indicate that Ctk1p and Ser2 CTD phosphorylation have a wide impact in termination of small non-coding RNAs but only affect a subset of mRNA coding genes.

  14. Modulating the bacterial surface with small RNAs: a new twist on PhoP/Q-mediated lipopolysaccharide modification

    DEFF Research Database (Denmark)

    Overgaard, Martin; Kallipolitis, Birgitte; Valentin-Hansen, Poul

    2009-01-01

    Summary In recent years, small non-coding RNAs have emerged as important regulatory components in bacterial stress responses and in bacterial virulence. Many of these are conserved in related species and act on target mRNAs by sequence complementarity. They are tightly controlled at the transcrip...... in enterobacteria and reinforces the idea that one central role of bacterial small regulatory RNAs is to modulate and fine-tune cell surface composition and structure....

  15. CRISPR transcript processing: a mechanism for generating a large number of small interfering RNAs

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    Djordjevic Marko

    2012-07-01

    Full Text Available Abstract Background CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated sequences is a recently discovered prokaryotic defense system against foreign DNA, including viruses and plasmids. CRISPR cassette is transcribed as a continuous transcript (pre-crRNA, which is processed by Cas proteins into small RNA molecules (crRNAs that are responsible for defense against invading viruses. Experiments in E. coli report that overexpression of cas genes generates a large number of crRNAs, from only few pre-crRNAs. Results We here develop a minimal model of CRISPR processing, which we parameterize based on available experimental data. From the model, we show that the system can generate a large amount of crRNAs, based on only a small decrease in the amount of pre-crRNAs. The relationship between the decrease of pre-crRNAs and the increase of crRNAs corresponds to strong linear amplification. Interestingly, this strong amplification crucially depends on fast non-specific degradation of pre-crRNA by an unidentified nuclease. We show that overexpression of cas genes above a certain level does not result in further increase of crRNA, but that this saturation can be relieved if the rate of CRISPR transcription is increased. We furthermore show that a small increase of CRISPR transcription rate can substantially decrease the extent of cas gene activation necessary to achieve a desired amount of crRNA. Conclusions The simple mathematical model developed here is able to explain existing experimental observations on CRISPR transcript processing in Escherichia coli. The model shows that a competition between specific pre-crRNA processing and non-specific degradation determines the steady-state levels of crRNA and is responsible for strong linear amplification of crRNAs when cas genes are overexpressed. The model further shows how disappearance of only a few pre-crRNA molecules normally present in the cell can lead to a large (two

  16. siRNA-like double-stranded RNAs are specifically protected against degradation in human cell extract.

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    John A H Hoerter

    Full Text Available RNA interference (RNAi is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence any gene of interest by the introduction of synthetic small-interfering (siRNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET. We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time.

  17. 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...

  18. The RNAi Universe in Fungi: A Varied Landscape of Small RNAs and Biological Functions.

    Science.gov (United States)

    Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M

    2017-09-08

    RNA interference (RNAi) is a conserved eukaryotic mechanism that uses small RNA molecules to suppress gene expression through sequence-specific messenger RNA degradation, translational repression, or transcriptional inhibition. In filamentous fungi, the protective function of RNAi in the maintenance of genome integrity is well known. However, knowledge of the regulatory role of RNAi in fungi has had to wait until the recent identification of different endogenous small RNA classes, which are generated by distinct RNAi pathways. In addition, RNAi research on new fungal models has uncovered the role of small RNAs and RNAi pathways in the regulation of diverse biological functions. In this review, we give an up-to-date overview of the different classes of small RNAs and RNAi pathways in fungi and their roles in the defense of genome integrity and regulation of fungal physiology and development, as well as in the interaction of fungi with biotic and abiotic environments.

  19. Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Wanzhong; Wang, Ping; Wang, Xin [Department of Otorhinolaryngology, Head and Neck Surgery, The First Clinical Hospital, Norman Bethune College of Medicine, Jilin University, Changchun (China); Song, Wenzhi [Department of Stomatology, China-Japan Friendship Hospital, Jilin University, Changchun (China); Cui, Xiangyan; Yu, Hong; Zhu, Wei [Department of Otorhinolaryngology, Head and Neck Surgery, The First Clinical Hospital, Norman Bethune College of Medicine, Jilin University, Changchun (China)

    2013-06-12

    Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.

  20. Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

    International Nuclear Information System (INIS)

    Yin, Wanzhong; Wang, Ping; Wang, Xin; Song, Wenzhi; Cui, Xiangyan; Yu, Hong; Zhu, Wei

    2013-01-01

    Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer

  1. Genome‐wide identification of novel small RNAs in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

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

    2012-01-01

    Bacterial small regulatory RNAs (sRNAs) function in post‐transcriptional control of gene expression and control a variety of processes including metabolic reactions, stress responses and pathogenesis in response to environmental signals. A variety of approaches have been used previously to identify...... with this approach. Although the use of three libraries increased the number of novel transcripts identified, there were significant differences in the subset of transcripts detected in each library, underscoring the importance of library preparation strategy and relative sRNA abundance for successful sRNA detection...... and that the approach described here may be applied to identify sRNAs in any bacterium under different growth and stress conditions....

  2. Identification of two small RNAs within the first 1.5-kb of the herpes simplex virus type 1-encoded latency-associated transcript.

    Science.gov (United States)

    Peng, Weiping; Vitvitskaia, Olga; Carpenter, Dale; Wechsler, Steven L; Jones, Clinton

    2008-01-01

    The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected neurons. In the rabbit or mouse ocular models of infection, expression of the first 1.5 kb of LAT coding sequences is sufficient for and necessary for wild-type levels of spontaneous reactivation from latency. The antiapoptosis functions of LAT, which maps to the same 1.5 kb of LAT, are important for the latency-reactivation cycle because replacement of LAT with other antiapoptosis genes (the baculovirus IAP gene or the bovine herpesvirus type 1 latency-related gene) restores wild-type levels of reactivation to a LAT null mutant. A recent study identified a micro-RNA within LAT that can inhibit apoptosis (Gupta et al, Nature 442: 82-85). In this study, the authors analyzed the first 1.5 kb of LAT for additional small RNAs that may have regulatory functions. Two LAT-specific small RNAs were detected in productively infected human neuroblastoma cells within the first 1.5 kb of LAT, in a region that is important for inhibiting apoptosis. Although these small RNAs possess extensive secondary structure and a stem-loop structure, bands migrating near 23 bases were not detected suggesting these small RNAs are not true micro-RNAs. Both of the small LAT-specific RNAs have the potential to base pair with the ICP4 mRNA. These two small LAT RNAs may play a role in the latency-reactivation cycle by reducing apoptosis and/or by reducing ICP4 RNA expression.

  3. An atlas of human long non-coding RNAs with accurate 5′ ends

    KAUST Repository

    Hon, Chung-Chau

    2017-02-28

    Long non-coding RNAs (lncRNAs) are largely heterogeneous and functionally uncharacterized. Here, using FANTOM5 cap analysis of gene expression (CAGE) data, we integrate multiple transcript collections to generate a comprehensive atlas of 27,919 human lncRNA genes with high-confidence 5′ ends and expression profiles across 1,829 samples from the major human primary cell types and tissues. Genomic and epigenomic classification of these lncRNAs reveals that most intergenic lncRNAs originate from enhancers rather than from promoters. Incorporating genetic and expression data, we show that lncRNAs overlapping trait-associated single nucleotide polymorphisms are specifically expressed in cell types relevant to the traits, implicating these lncRNAs in multiple diseases. We further demonstrate that lncRNAs overlapping expression quantitative trait loci (eQTL)-associated single nucleotide polymorphisms of messenger RNAs are co-expressed with the corresponding messenger RNAs, suggesting their potential roles in transcriptional regulation. Combining these findings with conservation data, we identify 19,175 potentially functional lncRNAs in the human genome.

  4. 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.

  5. 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.

  6. Inhibition of Reporter Genes by Small Interfering RNAs in Cell Culture and Living Fish

    DEFF Research Database (Denmark)

    Larashati, Sekar; Schyth, Brian Dall; Lorenzen, Niels

    2011-01-01

    be used to observe the knock down effect by siRNAs designed to target these reporters. One aim of this project is to verify the specific knock down effect of siRNAs in cell culture and in living fish and to establish easy-read out models for testing the effect especially in vivo. Cell culture from human...... coinjection and the assay is important in order to detect knock down by siRNA. Our experiment reveal in vivo knock down at 72 hours post injection of reporter gene and siRNA, but further dose-response experiments are required to confirm specifity....

  7. Computational Characterization of Exogenous MicroRNAs that Can Be Transferred into Human Circulation.

    Directory of Open Access Journals (Sweden)

    Jiang Shu

    Full Text Available MicroRNAs have been long considered synthesized endogenously until very recent discoveries showing that human can absorb dietary microRNAs from animal and plant origins while the mechanism remains unknown. Compelling evidences of microRNAs from rice, milk, and honeysuckle transported to human blood and tissues have created a high volume of interests in the fundamental questions that which and how exogenous microRNAs can be transferred into human circulation and possibly exert functions in humans. Here we present an integrated genomics and computational analysis to study the potential deciding features of transportable microRNAs. Specifically, we analyzed all publicly available microRNAs, a total of 34,612 from 194 species, with 1,102 features derived from the microRNA sequence and structure. Through in-depth bioinformatics analysis, 8 groups of discriminative features have been used to characterize human circulating microRNAs and infer the likelihood that a microRNA will get transferred into human circulation. For example, 345 dietary microRNAs have been predicted as highly transportable candidates where 117 of them have identical sequences with their homologs in human and 73 are known to be associated with exosomes. Through a milk feeding experiment, we have validated 9 cow-milk microRNAs in human plasma using microRNA-sequencing analysis, including the top ranked microRNAs such as bta-miR-487b, miR-181b, and miR-421. The implications in health-related processes have been illustrated in the functional analysis. This work demonstrates the data-driven computational analysis is highly promising to study novel molecular characteristics of transportable microRNAs while bypassing the complex mechanistic details.

  8. Computational Characterization of Exogenous MicroRNAs that Can Be Transferred into Human Circulation

    Science.gov (United States)

    Shu, Jiang; Chiang, Kevin; Zempleni, Janos; Cui, Juan

    2015-01-01

    MicroRNAs have been long considered synthesized endogenously until very recent discoveries showing that human can absorb dietary microRNAs from animal and plant origins while the mechanism remains unknown. Compelling evidences of microRNAs from rice, milk, and honeysuckle transported to human blood and tissues have created a high volume of interests in the fundamental questions that which and how exogenous microRNAs can be transferred into human circulation and possibly exert functions in humans. Here we present an integrated genomics and computational analysis to study the potential deciding features of transportable microRNAs. Specifically, we analyzed all publicly available microRNAs, a total of 34,612 from 194 species, with 1,102 features derived from the microRNA sequence and structure. Through in-depth bioinformatics analysis, 8 groups of discriminative features have been used to characterize human circulating microRNAs and infer the likelihood that a microRNA will get transferred into human circulation. For example, 345 dietary microRNAs have been predicted as highly transportable candidates where 117 of them have identical sequences with their homologs in human and 73 are known to be associated with exosomes. Through a milk feeding experiment, we have validated 9 cow-milk microRNAs in human plasma using microRNA-sequencing analysis, including the top ranked microRNAs such as bta-miR-487b, miR-181b, and miR-421. The implications in health-related processes have been illustrated in the functional analysis. This work demonstrates the data-driven computational analysis is highly promising to study novel molecular characteristics of transportable microRNAs while bypassing the complex mechanistic details. PMID:26528912

  9. Epigenetic regulation of normal human mammary cell type-specific miRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Vrba, Lukas [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Inst. of Plant Molecular Biology, Ceske Budejovice (Czech Republic). Biology Centre ASCR; Garbe, James C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Stampfer, Martha R. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Futscher, Bernard W. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center and Dept. of Pharmacology & Toxicology

    2011-08-26

    Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linked to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.

  10. Identification and Characterization of MicroRNAs in Small Brown Planthopper (Laodephax striatellus) by Next-Generation Sequencing

    Science.gov (United States)

    Lou, Yonggen; Cheng, Jia'an; Zhang, Hengmu; Xu, Jian-Hong

    2014-01-01

    MicroRNAs (miRNAs) are endogenous non-coding small RNAs that regulate gene expression at the post-transcriptional level and are thought to play critical roles in many metabolic activities in eukaryotes. The small brown planthopper (Laodephax striatellus Fallén), one of the most destructive agricultural pests, causes great damage to crops including rice, wheat, and maize. However, information about the genome of L. striatellus is limited. In this study, a small RNA library was constructed from a mixed L. striatellus population and sequenced by Solexa sequencing technology. A total of 501 mature miRNAs were identified, including 227 conserved and 274 novel miRNAs belonging to 125 and 250 families, respectively. Sixty-nine conserved miRNAs that are included in 38 families are predicted to have an RNA secondary structure typically found in miRNAs. Many miRNAs were validated by stem-loop RT-PCR. Comparison with the miRNAs in 84 animal species from miRBase showed that the conserved miRNA families we identified are highly conserved in the Arthropoda phylum. Furthermore, miRanda predicted 2701 target genes for 378 miRNAs, which could be categorized into 52 functional groups annotated by gene ontology. The function of miRNA target genes was found to be very similar between conserved and novel miRNAs. This study of miRNAs in L. striatellus will provide new information and enhance the understanding of the role of miRNAs in the regulation of L. striatellus metabolism and development. PMID:25057821

  11. Identification and characterization of microRNAs in small brown planthopper (Laodephax striatellus by next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Guoyan Zhou

    Full Text Available MicroRNAs (miRNAs are endogenous non-coding small RNAs that regulate gene expression at the post-transcriptional level and are thought to play critical roles in many metabolic activities in eukaryotes. The small brown planthopper (Laodephax striatellus Fallén, one of the most destructive agricultural pests, causes great damage to crops including rice, wheat, and maize. However, information about the genome of L. striatellus is limited. In this study, a small RNA library was constructed from a mixed L. striatellus population and sequenced by Solexa sequencing technology. A total of 501 mature miRNAs were identified, including 227 conserved and 274 novel miRNAs belonging to 125 and 250 families, respectively. Sixty-nine conserved miRNAs that are included in 38 families are predicted to have an RNA secondary structure typically found in miRNAs. Many miRNAs were validated by stem-loop RT-PCR. Comparison with the miRNAs in 84 animal species from miRBase showed that the conserved miRNA families we identified are highly conserved in the Arthropoda phylum. Furthermore, miRanda predicted 2701 target genes for 378 miRNAs, which could be categorized into 52 functional groups annotated by gene ontology. The function of miRNA target genes was found to be very similar between conserved and novel miRNAs. This study of miRNAs in L. striatellus will provide new information and enhance the understanding of the role of miRNAs in the regulation of L. striatellus metabolism and development.

  12. Linked biogenesis and degradation of human non-coding RNAs

    DEFF Research Database (Denmark)

    Andersen, Peter Refsing

    2012-01-01

    funktionelle roller majoriteten af disse transkripter spiller. De molekylære mekanismer bag dannelsen og nedbrydningen af både de nye klasser af ikke-kodende RNA transkripter og af flere etablerede klasser af ikke-kodende RNA transkripter er relativt ukendte i humane celler. Vi har undersøgt flere aspekter af......-5’ exoribonukleaseaktivitet i organismer så forskel¬lige som gær og mennesker. Gennem dette arbejde har vi vist at de fleste små RNAs molekyler, der oprinder fra humane protein-kodende gener (fraregnet mikroRNAer og introniske snoRNAer) repræsenterer RNA-nedbrydningssignaturer af specifikke molekylære processeringshændelser...... i dannelsen af pre-messenger RNA. Endvidere har vi fundet at 3’-forlængede humane introniske snoRNA-transkripter er substrater for RNA exosomet, men at produktionen af modne introniske snoRNAer ikke er afhængig af RNA exosomet, hvilket er ulig mekanismerne i gær, som man ellers have regnet med ville...

  13. Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins?

    Science.gov (United States)

    Nakanishi, Kotaro

    2016-09-01

    RNA silencing is a eukaryote-specific phenomenon in which microRNAs and small interfering RNAs degrade messenger RNAs containing a complementary sequence. To this end, these small RNAs need to be loaded onto an Argonaute protein (AGO protein) to form the effector complex referred to as RNA-induced silencing complex (RISC). RISC assembly undergoes multiple and sequential steps with the aid of Hsc70/Hsp90 chaperone machinery. The molecular mechanisms for this assembly process remain unclear, despite their significance for the development of gene silencing techniques and RNA interference-based therapeutics. This review dissects the currently available structures of AGO proteins and proposes models and hypotheses for RISC assembly, covering the conformation of unloaded AGO proteins, the chaperone-assisted duplex loading, and the slicer-dependent and slicer-independent duplex separation. The differences in the properties of RISC between prokaryotes and eukaryotes will also be clarified. WIREs RNA 2016, 7:637-660. doi: 10.1002/wrna.1356 For further resources related to this article, please visit the WIREs website. © 2016 The Authors. WIREs RNA published by Wiley Periodicals, Inc.

  14. Circular RNAs: Biogenesis, Function and Role in Human Diseases

    Directory of Open Access Journals (Sweden)

    John Greene

    2017-06-01

    Full Text Available Circular RNAs (circRNAs are currently classed as non-coding RNA (ncRNA that, unlike linear RNAs, form covalently closed continuous loops and act as gene regulators in mammals. They were originally thought to represent errors in splicing and considered to be of low abundance, however, there is now an increased appreciation of their important function in gene regulation. circRNAs are differentially generated by backsplicing of exons or from lariat introns. Unlike linear RNA, the 3′ and 5′ ends normally present in an RNA molecule have been joined together by covalent bonds leading to circularization. Interestingly, they have been found to be abundant, evolutionally conserved and relatively stable in the cytoplasm. These features confer numerous potential functions to circRNAs, such as acting as miRNA sponges, or binding to RNA-associated proteins to form RNA-protein complexes that regulate gene transcription. It has been proposed that circRNA regulate gene expression at the transcriptional or post-transcriptional level by interacting with miRNAs and that circRNAs may have a role in regulating miRNA function in cancer initiation and progression. circRNAs appear to be more often downregulated in tumor tissue compared to normal tissue and this may be due to (i errors in the back-splice machinery in malignant tissues, (ii degradation of circRNAs by deregulated miRNAs in tumor tissue, or (iii increasing cell proliferation leading to a reduction of circRNAs. circRNAs have been identified in exosomes and more recently, chromosomal translocations in cancer have been shown to generate aberrant fusion-circRNAs associated with resistance to drug treatments. In addition, though originally thought to be non-coding, there is now increasing evidence to suggest that select circRNAs can be translated into functional proteins. Although much remains to be elucidated about circRNA biology and mechanisms of gene regulation, these ncRNAs are quickly emerging as

  15. Small RNAs from Bemisia tabaci are transferred to Solanum lycopersicum phloem during feeding

    Directory of Open Access Journals (Sweden)

    Paula J.M. Van Kleeff

    2016-11-01

    Full Text Available The phloem-feeding whitefly Bemisia tabaci is a serious pest to a broad range of host plants, including many economically important crops such as tomato. These insects serve as a vector for various devastating plant viruses. It is known that whiteflies are capable of manipulating host-defense responses, potentially mediated by effector molecules in the whitefly saliva. We hypothesized that, beside putative effector proteins, small RNAs (sRNA are delivered by B. tabaci into the phloem, where they may play a role in manipulating host plant defenses. There is already evidence to suggest that sRNAs can mediate the host-pathogen dialogue. It has been shown that Botrytis cinerea, the causal agent of gray mold disease, takes advantage of the plant sRNA machinery to selectively silence host genes involved in defense signaling.Here we identified sRNAs originating from B. tabaci in the phloem of tomato plants on which they are feeding. sRNAs were isolated and sequenced from tomato phloem of whitefly-infested and control plants as well as from the nymphs themselves, control leaflets and from the infested leaflets. Using stem-loop RT-PCR, three whitefly sRNAs have been verified to be present in whitefly-infested leaflets that were also present in the whitefly-infested phloem sample. Our results show that whitefly sRNAs are indeed present in tomato tissues upon feeding, and they appear to be mobile in the phloem. Their role in the host-insect interaction can now be investigated.

  16. miRNAs in Human Subcutaneous Adipose Tissue

    DEFF Research Database (Denmark)

    Kristensen, Malene M.; Davidsen, Peter K.; Vigelso, Andreas

    2017-01-01

    Objective Obesity is central in the development of insulin resistance. However, the underlying mechanisms still need elucidation. Dysregulated microRNAs (miRNAs; post-transcriptional regulators) in adipose tissue may present an important link. Methods The miRNA expression in subcutaneous adipose ...

  17. Systematically profiling and annotating long intergenic non-coding RNAs in human embryonic stem cell.

    Science.gov (United States)

    Tang, Xing; Hou, Mei; Ding, Yang; Li, Zhaohui; Ren, Lichen; Gao, Ge

    2013-01-01

    While more and more long intergenic non-coding RNAs (lincRNAs) were identified to take important roles in both maintaining pluripotency and regulating differentiation, how these lincRNAs may define and drive cell fate decisions on a global scale are still mostly elusive. Systematical profiling and comprehensive annotation of embryonic stem cells lincRNAs may not only bring a clearer big picture of these novel regulators but also shed light on their functionalities. Based on multiple RNA-Seq datasets, we systematically identified 300 human embryonic stem cell lincRNAs (hES lincRNAs). Of which, one forth (78 out of 300) hES lincRNAs were further identified to be biasedly expressed in human ES cells. Functional analysis showed that they were preferentially involved in several early-development related biological processes. Comparative genomics analysis further suggested that around half of the identified hES lincRNAs were conserved in mouse. To facilitate further investigation of these hES lincRNAs, we constructed an online portal for biologists to access all their sequences and annotations interactively. In addition to navigation through a genome browse interface, users can also locate lincRNAs through an advanced query interface based on both keywords and expression profiles, and analyze results through multiple tools. By integrating multiple RNA-Seq datasets, we systematically characterized and annotated 300 hES lincRNAs. A full functional web portal is available freely at http://scbrowse.cbi.pku.edu.cn. As the first global profiling and annotating of human embryonic stem cell lincRNAs, this work aims to provide a valuable resource for both experimental biologists and bioinformaticians.

  18. Differential expression of small RNAs under chemical stress and fed-batch fermentation in E. coli

    DEFF Research Database (Denmark)

    Rau, Martin Holm; Nielsen, Alex Toftgaard; Long, Katherine

    2015-01-01

    Bacterial small RNAs (sRNAs) are recognized as posttranscriptional regulators involved in the control of bacterial lifestyle and adaptation to stressful conditions. Although chemical stress due to the toxicity of precursor and product compounds is frequently encountered in microbial bioprocessing...

  19. Characterizing ncRNAs in human pathogenic protists using high-throughput sequencing technology

    Directory of Open Access Journals (Sweden)

    Lesley Joan Collins

    2011-12-01

    Full Text Available ncRNAs are key genes in many human diseases including cancer and viral infection, as well as providing critical functions in pathogenic organisms such as fungi, bacteria, viruses and protists. Until now the identification and characterization of ncRNAs associated with disease has been slow or inaccurate requiring many years of testing to understand complicated RNA and protein gene relationships. High-throughput sequencing now offers the opportunity to characterize miRNAs, siRNAs, snoRNAs and long ncRNAs on a genomic scale making it faster and easier to clarify how these ncRNAs contribute to the disease state. However, this technology is still relatively new, and ncRNA discovery is not an application of high priority for streamlined bioinformatics. Here we summarize background concepts and practical approaches for ncRNA analysis using high-throughput sequencing, and how it relates to understanding human disease. As a case study, we focus on the parasitic protists Giardia lamblia and Trichomonas vaginalis, where large evolutionary distance has meant difficulties in comparing ncRNAs with those from model eukaryotes. A combination of biological, computational and sequencing approaches has enabled easier classification of ncRNA classes such as snoRNAs, but has also aided the identification of novel classes. It is hoped that a higher level of understanding of ncRNA expression and interaction may aid in the development of less harsh treatment for protist-based diseases.

  20. LncRNAWiki: harnessing community knowledge in collaborative curation of human long non-coding RNAs

    KAUST Repository

    Ma, L.; Li, A.; Zou, D.; Xu, X.; Xia, L.; Yu, J.; Bajic, Vladimir B.; Zhang, Z.

    2014-01-01

    Long non-coding RNAs (lncRNAs) perform a diversity of functions in numerous important biological processes and are implicated in many human diseases. In this report we present lncRNAWiki (http://lncrna.big.ac.cn), a wiki-based platform that is open

  1. The role of miRNAs in human papilloma virus (HPV)-associated cancers

    DEFF Research Database (Denmark)

    Lajer, C B; Garnæs, E; Friis-Hansen, L

    2012-01-01

    Although the role of human papilloma virus (HPV) in cervical squamous cell carcinoma (CSCC) is well established, the role in head and neck SCC (HNSCC) is less clear. MicroRNAs (miRNAs) have a role in the cancer development, and HPV status may affect the miRNA expression pattern in HNSCC. To explore...

  2. Specific and Novel microRNAs Are Regulated as Response to Fungal Infection in Human Dendritic Cells

    Science.gov (United States)

    Dix, Andreas; Czakai, Kristin; Leonhardt, Ines; Schäferhoff, Karin; Bonin, Michael; Guthke, Reinhard; Einsele, Hermann; Kurzai, Oliver; Löffler, Jürgen; Linde, Jörg

    2017-01-01

    Within the last two decades, the incidence of invasive fungal infections has been significantly increased. They are characterized by high mortality rates and are often caused by Candida albicans and Aspergillus fumigatus. The increasing number of infections underlines the necessity for additional anti-fungal therapies, which require extended knowledge of gene regulations during fungal infection. MicroRNAs are regulators of important cellular processes, including the immune response. By analyzing their regulation and impact on target genes, novel therapeutic and diagnostic approaches may be developed. Here, we examine the role of microRNAs in human dendritic cells during fungal infection. Dendritic cells represent the bridge between the innate and the adaptive immune systems. Therefore, analysis of gene regulation of dendritic cells is of particular significance. By applying next-generation sequencing of small RNAs, we quantify microRNA expression in monocyte-derived dendritic cells after 6 and 12 h of infection with C. albicans and A. fumigatus as well as treatment with lipopolysaccharides (LPS). We identified 26 microRNAs that are differentially regulated after infection by the fungi or LPS. Three and five of them are specific for fungal infections after 6 and 12 h, respectively. We further validated interactions of miR-132-5p and miR-212-5p with immunological relevant target genes, such as FKBP1B, KLF4, and SPN, on both RNA and protein level. Our results indicate that these microRNAs fine-tune the expression of immune-related target genes during fungal infection. Beyond that, we identified previously undiscovered microRNAs. We validated three novel microRNAs via qRT-PCR. A comparison with known microRNAs revealed possible relations with the miR-378 family and miR-1260a/b for two of them, while the third one features a unique sequence with no resemblance to known microRNAs. In summary, this study analyzes the effect of known microRNAs in dendritic cells during

  3. 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.

  4. High-throughput sequencing identification and characterization of potentially adhesion-related small RNAs in Streptococcus mutans.

    Science.gov (United States)

    Zhu, Wenhui; Liu, Shanshan; Liu, Jia; Zhou, Yan; Lin, Huancai

    2018-05-01

    Adherence capacity is one of the principal virulence factors of Streptococcus mutans, and adhesion virulence factors are controlled by small RNAs (sRNAs) at the post-transcriptional level in various bacteria. Here, we aimed to identify and decipher putative adhesion-related sRNAs in clinical strains of S. mutans. RNA deep-sequencing was performed to identify potential sRNAs under different adhesion conditions. The expression of sRNAs was analysed by quantitative real-time PCR (qRT-PCR), and bioinformatic methods were used to predict the functional characteristics of sRNAs. A total of 736 differentially expressed candidate sRNAs were predicted, and these included 352 sRNAs located on the antisense to mRNA (AM) and 384 sRNAs in intergenic regions (IGRs). The top 7 differentially expressed sRNAs were successfully validated by qRT-PCR in UA159, and 2 of these were further confirmed in 100 clinical isolates. Moreover, the sequences of two sRNAs were conserved in other Streptococcus species, indicating a conserved role in such closely related species. A good correlation between the expression of sRNAs and the adhesion of 100 clinical strains was observed, which, combined with GO and KEGG, provides a perspective for the comprehension of sRNA function annotation. This study revealed a multitude of novel putative adhesion-related sRNAs in S. mutans and contributed to a better understanding of information concerning the transcriptional regulation of adhesion in S. mutans.

  5. 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.

  6. Web-based tools for microRNAs involved in human cancer.

    Science.gov (United States)

    Mar-Aguilar, Fermín; Rodríguez-Padilla, Cristina; Reséndez-Pérez, Diana

    2016-06-01

    MicroRNAs (miRNAs/miRs) are a family of small, endogenous and evolutionarily-conserved non-coding RNAs that are involved in the regulation of several cellular and functional processes. miRNAs can act as oncogenes or tumor suppressors in all types of cancer, and could be used as prognostic and diagnostic biomarkers. Databases and computational algorithms are behind the majority of the research performed on miRNAs. These tools assemble and curate the relevant information on miRNAs and present it in a user-friendly manner. The current review presents 14 online databases that address every aspect of miRNA cancer research. Certain databases focus on miRNAs and a particular type of cancer, while others analyze the behavior of miRNAs in different malignancies at the same time. Additional databases allow researchers to search for mutations in miRNAs or their targets, and to review the naming history of a particular miRNA. All these databases are open-access, and are a valuable tool for those researchers working with these molecules, particularly those who lack access to an advanced computational infrastructure.

  7. Identification of novel growth phase- and media-dependent small non-coding RNAs in Streptococcus pyogenes M49 using intergenic tiling arrays

    Directory of Open Access Journals (Sweden)

    Patenge Nadja

    2012-10-01

    Full Text Available Abstract Background Small non-coding RNAs (sRNAs have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. Genome-wide screening methods have been successfully applied in Gram-negative bacteria to identify sRNA regulators. Many sRNAs are well characterized, including their target mRNAs and mode of action. In comparison, little is known about sRNAs in Gram-positive pathogens. In this study, we identified novel sRNAs in the exclusively human pathogen Streptococcus pyogenes M49 (Group A Streptococcus, GAS M49, employing a whole genome intergenic tiling array approach. GAS is an important pathogen that causes diseases ranging from mild superficial infections of the skin and mucous membranes of the naso-pharynx, to severe toxic and invasive diseases. Results We identified 55 putative sRNAs in GAS M49 that were expressed during growth. Of these, 42 were novel. Some of the newly-identified sRNAs belonged to one of the common non-coding RNA families described in the Rfam database. Comparison of the results of our screen with the outcome of two recently published bioinformatics tools showed a low level of overlap between putative sRNA genes. Previously, 40 potential sRNAs have been reported to be expressed in a GAS M1T1 serotype, as detected by a whole genome intergenic tiling array approach. Our screen detected 12 putative sRNA genes that were expressed in both strains. Twenty sRNA candidates appeared to be regulated in a medium-dependent fashion, while eight sRNA genes were regulated throughout growth in chemically defined medium. Expression of candidate genes was verified by reverse transcriptase-qPCR. For a subset of sRNAs, the transcriptional start was determined by 5′ rapid amplification of cDNA ends-PCR (RACE-PCR analysis. Conclusions In accord with the results of previous studies, we found little overlap between different screening methods, which underlines the fact that a comprehensive analysis of sRNAs

  8. 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.

  9. Small RNAs in plants: Recent development and application for crop improvement

    OpenAIRE

    Ayushi eKamthan; Abira eChaudhuri; Mohan eKamthan; Asis eDatta

    2015-01-01

    The phenomenon of RNA interference (RNAi) which involves sequence-specific gene regulation by small non-coding RNAs, i.e., small interfering RNA (siRNA) and microRNA (miRNA) has emerged as one of most powerful approaches for crop improvement. RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species. This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits. RN...

  10. Genomic dissection of small RNAs in wild rice (Oryza rufipogon): lessons for rice domestication.

    Science.gov (United States)

    Wang, Yu; Bai, Xuefei; Yan, Chenghai; Gui, Yiejie; Wei, Xinghua; Zhu, Qian-Hao; Guo, Longbiao; Fan, Longjiang

    2012-11-01

    The lack of a MIRNA set and genome sequence of wild rice (Oryza rufipogon) has prevented us from determining the role of MIRNA genes in rice domestication. In this study, a genome, three small RNA populations and a degradome of O. rufipogon were sequenced by Illumina platform and the expression levels of microRNAs (miRNAs) were investigated by miRNA chips. A de novo O. rufipogon genome was assembled using c. 55× coverage of raw sequencing data and a total of 387 MIRNAs were identified in the O. rufipogon genome based on c. 5.2 million unique small RNA reads from three different tissues of O. rufipogon. Of these, O. rufipogon MIRNAs, 259 were not found in the cultivated rice, suggesting a loss of these MIRNAs in the cultivated rice. We also found that 48 MIRNAs were novel in the cultivated rice, suggesting that they were potential targets of domestication selection. Some miRNAs showed significant expression differences between wild and cultivated rice, suggesting that expression of miRNA could also be a target of domestication, as demonstrated for the miR164 family. Our results illustrated that MIRNA genes, like protein-coding genes, might have been significantly shaped during rice domestication and could be one of the driving forces that contributed to rice domestication. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  11. Identification of Novel Long Non-coding and Circular RNAs in Human Papillomavirus-Mediated Cervical Cancer

    Directory of Open Access Journals (Sweden)

    Hongbo Wang

    2017-09-01

    Full Text Available Cervical cancer is the third most common cancer worldwide and the fourth leading cause of cancer-associated mortality in women. Accumulating evidence indicates that long non-coding RNAs (lncRNAs and circular RNAs (circRNAs may play key roles in the carcinogenesis of different cancers; however, little is known about the mechanisms of lncRNAs and circRNAs in the progression and metastasis of cervical cancer. In this study, we explored the expression profiles of lncRNAs, circRNAs, miRNAs, and mRNAs in HPV16 (human papillomavirus genotype 16 mediated cervical squamous cell carcinoma and matched adjacent non-tumor (ATN tissues from three patients with high-throughput RNA sequencing (RNA-seq. In total, we identified 19 lncRNAs, 99 circRNAs, 28 miRNAs, and 304 mRNAs that were commonly differentially expressed (DE in different patients. Among the non-coding RNAs, 3 lncRNAs and 44 circRNAs are novel to our knowledge. Functional enrichment analysis showed that DE lncRNAs, miRNAs, and mRNAs were enriched in pathways crucial to cancer as well as other gene ontology (GO terms. Furthermore, the co-expression network and function prediction suggested that all 19 DE lncRNAs could play different roles in the carcinogenesis and development of cervical cancer. The competing endogenous RNA (ceRNA network based on DE coding and non-coding RNAs showed that each miRNA targeted a number of lncRNAs and circRNAs. The link between part of the miRNAs in the network and cervical cancer has been validated in previous studies, and these miRNAs targeted the majority of the novel non-coding RNAs, thus suggesting that these novel non-coding RNAs may be involved in cervical cancer. Taken together, our study shows that DE non-coding RNAs could be further developed as diagnostic and therapeutic biomarkers of cervical cancer. The complex ceRNA network also lays the foundation for future research of the roles of coding and non-coding RNAs in cervical cancer.

  12. Identification of Novel Long Non-coding and Circular RNAs in Human Papillomavirus-Mediated Cervical Cancer

    Science.gov (United States)

    Wang, Hongbo; Zhao, Yingchao; Chen, Mingyue; Cui, Jie

    2017-01-01

    Cervical cancer is the third most common cancer worldwide and the fourth leading cause of cancer-associated mortality in women. Accumulating evidence indicates that long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) may play key roles in the carcinogenesis of different cancers; however, little is known about the mechanisms of lncRNAs and circRNAs in the progression and metastasis of cervical cancer. In this study, we explored the expression profiles of lncRNAs, circRNAs, miRNAs, and mRNAs in HPV16 (human papillomavirus genotype 16) mediated cervical squamous cell carcinoma and matched adjacent non-tumor (ATN) tissues from three patients with high-throughput RNA sequencing (RNA-seq). In total, we identified 19 lncRNAs, 99 circRNAs, 28 miRNAs, and 304 mRNAs that were commonly differentially expressed (DE) in different patients. Among the non-coding RNAs, 3 lncRNAs and 44 circRNAs are novel to our knowledge. Functional enrichment analysis showed that DE lncRNAs, miRNAs, and mRNAs were enriched in pathways crucial to cancer as well as other gene ontology (GO) terms. Furthermore, the co-expression network and function prediction suggested that all 19 DE lncRNAs could play different roles in the carcinogenesis and development of cervical cancer. The competing endogenous RNA (ceRNA) network based on DE coding and non-coding RNAs showed that each miRNA targeted a number of lncRNAs and circRNAs. The link between part of the miRNAs in the network and cervical cancer has been validated in previous studies, and these miRNAs targeted the majority of the novel non-coding RNAs, thus suggesting that these novel non-coding RNAs may be involved in cervical cancer. Taken together, our study shows that DE non-coding RNAs could be further developed as diagnostic and therapeutic biomarkers of cervical cancer. The complex ceRNA network also lays the foundation for future research of the roles of coding and non-coding RNAs in cervical cancer. PMID:28970820

  13. 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.

  14. Role of the Box C/D Motif in Localization of Small Nucleolar RNAs to Coiled Bodies and Nucleoli

    Science.gov (United States)

    Narayanan, Aarthi; Speckmann, Wayne; Terns, Rebecca; Terns, Michael P.

    1999-01-01

    Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C′, box D, and the 3′ terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs. PMID:10397754

  15. Analysis of Small RNAs in Streptococcus mutans under Acid Stress-A New Insight for Caries Research.

    Science.gov (United States)

    Liu, Shanshan; Tao, Ye; Yu, Lixia; Zhuang, Peilin; Zhi, Qinghui; Zhou, Yan; Lin, Huancai

    2016-09-14

    Streptococcus mutans (S. mutans) is the major clinical pathogen responsible for dental caries. Its acid tolerance has been identified as a significant virulence factor for its survival and cariogenicity in acidic conditions. Small RNAs (sRNAs) are recognized as key regulators of virulence and stress adaptation. Here, we constructed three libraries of sRNAs with small size exposed to acidic conditions for the first time, followed by verification using qRT-PCR. The levels of two sRNAs and target genes predicted to be bioinformatically related to acid tolerance were further evaluated under different acid stress conditions (pH 7.5, 6.5, 5.5, and 4.5) at three time points (0.5, 1, and 2 h). Meanwhile, bacterial growth characteristics and vitality were assessed. We obtained 1879 sRNAs with read counts of at least 100. One hundred and ten sRNAs were perfectly mapped to reported msRNAs in S. mutans. Ten out of 18 sRNAs were validated by qRT-PCR. The survival of bacteria declined as the acid was increased from pH 7.5 to 4.5 at each time point. The bacteria can proliferate under each pH except pH 4.5 with time. The levels of sRNAs gradually decreased from pH 7.5 to 5.5, and slightly increased in pH 4.5; however, the expression levels of target mRNAs were up-regulated in acidic conditions than in pH 7.5. These results indicate that some sRNAs are specially induced at acid stress conditions, involving acid adaptation, and provide a new insight into exploring the complex acid tolerance for S. mutans.

  16. Analysis of Small RNAs in Streptococcus mutans under Acid Stress—A New Insight for Caries Research

    Directory of Open Access Journals (Sweden)

    Shanshan Liu

    2016-09-01

    Full Text Available Streptococcus mutans (S. mutans is the major clinical pathogen responsible for dental caries. Its acid tolerance has been identified as a significant virulence factor for its survival and cariogenicity in acidic conditions. Small RNAs (sRNAs are recognized as key regulators of virulence and stress adaptation. Here, we constructed three libraries of sRNAs with small size exposed to acidic conditions for the first time, followed by verification using qRT-PCR. The levels of two sRNAs and target genes predicted to be bioinformatically related to acid tolerance were further evaluated under different acid stress conditions (pH 7.5, 6.5, 5.5, and 4.5 at three time points (0.5, 1, and 2 h. Meanwhile, bacterial growth characteristics and vitality were assessed. We obtained 1879 sRNAs with read counts of at least 100. One hundred and ten sRNAs were perfectly mapped to reported msRNAs in S. mutans. Ten out of 18 sRNAs were validated by qRT-PCR. The survival of bacteria declined as the acid was increased from pH 7.5 to 4.5 at each time point. The bacteria can proliferate under each pH except pH 4.5 with time. The levels of sRNAs gradually decreased from pH 7.5 to 5.5, and slightly increased in pH 4.5; however, the expression levels of target mRNAs were up-regulated in acidic conditions than in pH 7.5. These results indicate that some sRNAs are specially induced at acid stress conditions, involving acid adaptation, and provide a new insight into exploring the complex acid tolerance for S. mutans.

  17. Computational investigation of small RNAs in the establishment of root nodules and arbuscular mycorrhiza in leguminous plants.

    Science.gov (United States)

    Jin, Danfeng; Meng, Xianwen; Wang, Yue; Wang, Jingjing; Zhao, Yuhua; Chen, Ming

    2018-01-03

    Many small RNAs have been confirmed to play important roles in the development of root nodules and arbuscular mycorrhiza. In this study, we carried out the identification of certain small RNAs in leguminous plants (Medicago truncatula, soybean, peanut and common bean), such as miRNAs, tRFs and srRNAs, as well as the computational investigation of their regulations. Thirty miRNAs were predicted to be involved in establishing root nodules and mycorrhiza, and 12 of them were novel in common bean and peanut. The generation of tRFs in M. truncatula was not associated with tRNA gene frequencies and codon usage. Six tRFs exhibited different expressions in mycorrhiza and root nodules. Moreover, srRNA 5.8S in M. truncatula was generated from the regions with relatively low conservation at the rRNA 3' terminal. The protein-protein interactions between the proteins encoded by the target genes of miRNAs, tRFs and srRNAs were computed. The regulation of these three types of sRNAs in the symbiosis between leguminous plants and microorganisms is not a single regulation of certain signaling or metabolic pathways but a global regulation for the plants to own growth or specific events in symbiosis.

  18. Vitamin D activation of functionally distinct regulatory miRNAs in primary human osteoblasts.

    Science.gov (United States)

    Lisse, Thomas S; Chun, Rene F; Rieger, Sandra; Adams, John S; Hewison, Martin

    2013-06-01

    When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on posttranscriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOBs) identified human miRNAs that were differentially regulated by >1.5-fold following treatment with 1,25D (10 nM, 6 hours), which included miRNAs 637 and 1228. Quantitative reverse transcription PCR analyses showed that the host gene for miR-1228, low-density lipoprotein receptor-related protein 1 (LRP1), was coinduced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1-10 nM, 6 hours). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K), respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, small interfering RNA (siRNA) inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated downregulation of BMP2K protein expression. This was associated with suppression of prodifferentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed posttranscriptional mechanisms. Copyright © 2013 American Society for Bone and

  19. Expression profiling of microRNAs in human bone tissue from postmenopausal women.

    Science.gov (United States)

    De-Ugarte, Laura; Serra-Vinardell, Jenny; Nonell, Lara; Balcells, Susana; Arnal, Magdalena; Nogues, Xavier; Mellibovsky, Leonardo; Grinberg, Daniel; Diez-Perez, Adolfo; Garcia-Giralt, Natalia

    2018-01-01

    Bone tissue is composed of several cell types, which express their own microRNAs (miRNAs) that will play a role in cell function. The set of total miRNAs expressed in all cell types configures the specific signature of the bone tissue in one physiological condition. The aim of this study was to explore the miRNA expression profile of bone tissue from postmenopausal women. Tissue was obtained from trabecular bone and was analyzed in fresh conditions (n = 6). Primary osteoblasts were also obtained from trabecular bone (n = 4) and human osteoclasts were obtained from monocyte precursors after in vitro differentiation (n = 5). MicroRNA expression profiling was obtained for each sample by microarray and a global miRNA analysis was performed combining the data acquired in all the microarray experiments. From the 641 miRNAs detected in bone tissue samples, 346 (54%) were present in osteoblasts and/or osteoclasts. The other 46% were not identified in any of the bone cells analyzed. Intersection of osteoblast and osteoclast arrays identified 101 miRNAs shared by both cell types, which accounts for 30-40% of miRNAs detected in these cells. In osteoblasts, 266 miRNAs were detected, of which 243 (91%) were also present in the total bone array, representing 38% of all bone miRNAs. In osteoclasts, 340 miRNAs were detected, of which 196 (58%) were also present in the bone tissue array, representing 31% of all miRNAs detected in total bone. These analyses provide an overview of miRNAs expressed in bone tissue, broadening our knowledge in the microRNA field.

  20. 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.

  1. 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...

  2. Differential Expression Profile of lncRNAs from Primary Human Hepatocytes Following DEET and Fipronil Exposure

    Science.gov (United States)

    Wallace, Andrew D.; Hodgson, Ernest; Roe, R. Michael

    2017-01-01

    While the synthesis and use of new chemical compounds is at an all-time high, the study of their potential impact on human health is quickly falling behind, and new methods are needed to assess their impact. We chose to examine the effects of two common environmental chemicals, the insect repellent N,N-diethyl-m-toluamide (DEET) and the insecticide fluocyanobenpyrazole (fipronil), on transcript levels of long non-protein coding RNAs (lncRNAs) in primary human hepatocytes using a global RNA-Seq approach. While lncRNAs are believed to play a critical role in numerous important biological processes, many still remain uncharacterized, and their functions and modes of action remain largely unclear, especially in relation to environmental chemicals. RNA-Seq showed that 100 µM DEET significantly increased transcript levels for 2 lncRNAs and lowered transcript levels for 18 lncRNAs, while fipronil at 10 µM increased transcript levels for 76 lncRNAs and decreased levels for 193 lncRNAs. A mixture of 100 µM DEET and 10 µM fipronil increased transcript levels for 75 lncRNAs and lowered transcript levels for 258 lncRNAs. This indicates a more-than-additive effect on lncRNA transcript expression when the two chemicals were presented in combination versus each chemical alone. Differentially expressed lncRNA genes were mapped to chromosomes, analyzed by proximity to neighboring protein-coding genes, and functionally characterized via gene ontology and molecular mapping algorithms. While further testing is required to assess the organismal impact of changes in transcript levels, this initial analysis links several of the dysregulated lncRNAs to processes and pathways critical to proper cellular function, such as the innate and adaptive immune response and the p53 signaling pathway. PMID:28991164

  3. Incorporation of osteogenic and angiogenic small interfering RNAs into chitosan sponge for bone tissue engineering

    Directory of Open Access Journals (Sweden)

    Jia S

    2014-11-01

    Full Text Available Sen Jia,1,* Xinjie Yang,1,* Wen Song,2,* Lei Wang,1 Kaixiu Fang,3 Zhiqiang Hu,1,4 Zihui Yang,1 Chun Shan,1 Delin Lei,1 Bin Lu1 1Department of Oral and Maxillofacial Surgery, 2Department of Prosthetic Dentistry, 3Department of Implant Dentistry, School of Stomatology, State Key Laboratory of Military Stomatology, Fourth Military Medical University, Xi’an People’s Republic of China; 4Department of Otorhinolaryngology, No 113 Hospital of People’s Liberation Army, Ningbo, People’s Republic of China *These authors contributed to this paper equally and are considered to be joint first authors Abstract: Engineered bone substitutes are being extensively explored in response to growing demand. However, the angiogenesis that occurs during bone formation is often overlooked in scaffold design. In this novel study, we incorporated two small interfering RNAs (siRNAs, ie, small interfering RNA targets casein kinase 2 interaction protein 1 (siCkip-1 and small interfering RNA targets soluble VEGF receptor 1 (siFlt-1, which can promote osteogenesis and angiogenesis, into a chitosan sponge. This scaffold could maintain siRNAs for over 2 weeks in neutral phosphate-buffered saline and degraded rapidly in the presence of lysozyme. The chitosan sponge with siCkip-1 and siFlt-1 in vitro bioactivity was investigated using mesenchymal stem cells. Target genes were significantly suppressed, and osteocalcin, alkaline phosphatase, and vascular endothelial growth factor were significantly upregulated. Alizarin Red staining revealed that mineralization of the extracellular matrix was markedly enhanced by dual transfection. Further analysis by immunofluorescence confirmed that the siRNA-modified scaffold simultaneously improved the expression of osteocalcin and von Willebrand factor. In vivo testing in a skull critical-size defect model showed marked bone regeneration in rats treated with siCkip-1 and siFlt-1. In conclusion, chitosan sponge containing osteogenic and

  4. Synergistic and independent actions of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Xiaoyan Wang

    2015-04-01

    Full Text Available All types of small RNAs in plants, piwi-interacting RNAs (piRNAs in animals and a subset of siRNAs in Drosophila and C. elegans are subject to HEN1 mediated 3' terminal 2'-O-methylation. This modification plays a pivotal role in protecting small RNAs from 3' uridylation, trimming and degradation. In Arabidopsis, HESO1 is a major enzyme that uridylates small RNAs to trigger their degradation. However, U-tail is still present in null hen1 heso1 mutants, suggesting the existence of (an enzymatic activities redundant with HESO1. Here, we report that UTP: RNA uridylyltransferase (URT1 is a functional paralog of HESO1. URT1 interacts with AGO1 and plays a predominant role in miRNA uridylation when HESO1 is absent. Uridylation of miRNA is globally abolished in a hen1 heso1 urt1 triple mutant, accompanied by an extensive increase of 3'-to-5' trimming. In contrast, disruption of URT1 appears not to affect the heterochromatic siRNA uridylation. This indicates the involvement of additional nucleotidyl transferases in the siRNA pathway. Analysis of miRNA tailings in the hen1 heso1 urt1 triple mutant also reveals the existence of previously unknown enzymatic activities that can add non-uridine nucleotides. Importantly, we show HESO1 may also act redundantly with URT1 in miRNA uridylation when HEN1 is fully competent. Taken together, our data not only reveal a synergistic action of HESO1 and URT1 in the 3' uridylation of miRNAs, but also independent activities of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs and an antagonistic relationship between uridylation and trimming. Our results may provide further insight into the mechanisms of small RNA 3' end modification and stability control.

  5. Structured RNAs in the ENCODE selected regions of the human genome

    DEFF Research Database (Denmark)

    Washietl, Stefan; Pedersen, Jakob Skou; Korbel, Jan O

    2007-01-01

    Functional RNA structures play an important role both in the context of noncoding RNA transcripts as well as regulatory elements in mRNAs. Here we present a computational study to detect functional RNA structures within the ENCODE regions of the human genome. Since structural RNAs in general lack...... with the GENCODE annotation points to functional RNAs in all genomic contexts, with a slightly increased density in 3'-UTRs. While we estimate a significant false discovery rate of approximately 50%-70% many of the predictions can be further substantiated by additional criteria: 248 loci are predicted by both RNAz...

  6. Isolation of Exosome-Like Nanoparticles and Analysis of MicroRNAs Derived from Coconut Water Based on Small RNA High-Throughput Sequencing.

    Science.gov (United States)

    Zhao, Zhehao; Yu, Siran; Li, Min; Gui, Xin; Li, Ping

    2018-03-21

    In this study, the presence of microRNAs in coconut water was identified by real-time polymerase chain reaction (PCR) based on the results of high-throughput small RNA sequencing. In addition, the differences in microRNA content between immature and mature coconut water were compared. A total of 47 known microRNAs belonging to 25 families and 14 new microRNAs were identified in coconut endosperm. Through analysis using a target gene prediction software, potential microRNA target genes were identified in the human genome. Real-time PCR showed that the level of most microRNAs was higher in mature coconut water than in immature coconut water. Then, exosome-like nanoparticles were isolated from coconut water. After ultracentrifugation, some particle structures were seen in coconut water samples using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate fluorescence staining. Subsequent scanning electron microscopy observation and dynamic light scattering analysis also revealed some exosome-like nanoparticles in coconut water, and the mean diameters of the particles detected by the two methods were 13.16 and 59.72 nm, respectively. In conclusion, there are extracellular microRNAs in coconut water, and their levels are higher in mature coconut water than in immature coconut water. Some exosome-like nanoparticles were isolated from coconut water, and the diameter of these particles was smaller than that of animal-derived exosomes.

  7. AnnoLnc: a web server for systematically annotating novel human lncRNAs.

    Science.gov (United States)

    Hou, Mei; Tang, Xing; Tian, Feng; Shi, Fangyuan; Liu, Fenglin; Gao, Ge

    2016-11-16

    Long noncoding RNAs (lncRNAs) have been shown to play essential roles in almost every important biological process through multiple mechanisms. Although the repertoire of human lncRNAs has rapidly expanded, their biological function and regulation remain largely elusive, calling for a systematic and integrative annotation tool. Here we present AnnoLnc ( http://annolnc.cbi.pku.edu.cn ), a one-stop portal for systematically annotating novel human lncRNAs. Based on more than 700 data sources and various tool chains, AnnoLnc enables a systematic annotation covering genomic location, secondary structure, expression patterns, transcriptional regulation, miRNA interaction, protein interaction, genetic association and evolution. An intuitive web interface is available for interactive analysis through both desktops and mobile devices, and programmers can further integrate AnnoLnc into their pipeline through standard JSON-based Web Service APIs. To the best of our knowledge, AnnoLnc is the only web server to provide on-the-fly and systematic annotation for newly identified human lncRNAs. Compared with similar tools, the annotation generated by AnnoLnc covers a much wider spectrum with intuitive visualization. Case studies demonstrate the power of AnnoLnc in not only rediscovering known functions of human lncRNAs but also inspiring novel hypotheses.

  8. Onco-GPCR signaling and dysregulated expression of microRNAs in human cancer.

    Science.gov (United States)

    Nohata, Nijiro; Goto, Yusuke; Gutkind, J Silvio

    2017-01-01

    The G-protein-coupled receptor (GPCR) family is the largest family of cell-surface receptors involved in signal transduction. Aberrant expression of GPCRs and G proteins are frequently associated with prevalent human diseases, including cancer. In fact, GPCRs represent the therapeutic targets of more than a quarter of the clinical drugs currently on the market. MiRNAs (miRNAs) are also aberrantly expressed in many human cancers, and they have significant roles in the initiation, development and metastasis of human malignancies. Recent studies have revealed that dysregulation of miRNAs and their target genes expression are associated with cancer progression. The emerging information suggests that miRNAs play an important role in the fine tuning of many signaling pathways, including GPCR signaling. We summarize our current knowledge of the individual functions of miRNAs regulated by GPCRs and GPCR signaling-associated molecules, and miRNAs that regulate the expression and activity of GPCRs, their endogenous ligands and their coupled heterotrimeric G proteins in human cancer.

  9. Finding for a Needle in a Haystack: Trips of Small RNAs from in silico to in vitro

    International Nuclear Information System (INIS)

    Bermudez Santana, Clara Isabel

    2011-01-01

    Efforts to study the transcriptome have led quantitative and qualitative analyses of all the functional RNA molecules products of transcription. Most of the studies have been focused on the fraction of coding RNAs and have been broadly published. However, the comprehension of the fraction associated to non-coding RNAs that are not translated into proteins but instead, shows a critical role for RNAs in cellular function, it is nowadays one field of Genetics that has in turn led to the transformation of technologies in both experimental and computational research. The characterization of small RNAs associated to the RNA interference pathway (whereby RNA can regulate gene expression) corresponds to one example in which frontiers of knowledge have been expanded not only to increase our comprehension of expression regulation, but also to allow interdisciplinary work among experimentalists and theoreticians. As follow it is presented an example based on small RNA biology to link next generation sequencing technologies and computational research.

  10. 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.

  11. Two Virus-Induced MicroRNAs Known Only from Teleost Fishes Are Orthologues of MicroRNAs Involved in Cell Cycle Control in Humans

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Bela-Ong, Dennis; Jalali, Seyed Amir Hossein

    2015-01-01

    MicroRNAs (miRNAs) are similar to 22 base pair-long non-coding RNAs which regulate gene expression in the cytoplasm of eukaryotic cells by binding to specific target regions in mRNAs to mediate transcriptional blocking or mRNA cleavage. Through their fundamental roles in cellular pathways, gene...... regulation mediated by miRNAs has been shown to be involved in almost all biological phenomena, including development, metabolism, cell cycle, tumor formation, and host-pathogen interactions. To address the latter in a primitive vertebrate host, we here used an array platform to analyze the miRNA response...... regulation. Stimulation of fish cell cultures with the IFN inducer poly I:C accordingly upregulated the expression of miR-462 and miR-731, while no stimulatory effect on miR-191 and miR-425 expression was observed in human cell lines. Despite high sequence conservation, evolution has thus resulted...

  12. MicroRNAs regulate human adipocyte lipolysis: effects of miR-145 are linked to TNF-α.

    Directory of Open Access Journals (Sweden)

    Silvia Lorente-Cebrián

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α. Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.

  13. Long Non-Coding RNAs Associated with Metabolic Traits in Human White Adipose Tissue

    Directory of Open Access Journals (Sweden)

    Hui Gao

    2018-04-01

    Full Text Available Long non-coding RNAs (lncRNAs belong to a recently discovered class of molecules proposed to regulate various cellular processes. Here, we systematically analyzed their expression in human subcutaneous white adipose tissue (WAT and found that a limited set was differentially expressed in obesity and/or the insulin resistant state. Two lncRNAs herein termed adipocyte-specific metabolic related lncRNAs, ASMER-1 and ASMER-2 were enriched in adipocytes and regulated by both obesity and insulin resistance. Knockdown of either ASMER-1 or ASMER-2 by antisense oligonucleotides in in vitro differentiated human adipocytes revealed that both genes regulated adipogenesis, lipid mobilization and adiponectin secretion. The observed effects could be attributed to crosstalk between ASMERs and genes within the master regulatory pathways for adipocyte function including PPARG and INSR. Altogether, our data demonstrate that lncRNAs are modulators of the metabolic and secretory functions in human fat cells and provide an emerging link between WAT and common metabolic conditions. Keywords: White adipose tissue, Adipocytes, Long non-coding RNAs, Metabolic traits, Lipolysis, Adiponectin

  14. Yeast endoribonuclease stimulated by Novikoff Hepatoma small nuclear RNAS U1 and U2

    International Nuclear Information System (INIS)

    Stevens, A.

    1982-01-01

    Using [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from yeast as a substrate, an endoribonuclease has been detected in enzyme fractions derived from a high salt wash of ribonucleoprotein particles of Saccharomyces cerevisiae. The [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) seems to be a preferred substrate since other polyribonucleotides are hydrolyzed more slowly, if at all. The enzyme is inhibited by ethidium bromide, but fully double-stranded polyribonucleotides are not hydrolyzed. The hydrolysis of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) is stimulated about 2.5-fold by the addition of small nuclear RNAs U1 and U2 of Novikoff hepatoma cells. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA

  15. A yeast endoribonuclease stimulated by Novikoff hepatoma small nuclear RNAs U1 and U2

    International Nuclear Information System (INIS)

    Stevens, A.

    1982-01-01

    Using [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from yeast as a substrate, an endoribonuclease has been detected in enzyme fractions derived from a high salt wash of ribonucleoprotein particles of Saccharomyces cerevisiae. The [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) seems to be a preferred substrate since other polyribonucleotides are hydrolyzed more slowly, if at all. The enzyme is inhibited by ethidium bromide, but fully double-stranded polyribonucleotides are not hydrolyzed. The hydrolysis of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) is stimulated about 2.5-fold by the addition of small nuclear RNAs U1 and U2 of Novikoff hepatoma cells. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA

  16. Small regulatory RNAs control the multi-cellular adhesive lifestyle of Escherichia coli

    DEFF Research Database (Denmark)

    Jørgensen, Mikkel Girke; Nielsen, Jesper Sejrup; Boysen, Anders

    2012-01-01

    Small regulatory RNA molecules have recently been recognized as important regulatory elements of developmental processes in both eukaryotes and bacteria. We here describe a striking example in Escherichia coli that can switch between a single-cell motile lifestyle and a multi-cellular, sessile....... Our demonstration that basal expression of each of the three RNA species is sufficient to downregulate CsgD synthesis and prevent curli formation indicates that all play a prominent role in the curli regulatory network. Our findings provide the first clue as to how the Rcs signalling pathway...... negatively regulates curli synthesis and increase the number of small regulatory RNAs that act directly on the csgD mRNA to five....

  17. Identification of viral microRNAs expressed in human sacral ganglia latently infected with herpes simplex virus 2.

    Science.gov (United States)

    Umbach, Jennifer L; Wang, Kening; Tang, Shuang; Krause, Philip R; Mont, Erik K; Cohen, Jeffrey I; Cullen, Bryan R

    2010-01-01

    Deep sequencing of small RNAs isolated from human sacral ganglia latently infected with herpes simplex virus 2 (HSV-2) was used to identify HSV-2 microRNAs (miRNAs) expressed during latent infection. This effort resulted in the identification of five distinct HSV-2 miRNA species, two of which, miR-H3/miR-I and miR-H4/miR-II, have been previously reported. Three novel HSV-2 miRNAs were also identified, and two of these, miR-H7 and miR-H9, are derived from the latency-associated transcript (LAT) and are located antisense to the viral transcript encoding transactivator ICP0. A third novel HSV-2 miRNA, miR-H10, is encoded within the unique long (U(L)) region of the genome, 3' to the U(L)15 open reading frame, and is presumably excised from a novel, latent HSV-2 transcript distinct from LAT.

  18. Identification of Viral MicroRNAs Expressed in Human Sacral Ganglia Latently Infected with Herpes Simplex Virus 2▿

    Science.gov (United States)

    Umbach, Jennifer L.; Wang, Kening; Tang, Shuang; Krause, Philip R.; Mont, Erik K.; Cohen, Jeffrey I.; Cullen, Bryan R.

    2010-01-01

    Deep sequencing of small RNAs isolated from human sacral ganglia latently infected with herpes simplex virus 2 (HSV-2) was used to identify HSV-2 microRNAs (miRNAs) expressed during latent infection. This effort resulted in the identification of five distinct HSV-2 miRNA species, two of which, miR-H3/miR-I and miR-H4/miR-II, have been previously reported. Three novel HSV-2 miRNAs were also identified, and two of these, miR-H7 and miR-H9, are derived from the latency-associated transcript (LAT) and are located antisense to the viral transcript encoding transactivator ICP0. A third novel HSV-2 miRNA, miR-H10, is encoded within the unique long (UL) region of the genome, 3′ to the UL15 open reading frame, and is presumably excised from a novel, latent HSV-2 transcript distinct from LAT. PMID:19889786

  19. Expression of microRNAs in bovine and human pre-implantation embryo culture media

    Science.gov (United States)

    Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

    2014-01-01

    MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

  20. Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas.

    Science.gov (United States)

    Calin, George A; Liu, Chang-gong; Ferracin, Manuela; Hyslop, Terry; Spizzo, Riccardo; Sevignani, Cinzia; Fabbri, Muller; Cimmino, Amelia; Lee, Eun Joo; Wojcik, Sylwia E; Shimizu, Masayoshi; Tili, Esmerina; Rossi, Simona; Taccioli, Cristian; Pichiorri, Flavia; Liu, Xiuping; Zupo, Simona; Herlea, Vlad; Gramantieri, Laura; Lanza, Giovanni; Alder, Hansjuerg; Rassenti, Laura; Volinia, Stefano; Schmittgen, Thomas D; Kipps, Thomas J; Negrini, Massimo; Croce, Carlo M

    2007-09-01

    Noncoding RNA (ncRNA) transcripts are thought to be involved in human tumorigenesis. We report that a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Genome-wide profiling revealed that UCRs have distinct signatures in human leukemias and carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in cancers. We identified certain UCRs whose expression may be regulated by microRNAs abnormally expressed in human chronic lymphocytic leukemia, and we proved that the inhibition of an overexpressed UCR induces apoptosis in colon cancer cells. Our findings argue that ncRNAs and interaction between noncoding genes are involved in tumorigenesis to a greater extent than previously thought.

  1. Recurrent chimeric RNAs enriched in human prostate cancer identified by deep sequencing

    Science.gov (United States)

    Kannan, Kalpana; Wang, Liguo; Wang, Jianghua; Ittmann, Michael M.; Li, Wei; Yen, Laising

    2011-01-01

    Transcription-induced chimeric RNAs, possessing sequences from different genes, are expected to increase the proteomic diversity through chimeric proteins or altered regulation. Despite their importance, few studies have focused on chimeric RNAs especially regarding their presence/roles in human cancers. By deep sequencing the transcriptome of 20 human prostate cancer and 10 matched benign prostate tissues, we obtained 1.3 billion sequence reads, which led to the identification of 2,369 chimeric RNA candidates. Chimeric RNAs occurred in significantly higher frequency in cancer than in matched benign samples. Experimental investigation of a selected 46 set led to the confirmation of 32 chimeric RNAs, of which 27 were highly recurrent and previously undescribed in prostate cancer. Importantly, a subset of these chimeras was present in prostate cancer cell lines, but not detectable in primary human prostate epithelium cells, implying their associations with cancer. These chimeras contain discernable 5′ and 3′ splice sites at the RNA junction, indicating that their formation is mediated by splicing. Their presence is also largely independent of the expression of parental genes, suggesting that other factors are involved in their production and regulation. One chimera, TMEM79-SMG5, is highly differentially expressed in human cancer samples and therefore a potential biomarker. The prevalence of chimeric RNAs may allow the limited number of human genes to encode a substantially larger number of RNAs and proteins, forming an additional layer of cellular complexity. Together, our results suggest that chimeric RNAs are widespread, and increased chimeric RNA events could represent a unique class of molecular alteration in cancer. PMID:21571633

  2. Comparative analysis of virus-derived small RNAs within cassava (Manihot esculenta Crantz) infected with cassava brown streak viruses.

    Science.gov (United States)

    Ogwok, Emmanuel; Ilyas, Muhammad; Alicai, Titus; Rey, Marie E C; Taylor, Nigel J

    2016-04-02

    Infection of plant cells by viral pathogens triggers RNA silencing, an innate antiviral defense mechanism. In response to infection, small RNAs (sRNAs) are produced that associate with Argonaute (AGO)-containing silencing complexes which act to inactivate viral genomes by posttranscriptional gene silencing (PTGS). Deep sequencing was used to compare virus-derived small RNAs (vsRNAs) in cassava genotypes NASE 3, TME 204 and 60444 infected with the positive sense single-stranded RNA (+ssRNA) viruses cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), the causal agents of cassava brown streak disease (CBSD). An abundance of 21-24nt vsRNAs was detected and mapped, covering the entire CBSV and UCBSV genomes. The 21nt vsRNAs were most predominant, followed by the 22 nt class with a slight bias toward sense compared to antisense polarity, and a bias for adenine and uracil bases present at the 5'-terminus. Distribution and frequency of vsRNAs differed between cassava genotypes and viral genomes. In susceptible genotypes TME 204 and 60444, CBSV-derived sRNAs were seen in greater abundance than UCBSV-derived sRNAs. NASE 3, known to be resistant to UCBSV, accumulated negligible UCBSV-derived sRNAs but high populations of CBSV-derived sRNAs. Transcript levels of cassava homologues of AGO2, DCL2 and DCL4, which are central to the gene-silencing complex, were found to be differentially regulated in CBSV- and UCBSV-infected plants across genotypes, suggesting these proteins play a role in antiviral defense. Irrespective of genotype or viral pathogen, maximum populations of vsRNAs mapped to the cytoplasmic inclusion, P1 and P3 protein-encoding regions. Our results indicate disparity between CBSV and UCBSV host-virus interaction mechanisms, and provide insight into the role of virus-induced gene silencing as a mechanism of resistance to CBSD. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  3. 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.

  4. 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.

  5. Small non-coding RNAs: new insights in modulation of host immune response by intracellular bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Waqas Ahmed

    2016-10-01

    Full Text Available Pathogenic bacteria possess intricate regulatory networks that temporally control the production of virulence factors, and enable the bacteria to survive and proliferate within host cell. Small non-coding RNAs (sRNAs have been identified as important regulators of gene expression in diverse biological contexts. Recent research has shown bacterial sRNAs involved in growth and development, cell proliferation, differentiation, metabolism, cell signaling and immune response through regulating protein–protein interactions or via their ability to base pair with RNA and DNA. In this review, we provide a brief overview of mechanism of action employed by immune-related sRNAs, their known functions in immunity, and how they can be integrated into regulatory circuits that govern virulence, which will facilitates to understand pathogenesis and the development of novel, more effective therapeutic approaches to treat infections caused by intracellular bacterial pathogens.

  6. Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Sarah Inwood

    2018-01-01

    Full Text Available The ability to produce recombinant proteins by utilizing different “cell factories” revolutionized the biotherapeutic and pharmaceutical industry. Chinese hamster ovary (CHO cells are the dominant industrial producer, especially for antibodies. Human embryonic kidney cells (HEK, while not being as widely used as CHO cells, are used where CHO cells are unable to meet the needs for expression, such as growth factors. Therefore, improving recombinant protein expression from mammalian cells is a priority, and continuing effort is being devoted to this topic. Non-coding RNAs are RNA segments that are not translated into a protein and often have a regulatory role. Since their discovery, major progress has been made towards understanding their functions. Non-coding RNA has been investigated extensively in relation to disease, especially cancer, and recently they have also been used as a method for engineering cells to improve their protein expression capability. In this review, we provide information about methods used to identify non-coding RNAs with the potential of improving recombinant protein expression in mammalian cell lines.

  7. 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.

  8. Differentially expressed wound healing-related microRNAs in the human diabetic cornea.

    Science.gov (United States)

    Funari, Vincent A; Winkler, Michael; Brown, Jordan; Dimitrijevich, Slobodan D; Ljubimov, Alexander V; Saghizadeh, Mehrnoosh

    2013-01-01

    MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples. HCEC were transfected with human pre-miR™miRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.

  9. Differentially expressed wound healing-related microRNAs in the human diabetic cornea.

    Directory of Open Access Journals (Sweden)

    Vincent A Funari

    Full Text Available MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6 and diabetic (n=6 central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR and by in situ hybridization (ISH in independent samples. HCEC were transfected with human pre-miR™miRNA precursors (h-miR or their inhibitors (antagomirs using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.

  10. MSDD: a manually curated database of experimentally supported associations among miRNAs, SNPs and human diseases

    OpenAIRE

    Yue, Ming; Zhou, Dianshuang; Zhi, Hui; Wang, Peng; Zhang, Yan; Gao, Yue; Guo, Maoni; Li, Xin; Wang, Yanxia; Zhang, Yunpeng; Ning, Shangwei; Li, Xia

    2017-01-01

    Abstract The MiRNA SNP Disease Database (MSDD, http://www.bio-bigdata.com/msdd/) is a manually curated database that provides comprehensive experimentally supported associations among microRNAs (miRNAs), single nucleotide polymorphisms (SNPs) and human diseases. SNPs in miRNA-related functional regions such as mature miRNAs, promoter regions, pri-miRNAs, pre-miRNAs and target gene 3′-UTRs, collectively called ‘miRSNPs’, represent a novel category of functional molecules. miRSNPs can lead to m...

  11. Differential RISC association of endogenous human microRNAs predicts their inhibitory potential.

    Science.gov (United States)

    Flores, Omar; Kennedy, Edward M; Skalsky, Rebecca L; Cullen, Bryan R

    2014-04-01

    It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function.

  12. Sequence comparison of six human microRNAs genes between tuberculosis patients and healthy individuals.

    Science.gov (United States)

    Amila, A; Acosta, A; Sarmiento, M E; Suraiya, Siti; Zafarina, Z; Panneerchelvam, S; Norazmi, M N

    2015-12-01

    MicroRNAs (miRNAs) play an important role in diseases development. Therefore, human miRNAs may be able to inhibit the survival of Mycobacterium tuberculosis (Mtb) in the human host by targeting critical genes of the pathogen. Mutations within miRNAs can alter their target selection, thereby preventing them from inhibiting Mtb genes, thus increasing host susceptibility to the disease. This study was undertaken to investigate the genetic association of pulmonary tuberculosis (TB) with six human miRNAs genes, namely, hsa-miR-370, hsa-miR-520d, hsa-miR-154, hsa-miR-497, hsa-miR-758, and hsa-miR-593, which have been predicted to interact with Mtb genes. The objective of the study was to determine the possible sequence variation of selected miRNA genes that are potentially associated with the inhibition of critical Mtb genes in TB patients. The study did not show differences in the sequences compared with healthy individuals without antecedents of TB. This result could have been influenced by the sample size and the selection of miRNA genes, which need to be addressed in future studies. Copyright © 2015 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

  13. Intronic microRNAs

    International Nuclear Information System (INIS)

    Ying, S.-Y.; Lin, S.-L.

    2005-01-01

    MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular mRNAs that contain partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. MiRNA was originally discovered in the intergenic regions of the Caenorhabditis elegans genome as native RNA fragments that modulate a wide range of genetic regulatory pathways during animal development. However, neither RNA promoter nor polymerase responsible for miRNA biogenesis was determined. Recent findings of intron-derived miRNA in C. elegans, mouse, and human have inevitably led to an alternative pathway for miRNA biogenesis, which relies on the coupled interaction of Pol-II-mediated pre-mRNA transcription and intron excision, occurring in certain nuclear regions proximal to genomic perichromatin fibrils

  14. A 6-Nucleotide Regulatory Motif within the AbcR Small RNAs of Brucella abortus Mediates Host-Pathogen Interactions.

    Science.gov (United States)

    Sheehan, Lauren M; Caswell, Clayton C

    2017-06-06

    In Brucella abortus , two small RNAs (sRNAs), AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems. AbcR1 and AbcR2 are required for Brucella virulence, as a double chromosomal deletion of both sRNAs results in attenuation in mice. Although these sRNAs are responsible for targeting transcripts for degradation, the mechanism utilized by the AbcR sRNAs to regulate mRNA in Brucella has not been described. Here, two motifs (M1 and M2) were identified in AbcR1 and AbcR2, and complementary motif sequences were defined in AbcR-regulated transcripts. Site-directed mutagenesis of M1 or M2 or of both M1 and M2 in the sRNAs revealed transcripts to be targeted by one or both motifs. Electrophoretic mobility shift assays revealed direct, concentration-dependent binding of both AbcR sRNAs to a target mRNA sequence. These experiments genetically and biochemically characterized two indispensable motifs within the AbcR sRNAs that bind to and regulate transcripts. Additionally, cellular and animal models of infection demonstrated that only M2 in the AbcR sRNAs is required for Brucella virulence. Furthermore, one of the M2-regulated targets, BAB2_0612, was found to be critical for the virulence of B. abortus in a mouse model of infection. Although these sRNAs are highly conserved among Alphaproteobacteria , the present report displays how gene regulation mediated by the AbcR sRNAs has diverged to meet the intricate regulatory requirements of each particular organism and its unique biological niche. IMPORTANCE Small RNAs (sRNAs) are important components of bacterial regulation, allowing organisms to quickly adapt to changes in their environments. The AbcR sRNAs are highly conserved throughout the Alphaproteobacteria and negatively regulate myriad transcripts, many encoding ABC-type transport systems. In Brucella abortus , AbcR1 and AbcR2 are functionally redundant, as only a double abcR1 abcR2 ( abcR1 / 2 ) deletion results in attenuation in

  15. Analysis of sucrose-induced small RNAs in Streptococcus mutans in the presence of different sucrose concentrations.

    Science.gov (United States)

    Liu, Shan Shan; Zhu, Wen Hui; Zhi, Qing Hui; Liu, Jia; Wang, Yan; Lin, Huan Cai

    2017-07-01

    Streptococcus mutans (S. mutans) is the major pathogen contributing to dental caries. Sucrose is an important carbohydrate source for S. mutans and is crucial for dental caries. Small RNAs (sRNAs) are key post-transcriptional regulators of stress adaptation and virulence in bacteria. Here, for the first time, we created three replicate RNA libraries exposed to either 1 or 5% sucrose. The expression levels of sRNAs and target genes (gtfB, gtfC, and spaP) related to virulence were assessed. In addition, some phenotypic traits were evaluated. We obtained 2125 sRNA candidates with at least 100 average reads in 1% sucrose or 5% sucrose. Of these candidates, 2 were upregulated and 20 were downregulated in 1% sucrose. Six of these 22 differentially expressed sRNAs were validated by qRT-PCR. The expression level of target gene gtfB was higher in 1% sucrose. The adherence ratio of S. mutans was higher in 1% sucrose than in 5% sucrose. The synthesis of water-insoluble glucans (WIGs) was significantly higher in 5% sucrose than in 1% sucrose. These data suggest that a series of sRNAs can be induced in response to sucrose, and that some sRNAs might be involved in the regulation of phenotypes, providing new insight into the prevention of caries.

  16. A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets.

    Science.gov (United States)

    Yasuda, Shinsuke; Wada, Yuko; Kakizaki, Tomohiro; Tarutani, Yoshiaki; Miura-Uno, Eiko; Murase, Kohji; Fujii, Sota; Hioki, Tomoya; Shimoda, Taiki; Takada, Yoshinobu; Shiba, Hiroshi; Takasaki-Yasuda, Takeshi; Suzuki, Go; Watanabe, Masao; Takayama, Seiji

    2016-12-22

    In diploid organisms, phenotypic traits are often biased by effects known as Mendelian dominant-recessive interactions between inherited alleles. Phenotypic expression of SP11 alleles, which encodes the male determinants of self-incompatibility in Brassica rapa, is governed by a complex dominance hierarchy 1-3 . Here, we show that a single polymorphic 24 nucleotide small RNA, named SP11 methylation inducer 2 (Smi2), controls the linear dominance hierarchy of the four SP11 alleles (S 44 > S 60 > S 40 > S 29 ). In all dominant-recessive interactions, small RNA variants derived from the linked region of dominant SP11 alleles exhibited high sequence similarity to the promoter regions of recessive SP11 alleles and acted in trans to epigenetically silence their expression. Together with our previous study 4 , we propose a new model: sequence similarity between polymorphic small RNAs and their target regulates mono-allelic gene expression, which explains the entire five-phased linear dominance hierarchy of the SP11 phenotypic expression in Brassica.

  17. Systematic identification of edited microRNAs in the human brain

    Science.gov (United States)

    Alon, Shahar; Mor, Eyal; Vigneault, Francois; Church, George M.; Locatelli, Franco; Galeano, Federica; Gallo, Angela; Shomron, Noam; Eisenberg, Eli

    2012-01-01

    Adenosine-to-inosine (A-to-I) editing modifies RNA transcripts from their genomic blueprint. A prerequisite for this process is a double-stranded RNA (dsRNA) structure. Such dsRNAs are formed as part of the microRNA (miRNA) maturation process, and it is therefore expected that miRNAs are affected by A-to-I editing. Editing of miRNAs has the potential to add another layer of complexity to gene regulation pathways, especially if editing occurs within the miRNA–mRNA recognition site. Thus, it is of interest to study the extent of this phenomenon. Current reports in the literature disagree on its extent; while some reports claim that it may be widespread, others deem the reported events as rare. Utilizing a next-generation sequencing (NGS) approach supplemented by an extensive bioinformatic analysis, we were able to systematically identify A-to-I editing events in mature miRNAs derived from human brain tissues. Our algorithm successfully identified many of the known editing sites in mature miRNAs and revealed 17 novel human sites, 12 of which are in the recognition sites of the miRNAs. We confirmed most of the editing events using in vitro ADAR overexpression assays. The editing efficiency of most sites identified is very low. Similar results are obtained for publicly available data sets of mouse brain-regions tissues. Thus, we find that A-to-I editing does alter several miRNAs, but it is not widespread. PMID:22499667

  18. Downregulation of six microRNAs is associated with advanced stage, lymph node metastasis and poor prognosis in small cell carcinoma of the cervix.

    Directory of Open Access Journals (Sweden)

    Long Huang

    Full Text Available BACKGROUND: Small cell carcinoma of the cervix (SCCC is very rare, and due to the long time period required to recruit sufficient numbers of patients, there is a paucity of information regarding the prognostic factors associated with survival. MicroRNAs (miRNAs have been used as cancer-related biomarkers in a variety of tumor types, and the objective of this study was to determine whether microRNA expression profiles can predict clinical outcome in SCCC. METHODOLOGY/PRINCIPAL FINDINGS: Forty-four patients with SCCC who underwent radical hysterectomy between January 2000 and October 2009 were enrolled. Using the GeneCopoeia All-in-One™ Customized Human qPCR Primer Array, the expression profiles of 30 miRNAs associated with tumor metastasis was obtained from the formalin-fixed paraffin embedded samples of all 44 patients. Seven miRNAs, has-let-7c, has-miR-10b, has-miR-100, has-miR-125b, has-miR-143, has-miR-145 and has-miR-199a-5p were significantly down-regulated in advanced stage SCCC patients (FIGO IB2-IV compared to early stage SCCC patients (FIGOIB1. Among, downregulation of six miRNAs, has-let-7c, has-miR-100, has-miR-125b, has-miR-143, has-miR-145 and has-miR-199a-5p were significantly associated with lymph node metastasis and reduced survival in SCCC. Kaplan-Meier survival analyses revealed that SCCC patients with low expression of has-miR-100 (P = 0.019 and has-miR-125b (P = 0.020 projected a significant tendency towards poorer prognosis. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that downregulation of 7 miRNA associated with advanced stage, 6 miRNAs with metastasis and 2 with poor prognosis in SCCC. Functional analysis of these miRNAs may enhance our understanding of SCCC, as altered expression of specific miRNAs may regulate the metastatic pathway and provide novel targets for therapy.

  19. Small and Long Regulatory RNAs in the Immune System and Immune Diseases

    OpenAIRE

    Stachurska, Anna; Zorro, Maria M.; van der Sijde, Marijke R.; Withoff, Sebo

    2014-01-01

    Cellular differentiation is regulated on the level of gene expression, and it is known that dysregulation of gene expression can lead to deficiencies in differentiation that contribute to a variety of diseases, particularly of the immune system. Until recently, it was thought that the dysregulation was governed by changes in the binding or activity of a class of proteins called transcription factors. However, the discovery of micro-RNAs and recent descriptions of long non-coding RNAs (lncRNAs...

  20. How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing.

    Science.gov (United States)

    Koralewska, Natalia; Hoffmann, Weronika; Pokornowska, Maria; Milewski, Marek; Lipinska, Andrea; Bienkowska-Szewczyk, Krystyna; Figlerowicz, Marek; Kurzynska-Kokorniak, Anna

    2016-01-01

    Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

  1. Differential Expression of Tomato Spotted Wilt Virus-Derived Viral Small RNAs in Infected Commercial and Experimental Host Plants

    Science.gov (United States)

    Mitter, Neena; Koundal, Vikas; Williams, Sarah; Pappu, Hanu

    2013-01-01

    Background Viral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide. Principal Findings Tomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV. Significance Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral

  2. Differential expression of tomato spotted wilt virus-derived viral small RNAs in infected commercial and experimental host plants.

    Directory of Open Access Journals (Sweden)

    Neena Mitter

    Full Text Available BACKGROUND: Viral small RNAs (vsiRNAs in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV, a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide. PRINCIPAL FINDINGS: Tomato spotted wilt virus (TSWV-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1 higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV. SIGNIFICANCE: Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs

  3. Deep RNA sequencing reveals dynamic regulation of myocardial noncoding RNAs in failing human heart and remodeling with mechanical circulatory support.

    Science.gov (United States)

    Yang, Kai-Chien; Yamada, Kathryn A; Patel, Akshar Y; Topkara, Veli K; George, Isaac; Cheema, Faisal H; Ewald, Gregory A; Mann, Douglas L; Nerbonne, Jeanne M

    2014-03-04

    Microarrays have been used extensively to profile transcriptome remodeling in failing human heart, although the genomic coverage provided is limited and fails to provide a detailed picture of the myocardial transcriptome landscape. Here, we describe sequencing-based transcriptome profiling, providing comprehensive analysis of myocardial mRNA, microRNA (miRNA), and long noncoding RNA (lncRNA) expression in failing human heart before and after mechanical support with a left ventricular (LV) assist device (LVAD). Deep sequencing of RNA isolated from paired nonischemic (NICM; n=8) and ischemic (ICM; n=8) human failing LV samples collected before and after LVAD and from nonfailing human LV (n=8) was conducted. These analyses revealed high abundance of mRNA (37%) and lncRNA (71%) of mitochondrial origin. miRNASeq revealed 160 and 147 differentially expressed miRNAs in ICM and NICM, respectively, compared with nonfailing LV. Among these, only 2 (ICM) and 5 (NICM) miRNAs are normalized with LVAD. RNASeq detected 18 480, including 113 novel, lncRNAs in human LV. Among the 679 (ICM) and 570 (NICM) lncRNAs differentially expressed with heart failure, ≈10% are improved or normalized with LVAD. In addition, the expression signature of lncRNAs, but not miRNAs or mRNAs, distinguishes ICM from NICM. Further analysis suggests that cis-gene regulation represents a major mechanism of action of human cardiac lncRNAs. The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

  4. Automatic extraction and processing of small RNAs on a multi-well/multi-channel (M&M) chip.

    Science.gov (United States)

    Zhong, Runtao; Flack, Kenneth; Zhong, Wenwan

    2012-12-07

    The study of the regulatory roles in small RNAs can be accelerated by techniques that permit simple, low-cost, and rapid extraction of small RNAs from a small number of cells. In order to ensure highly specific and sensitive detection, the extracted RNAs should be free of the background nucleic acids and present stably in a small volume. To meet these criteria, we designed a multi-well/multi-channel (M&M) chip to carry out automatic and selective isolation of small RNAs via solid-phase extraction (SPE), followed by reverse-transcription (RT) to convert them to the more stable cDNAs in a final volume of 2 μL. Droplets containing buffers for RNA binding, washing, and elution were trapped in microwells, which were connected by one channel, and suspended in mineral oil. The silica magnetic particles (SMPs) for SPE were moved along the channel from well to well, i.e. in between droplets, by a fixed magnet and a translation stage, allowing the nucleic acid fragments to bind to the SMPs, be washed, and then be eluted for RT reaction within 15 minutes. RNAs shorter than 63 nt were selectively enriched from cell lysates, with recovery comparable to that of a commercial kit. Physical separation of the droplets on our M&M chip allowed the usage of multiple channels for parallel processing of multiple samples. It also permitted smooth integration with on-chip RT-PCR, which simultaneously detected the target microRNA, mir-191, expressed in fewer than 10 cancer cells. Our results have demonstrated that the M&M chip device is a valuable and cost-saving platform for studying small RNA expression patterns in a limited number of cells with reasonable sample throughput.

  5. Identification of antisense long noncoding RNAs that function as SINEUPs in human cells.

    Science.gov (United States)

    Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari; Gustincich, Stefano; Carninci, Piero

    2016-09-20

    Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5' UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3' end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species.

  6. Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

    Science.gov (United States)

    Cooper, Edwin L.; Overstreet, Nicola

    2014-03-01

    Recent evidence supports that prokaryotes exhibit adaptive immunity in the form of CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) and Cas (CRISPR associated proteins). The CRISPR-Cas system confers resistance to exogenous genetic elements such as phages and plasmids by allowing for the recognition and silencing of these genetic elements. Moreover, CRISPR-Cas serves as a memory of past exposures. This suggests that the evolution of the immune system has counterparts among the prokaryotes, not exclusively among eukaryotes. Mathematical models have been proposed which simulate the evolutionary patterns of CRISPR, however large gaps in our understanding of CRISPR-Cas function and evolution still exist. The CRISPR-Cas system is analogous to small RNAs involved in resistance mechanisms throughout the tree of life, and a deeper understanding of the evolution of small RNA pathways is necessary before the relationship between these convergent systems is to be determined. Presented in this review are novel RNAi therapies based on CRISPR-Cas analogs and the potential for future therapies based on CRISPR-Cas system components.

  7. Nanoparticle (MPG)-mediated delivery of small RNAs into human ...

    African Journals Online (AJOL)

    Aghomotsegin

    Divita and co-workers reported that MPG delivers active macromolecules permitting the control of the release of the cargo in the appropriate target subcellular compart- ment. Therefore, by tampering with the NLS sequence of. MPG, delivery between the nucleus and the cytoplasm can be discriminated and MPG containing ...

  8. The different expression profiles of microRNAs in elderly and young human dental pulp and the role of miR-433 in human dental pulp cells.

    Science.gov (United States)

    Wang, Kejing; Li, Li; Wu, Junjun; Qiu, Qin; Zhou, Fengjuan; Wu, Hongkun

    2015-03-01

    As a kind of endogenous noncoding small RNA, MicroRNA (miRNA) plays important roles of regulation to various physiological functions, while its affections on senescence of human dental pulp cell (HDPCs) are still unknown. Thus, we identified the senescence-associated miRNAs in HDPCs by microarray analysis, predicted their targets and regulatory signaling pathway by gene ontology and Kyoto encyclopedia of genes and genomes pathway database analysis. After validated, the senescence-associated miRNAs' expression level was up- and down-regulated using lentivirus package and cell transfection to find its role in HDPCs' morphology, proliferation, apoptosis, and mineralization. The results showed 27 miRNAs differentially expressed at least 1.5-fold, of which 16 were up-regulated and 11 down-regulated, the function of their targets was mainly focused on signal transduction, cell proliferation, apoptosis, and transcription regulation. According to the change fold, we speculated that miR-433 could be one of the vital senescence-associated miRNAs of HDPCs and found its target (GRB2), validated that miR-433 could negatively regulate GRB2 and the RAS-MAPK signaling pathway, leading to the decline of proliferation and mineralization ability of HDPCs and the acceleration of cell apoptosis, suggesting the regulation of miR-433 might be the potential target to promote repair and regeneration of HDPCs in the elderly. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. 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.

  10. NGS Analysis of Human Embryo Culture Media Reveals miRNAs of Extra Embryonic Origin.

    Science.gov (United States)

    Sánchez-Ribas, Immaculada; Diaz-Gimeno, Patricia; Quiñonero, Alicia; Ojeda, María; Larreategui, Zaloa; Ballesteros, Agustín; Domínguez, Francisco

    2018-01-01

    Our objective in this work was to isolate, identify, and compare micro-RNAs (miRNAs) found in spent culture media of euploid and aneuploid in vitro fertilization (IVF) embryos. Seventy-two embryos from 62 patients were collected, and their spent media were retained. A total of 108 spent conditioned media samples were analyzed (n = 36 day 3 euploid embryos, n = 36 day 3 aneuploid embryos, and n = 36 matched control media). Fifty hed-control media embryos were analyzed using next-generation sequencing (NGS) technology. We detected 53 known human miRNAs present in the spent conditioned media of euploid and aneuploid IVF embryos. miR-181b-5p and miR-191-5p were found the most represented. We validated our results by quantitative polymerase chain reaction (qPCR), but no significant results were obtained between the groups. In conclusion, we obtained the list of miRNAs present in the spent conditioned media from euploid and aneuploid IVF embryos, but our data suggest that these miRNAs could have a nonembryonic origin.

  11. Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC

    Science.gov (United States)

    Castanotto, Daniela; Sakurai, Kumi; Lingeman, Robert; Li, Haitang; Shively, Louise; Aagaard, Lars; Soifer, Harris; Gatignol, Anne; Riggs, Arthur; Rossi, John J.

    2007-01-01

    Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs. PMID:17660190

  12. An atlas of human long non-coding RNAs with accurate 5′ ends

    KAUST Repository

    Hon, Chung-Chau; Ramilowski, Jordan A.; Harshbarger, Jayson; Bertin, Nicolas; Rackham, Owen J. L.; Gough, Julian; Denisenko, Elena; Schmeier, Sebastian; Poulsen, Thomas M.; Severin, Jessica; Lizio, Marina; Kawaji, Hideya; Kasukawa, Takeya; Itoh, Masayoshi; Burroughs, A. Maxwell; Noma, Shohei; Djebali, Sarah; Alam, Tanvir; Medvedeva, Yulia A.; Testa, Alison C.; Lipovich, Leonard; Yip, Chi-Wai; Abugessaisa, Imad; Mendez, Mickaë l; Hasegawa, Akira; Tang, Dave; Lassmann, Timo; Heutink, Peter; Babina, Magda; Wells, Christine A.; Kojima, Soichi; Nakamura, Yukio; Suzuki, Harukazu; Daub, Carsten O.; Hoon, Michiel J. L. de; Arner, Erik; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R. R.

    2017-01-01

    RNAs in multiple diseases. We further demonstrate that lncRNAs overlapping expression quantitative trait loci (eQTL)-associated single nucleotide polymorphisms of messenger RNAs are co-expressed with the corresponding messenger RNAs, suggesting their potential

  13. Opposite responses of rabbit and human globin mRNAs to translational inhibition by cap analogues

    International Nuclear Information System (INIS)

    Shakin, S.H.; Liebhaber, S.A.

    1987-01-01

    The translational efficiency of an mRNA may be determined at the step of translational initiation by the efficiency of its interaction with the cap binding protein complex. To further investigate the role of these interactions in translational control, the authors compare in vitro the relative sensitivities of rabbit and human α- and β-globin mRNAs to translational inhibition by cap analogues. They find that rabbit β-globin mRNA is more resistant to translational inhibition by cap analogues than rabbit α-globin mRNA, while in contrast, human β-globin mRNA is more sensitive to cap analogue inhibition than human α- and β-globin mRNAs is unexpected as direct in vivo and in vitro comparisons of polysome profiles reveal parallel translational handling of the α- and β-globin mRNAs from these two species. This discordance between the relative translational sensitivities of these mRNAs to cap analogues and their relative ribosome loading activities suggests that cap-dependent events may not be rate limiting in steady-state globin translation

  14. 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.

  15. A study of the evolution of human microRNAs by their apparent repression effectiveness on target genes.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available BACKGROUND: Even though the genomes of many model species have already been sequenced, our knowledge of gene regulation in evolution is still very limited. One big obstacle is that it is hard to predict the target genes of transcriptional factors accurately from sequences. In this respect, microRNAs (miRNAs are different from transcriptional factors, as target genes of miRNAs can be readily predicted from sequences. This feature of miRNAs offers an unprecedented vantage point for evolutionary analysis of gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we analyzed a particular aspect of miRNA evolution, the differences in the "apparent repression effectiveness (ARE" between human miRNAs of different conservational levels. ARE is a measure we designed to evaluate the repression effect of miRNAs on target genes based on publicly available gene expression data in normal tissues and miRNA targeting and expression data. We found that ARE values of more conserved miRNAs are significantly higher than those of less conserved miRNAs in general. We also found the gain in expression abundance and broadness of miRNAs in evolution contributed to the gain in ARE. CONCLUSIONS/SIGNIFICANCE: The ARE measure quantifies the repressive effects of miRNAs and enables us to study the influences of many factors on miRNA-mediated repression, such as conservational levels and expression levels of miRNAs. The gain in ARE can be explained by the existence of a trend of miRNAs in evolution to effectively control more target genes, which is beneficial to the miRNAs but not necessarily to the organism at all times. Our results from miRNAs gave us an insight of the complex interplay between regulators and target genes in evolution.

  16. 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.

  17. 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.

  18. A universal protocol for the combined isolation of metabolites, DNA, long RNAs, small RNAs, and proteins from plants and microorganisms

    Czech Academy of Sciences Publication Activity Database

    Valledor, Luis; Escandón, M.; Meijón, M.; Nukarinen, E.; Jesús Cañal, M.; Weckwerth, W.

    2014-01-01

    Roč. 79, č. 1 (2014), s. 173-180 ISSN 0960-7412 R&D Projects: GA MŠk(CZ) EE2.3.20.0256 Institutional support: RVO:67179843 Keywords : systems biology * combined isolation * RNA * small RNA * proteins * metabolites * Chlamydomonas reinhardtii * Arabidopsis thaliana * Populus sp. * Pinus sp. * technical advance Subject RIV: EI - Biotechnology ; Bionics Impact factor: 5.972, year: 2014

  19. Potential Diagnostic, Prognostic and Therapeutic Targets of MicroRNAs in Human Gastric Cancer

    Directory of Open Access Journals (Sweden)

    Ming-Ming Tsai

    2016-06-01

    Full Text Available Human gastric cancer (GC is characterized by a high incidence and mortality rate, largely because it is normally not identified until a relatively advanced stage owing to a lack of early diagnostic biomarkers. Gastroscopy with biopsy is the routine method for screening, and gastrectomy is the major therapeutic strategy for GC. However, in more than 30% of GC surgical patients, cancer has progressed too far for effective medical resection. Thus, useful biomarkers for early screening or detection of GC are essential for improving patients’ survival rate. MicroRNAs (miRNAs play an important role in tumorigenesis. They contribute to gastric carcinogenesis by altering the expression of oncogenes and tumor suppressors. Because of their stability in tissues, serum/plasma and other body fluids, miRNAs have been suggested as novel tumor biomarkers with suitable clinical potential. Recently, aberrantly expressed miRNAs have been identified and tested for clinical application in the management of GC. Aberrant miRNA expression profiles determined with miRNA microarrays, quantitative reverse transcription-polymerase chain reaction and next-generation sequencing approaches could be used to establish sample specificity and to identify tumor type. Here, we provide an up-to-date summary of tissue-based GC-associated miRNAs, describing their involvement and that of their downstream targets in tumorigenic and biological processes. We examine correlations among significant clinical parameters and prognostic indicators, and discuss recurrence monitoring and therapeutic options in GC. We also review plasma/serum-based, GC-associated, circulating miRNAs and their clinical applications, focusing especially on early diagnosis. By providing insights into the mechanisms of miRNA-related tumor progression, this review will hopefully aid in the identification of novel potential therapeutic targets.

  20. Innate Immunity and Human Milk MicroRNAs Content: A New Perspective for Premature Newborns

    Directory of Open Access Journals (Sweden)

    Erika Cione

    2017-02-01

    Full Text Available Context The premature newborns are prone to develop both early onset and late onset neonatal sepsis. The major causes of this phenomenon rely on the immaturity of the immune system, which has reduced capability to respond adequately to pathogens. Evidence Acquisition Titles and abstracts of previous papers were scanned before reading the full-text, in order to retrieve appropriate information. The databases used for searching were PubMed, Cochrane, and Embase for articles published before 1st of July, 2016. Secondary search for articles cited in reference lists were identified by the primary search. This review focused on neonatal sepsis incidence and the associated immune response with regards to microRNAs of human milk as a new microelement that enables regulation of innate immunity functions. Results Since human milk is a valuable source of microRNAs, a better understanding of its content will open a new therapeutic avenue for the clinical management of infectious diseases affecting premature newborns. The variation in miRNAs quantity in human milk needs to be considered. Mother’s milk can have different amounts of miRNAs and the identification of a microMilk batch richer of miRNAs can be a nutrition intervention method for modulating innate immunity in clinical management of premature newborns. Conclusions Routine translation of the microMilk concept for neonatal intensive care unit (NICU, in the management of premature newborns could be a way of defending premature newborns and Very Low Birth Weight (VLBW infants from both early and late sepsis.

  1. MicroRNAs at the human 14q32 locus have prognostic significance in osteosarcoma

    Directory of Open Access Journals (Sweden)

    Sarver Aaron L

    2013-01-01

    Full Text Available Abstract Background Deregulation of microRNA (miRNA transcript levels has been observed in many types of tumors including osteosarcoma. Molecular pathways regulated by differentially expressed miRNAs may contribute to the heterogeneous tumor behaviors observed in naturally occurring cancers. Thus, tumor-associated miRNA expression may provide informative biomarkers for disease outcome and metastatic potential in osteosarcoma patients. We showed previously that clusters of miRNAs at the 14q32 locus are downregulated in human osteosarcoma. Methods Human and canine osteosarcoma patient’s samples with clinical follow-up data were used in this study. We used bioinformatics and comparative genomics approaches to identify miRNA based prognostic biomarkers in osteosarcoma. Kaplan-Meier survival curves and Whitney Mann U tests were conducted for validating the statistical significance. Results Here we show that an inverse correlation exists between aggressive tumor behavior (increased metastatic potential and accelerated time to death and the residual expression of 14q32 miRNAs (using miR-382 as a representative of 14q32 miRNAs in a series of clinically annotated samples from human osteosarcoma patients. We also show a comparable decrease in expression of orthologous 14q32 miRNAs in canine osteosarcoma samples, with conservation of the inverse correlation between aggressive behavior and expression of orthologous miRNA miR-134 and miR-544. Conclusions We conclude that downregulation of 14q32 miRNA expression is an evolutionarily conserved mechanism that contributes to the biological behavior of osteosarcoma, and that quantification of representative transcripts from this family, such as miR-382, miR-134, and miR-544, provide prognostic and predictive markers that can assist in the management of patients with this disease.

  2. MicroRNAs at the human 14q32 locus have prognostic significance in osteosarcoma

    Science.gov (United States)

    2013-01-01

    Background Deregulation of microRNA (miRNA) transcript levels has been observed in many types of tumors including osteosarcoma. Molecular pathways regulated by differentially expressed miRNAs may contribute to the heterogeneous tumor behaviors observed in naturally occurring cancers. Thus, tumor-associated miRNA expression may provide informative biomarkers for disease outcome and metastatic potential in osteosarcoma patients. We showed previously that clusters of miRNAs at the 14q32 locus are downregulated in human osteosarcoma. Methods Human and canine osteosarcoma patient’s samples with clinical follow-up data were used in this study. We used bioinformatics and comparative genomics approaches to identify miRNA based prognostic biomarkers in osteosarcoma. Kaplan-Meier survival curves and Whitney Mann U tests were conducted for validating the statistical significance. Results Here we show that an inverse correlation exists between aggressive tumor behavior (increased metastatic potential and accelerated time to death) and the residual expression of 14q32 miRNAs (using miR-382 as a representative of 14q32 miRNAs) in a series of clinically annotated samples from human osteosarcoma patients. We also show a comparable decrease in expression of orthologous 14q32 miRNAs in canine osteosarcoma samples, with conservation of the inverse correlation between aggressive behavior and expression of orthologous miRNA miR-134 and miR-544. Conclusions We conclude that downregulation of 14q32 miRNA expression is an evolutionarily conserved mechanism that contributes to the biological behavior of osteosarcoma, and that quantification of representative transcripts from this family, such as miR-382, miR-134, and miR-544, provide prognostic and predictive markers that can assist in the management of patients with this disease. PMID:23311495

  3. Microarray data re-annotation reveals specific lncRNAs and their potential functions in non-small cell lung cancer subtypes

    OpenAIRE

    Zhou, Dongbo; Xie, Mingxuan; He, Baimei; Gao, Ying; Yu, Qiao; He, Bixiu; Chen, Qiong

    2017-01-01

    Non-small-cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. The most common subtypes of NSCLC are adenocarcinoma (AC) and squamous cell carcinoma (SCC). However, the pathophysiological mechanisms contributing to AC and SCC are still largely unknown, especially the roles of long non-coding RNAs (lncRNAs). The present study identified differentially expressed lncRNAs between lung AC and SCC by re-annotation of NSCLC microarray data analysis profiling. The potential func...

  4. Identification of crucial microRNAs and genes in hypoxia-induced human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Geng Y

    2016-07-01

    Full Text Available Ying Geng,1,* Lili Deng,2,* Dongju Su,1 Jinling Xiao,1 Dongjie Ge,3 Yongxia Bao,1 Hui Jing4 1Department of Respiratory, 2Department of Oncology, The Second Affiliated Hospital of Harbin Medical University, 3Department of Respiratory, The First Hospital of Harbin, 4Department of Emergency, The Second Affiliated Hospital of Harbin Medical University Harbin, Heilongjiang, People’s Republic of China *These authors contributed equally to this work Background: Variations of microRNA (miRNA expression profile in hypoxic lung cancer cells have not been studied so far. Therefore, using miRNA microarray technology, this study aimed to study the miRNA expression profile and investigate the potential crucial miRNAs and their target genes in hypoxia-induced human lung adenocarcinoma cells.Materials and methods: Based on miRNA microarray, miRNA expression profiling of hypoxia-induced lung adenocarcinoma A549 cells was obtained. After identification of differentially expressed miRNAs (DE-miRNAs in hypoxic cells, target genes of DE-miRNAs were predicted, and functional enrichment analysis of targets was conducted. Furthermore, the expression levels of DE-miRNAs and their target genes were validated by real-time quantitative polymerase chain reaction. In addition, using miRNA mimics, the effect of overexpressed DE-miRNAs on A549 cell behaviors (cell proliferation, cell cycle, and apoptosis was evaluated.Results: In total, 14 DE-miRNAs (nine upregulated miRNAs and five downregulated miRNAs were identified in hypoxic cells, compared with normoxic cells. Target genes of both upregulated and downregulated miRNAs were enriched in the functions such as chromatin modification, and pathways such as Wnt signaling pathway and transforming growth factor (TGF-β signaling pathway. The expression levels of several miRNAs and their target genes were confirmed, including hsa-miR-301b/FOXF2, hsa-miR-148b-3p/WNT10B, hsa-miR-769-5p/(SMAD2, ARID1A, and hsa-miR-622. Among them

  5. MicroRNAs control transcription factor NF-kB (p65) expression in human ovarian cells.

    Science.gov (United States)

    Sirotkin, Alexander V; Alexa, Richard; Kišová, Gabriela; Harrath, Abdel Halim; Alwasel, Saleh; Ovcharenko, Dmitriy; Mlynček, Miloš

    2015-05-01

    MicroRNAs (miRNAs) are known to influence ovarian cell proliferation, apoptosis and hormone release, but it remains unknown whether miRNAs affect ovarian functions via transcription factors. We examined the effect of miRNAs on nuclear factor-κappaB (NF-kB) (p65) expression in human ovarian luteinized granulosa cells. We transfected cultured primary human ovarian luteinized granulosa cells with 80 different constructs encoding human pre-miRNAs and then evaluated NF-kB (p65) expression (percentage of cells containing p65) by immunocytochemistry. We found that 21 of the constructs stimulated NF-kB (p65) expression and 18 of the constructs inhibited NF-kB (p65) expression. This is the first direct demonstration that miRNAs affect NF-kB (p65) expression and the first genome-scale miRNA screen to identify upregulation and downregulation of NF-kB accumulation by miRNAs in the ovary. Novel miRNAs that affect the NF-kB signalling pathway could be useful for the control of NF-kB-dependent reproductive processes and the treatment of NF-kB-dependent reproductive disorders.

  6. Implications of MicroRNAs in the Treatment of Gefitinib-Resistant Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Thomas K. Sin

    2016-02-01

    Full Text Available Non-small cell lung cancer (NSCLC represents about 85% of the reported cases of lung cancer. Acquired resistance to targeted therapy with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs, such as gefitinib, is not uncommon. It is thus vital to explore novel strategies to restore sensitivity to gefitinib. Provided that microRNAs (miRNAs negatively regulate their gene targets at the transcriptional level, it is speculated that miRNA mimetics may reduce the expression, activity and signal transduction of EGFR so that sensitization of tumour sites to gefitinib-induced cytotoxicity can be achieved. Indeed, a growing body of evidence has shown that the manipulation of endogenous levels of miRNA not only attenuates the EGFR/PI3K/Akt phosphorylation cascade, but also restores apoptotic cell death in in vitro models of experimentally-induced gefitinib resistance and provoked tumour regression/shrinkage in xenograft models. These data are in concordant with the clinical data showing that the differential expression profiles of miRNA in tumour tissues and blood associate strongly with drug response and overall survival. Furthermore, another line of studies indicate that the chemopreventive effects of a variety of natural compounds may involve miRNAs. The present review aims to discuss the therapeutic capacity of miRNAs in relation to recent discoveries on EGFR-TKI resistance, including chronic drug exposure and mutations.

  7. MicroRNAs, Regulatory Networks, and Comorbidities

    DEFF Research Database (Denmark)

    Russo, Francesco; Belling, Kirstine; Jensen, Anders Boeck

    2017-01-01

    MicroRNAs (miRNAs) are small noncoding RNAs involved in the posttranscriptional regulation of messenger RNAs (mRNAs). Each miRNA targets a specific set of mRNAs. Upon binding the miRNA inhibits mRNA translation or facilitate mRNA degradation. miRNAs are frequently deregulated in several pathologies...

  8. Long Intergenic Noncoding RNAs Mediate the Human Chondrocyte Inflammatory Response and Are Differentially Expressed in Osteoarthritis Cartilage.

    Science.gov (United States)

    Pearson, Mark J; Philp, Ashleigh M; Heward, James A; Roux, Benoit T; Walsh, David A; Davis, Edward T; Lindsay, Mark A; Jones, Simon W

    2016-04-01

    To identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA. OA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non-OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase-polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex. RNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin-1β (IL-1β) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50-associated cyclooxygenase 2-extragenic RNA (PACER) and 2 novel chondrocyte inflammation-associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non-OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL-1-stimulated secretion of proinflammatory cytokines. The inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role in mediating inflammation-driven cartilage degeneration in

  9. The overexpression of insect endogenous small RNAs in transgenic rice inhibits growth and delays pupation of striped stem borer (Chilo suppressalis).

    Science.gov (United States)

    Jiang, Shan; Wu, Hao; Liu, Haoju; Zheng, Jie; Lin, Yongjun; Chen, Hao

    2017-07-01

    The striped stem borer (SSB), Chilo suppressalis Walker, is a major rice insect pest worldwide. RNA interference (RNAi) has become a promising strategy for developing insect-resistant crops. In a previous study, five double-stranded RNAs (dsRNAs) targeting important SSB housekeeping genes were overexpressed in rice, but none of the acquired dsRNA-transgenic rice plants showed significant effects on SSB. Thirteen selected SSB endogenous small RNAs, predicted as SSB novel microRNAs (miRNAs), were overexpressed in rice using artificial miRNA (amiRNA) expression technology. Feeding tests showed that two out of 13 selected SSB novel miRNAs caused significant growth inhibition for feeding SSB larvae based on transgenic rice expression. Pupation was delayed 4 days when SSB larvae consecutively fed on transgenic rice expressing the SSB novel miRNA candidate csu-novel-miR15 (csu-15 rice). Gene expression analysis confirmed that the expression levels of at least six SSB unigenes significantly changed (i.e., were up- or down-regulated) after feeding on csu-15 rice. Our research demonstrated a novel RNAi strategy using SSB endogenous small RNAs to develop RNAi crops for pest management; this strategy is different from the common RNAi resulting from transgenic dsRNAs or amiRNAs targeting certain insect endogenous genes. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  10. Senescence-associated microRNAs target cell cycle regulatory genes in normal human lung fibroblasts.

    Science.gov (United States)

    Markopoulos, Georgios S; Roupakia, Eugenia; Tokamani, Maria; Vartholomatos, George; Tzavaras, Theodore; Hatziapostolou, Maria; Fackelmayer, Frank O; Sandaltzopoulos, Raphael; Polytarchou, Christos; Kolettas, Evangelos

    2017-10-01

    Senescence recapitulates the ageing process at the cell level. A senescent cell stops dividing and exits the cell cycle. MicroRNAs (miRNAs) acting as master regulators of transcription, have been implicated in senescence. In the current study we investigated and compared the expression of miRNAs in young versus senescent human fibroblasts (HDFs), and analysed the role of mRNAs expressed in replicative senescent HFL-1 HDFs. Cell cycle analysis confirmed that HDFs accumulated in G 1 /S cell cycle phase. Nanostring analysis of isolated miRNAs from young and senescent HFL-1 showed that a distinct set of 15 miRNAs were significantly up-regulated in senescent cells including hsa-let-7d-5p, hsa-let-7e-5p, hsa-miR-23a-3p, hsa-miR-34a-5p, hsa-miR-122-5p, hsa-miR-125a-3p, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-181a-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-503-5p, hsa-miR-574-3p, hsa-miR-574-5p and hsa-miR-4454. Importantly, pathway analysis of miRNA target genes down-regulated during replicative senescence in a public RNA-seq data set revealed a significant high number of genes regulating cell cycle progression, both G 1 /S and G 2 /M cell cycle phase transitions and telomere maintenance. The reduced expression of selected miRNA targets, upon replicative and oxidative-stress induced senescence, such as the cell cycle effectors E2F1, CcnE, Cdc6, CcnB1 and Cdc25C was verified at the protein and/or RNA levels. Induction of G1/S cell cycle phase arrest and down-regulation of cell cycle effectors correlated with the up-regulation of miR-221 upon both replicative and oxidative stress-induced senescence. Transient expression of miR-221/222 in HDFs promoted the accumulation of HDFs in G1/S cell cycle phase. We propose that miRNAs up-regulated during replicative senescence may act in concert to induce cell cycle phase arrest and telomere erosion, establishing a senescent phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Role of transposon-derived small RNAs in the interplay between genomes and parasitic DNA in rice.

    Directory of Open Access Journals (Sweden)

    Misuzu Nosaka

    2012-09-01

    Full Text Available RNA silencing is a defense system against "genomic parasites" such as transposable elements (TE, which are potentially harmful to host genomes. In plants, transcripts from TEs induce production of double-stranded RNAs (dsRNAs and are processed into small RNAs (small interfering RNAs, siRNAs that suppress TEs by RNA-directed DNA methylation. Thus, the majority of TEs are epigenetically silenced. On the other hand, most of the eukaryotic genome is composed of TEs and their remnants, suggesting that TEs have evolved countermeasures against host-mediated silencing. Under some circumstances, TEs can become active and increase in copy number. Knowledge is accumulating on the mechanisms of TE silencing by the host; however, the mechanisms by which TEs counteract silencing are poorly understood. Here, we show that a class of TEs in rice produces a microRNA (miRNA to suppress host silencing. Members of the microRNA820 (miR820 gene family are located within CACTA DNA transposons in rice and target a de novo DNA methyltransferase gene, OsDRM2, one of the components of epigenetic silencing. We confirmed that miR820 negatively regulates the expression of OsDRM2. In addition, we found that expression levels of various TEs are increased quite sensitively in response to decreased OsDRM2 expression and DNA methylation at TE loci. Furthermore, we found that the nucleotide sequence of miR820 and its recognition site within the target gene in some Oryza species have co-evolved to maintain their base-pairing ability. The co-evolution of these sequences provides evidence for the functionality of this regulation. Our results demonstrate how parasitic elements in the genome escape the host's defense machinery. Furthermore, our analysis of the regulation of OsDRM2 by miR820 sheds light on the action of transposon-derived small RNAs, not only as a defense mechanism for host genomes but also as a regulator of interactions between hosts and their parasitic elements.

  12. Modeling post-transcriptional regulation activity of small non-coding RNAs in Escherichia coli.

    Science.gov (United States)

    Wang, Rui-Sheng; Jin, Guangxu; Zhang, Xiang-Sun; Chen, Luonan

    2009-04-29

    Transcriptional regulation is a fundamental process in biological systems, where transcription factors (TFs) have been revealed to play crucial roles. In recent years, in addition to TFs, an increasing number of non-coding RNAs (ncRNAs) have been shown to mediate post-transcriptional processes and regulate many critical pathways in both prokaryotes and eukaryotes. On the other hand, with more and more high-throughput biological data becoming available, it is possible and imperative to quantitatively study gene regulation in a systematic and detailed manner. Most existing studies for inferring transcriptional regulatory interactions and the activity of TFs ignore the possible post-transcriptional effects of ncRNAs. In this work, we propose a novel framework to infer the activity of regulators including both TFs and ncRNAs by exploring the expression profiles of target genes and (post)transcriptional regulatory relationships. We model the integrated regulatory system by a set of biochemical reactions which lead to a log-bilinear problem. The inference process is achieved by an iterative algorithm, in which two linear programming models are efficiently solved. In contrast to available related studies, the effects of ncRNAs on transcription process are considered in this work, and thus more reasonable and accurate reconstruction can be expected. In addition, the approach is suitable for large-scale problems from the viewpoint of computation. Experiments on two synthesized data sets and a model system of Escherichia coli (E. coli) carbon source transition from glucose to acetate illustrate the effectiveness of our model and algorithm. Our results show that incorporating the post-transcriptional regulation of ncRNAs into system model can mine the hidden effects from the regulation activity of TFs in transcription processes and thus can uncover the biological mechanisms in gene regulation in a more accurate manner. The software for the algorithm in this paper is available

  13. Determination of the potential bioavailability of plant microRNAs using a simulated human digestion process.

    Science.gov (United States)

    Philip, Anna; Ferro, Valerie A; Tate, Rothwelle J

    2015-10-01

    The "dietary xenomiR hypothesis" proposes that microRNAs (miRNAs) in foodstuffs survive transit through the mammalian gastrointestinal tract and pass into cells intact to affect gene regulation. However, debate continues as to whether dietary intake poses a feasible route for such exogenous gene regulators. Understanding on miRNA levels during pretreatments of human diet is essential to test their bioavailability during digestion. This study makes the novel first use of an in vitro method to eliminate the inherent complexities and variability of in vivo approaches used to test this hypothesis. Plant miRNA levels in soybean and rice were measured during storage, processing, cooking, and early digestion using real-time PCR. We have demonstrated for the first time that storage, processing, and cooking does not abolish the plant miRNAs present in the foodstuffs. In addition, utilizing a simulated human digestion system revealed significant plant miRNA bioavailability after early stage digestion for 75 min. Attenuation of plant messenger RNA and synthetic miRNA was observed under these conditions. Even after an extensive pretreatment, plant-derived miRNA, delivered by typical dietary ingestion, has a robustness that could make them bioavailable for uptake during early digestion. The potential benefit of these regulatory molecules in pharma nutrition could be explored further. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Lnc2Cancer: a manually curated database of experimentally supported lncRNAs associated with various human cancers.

    Science.gov (United States)

    Ning, Shangwei; Zhang, Jizhou; Wang, Peng; Zhi, Hui; Wang, Jianjian; Liu, Yue; Gao, Yue; Guo, Maoni; Yue, Ming; Wang, Lihua; Li, Xia

    2016-01-04

    Lnc2Cancer (http://www.bio-bigdata.net/lnc2cancer) is a manually curated database of cancer-associated long non-coding RNAs (lncRNAs) with experimental support that aims to provide a high-quality and integrated resource for exploring lncRNA deregulation in various human cancers. LncRNAs represent a large category of functional RNA molecules that play a significant role in human cancers. A curated collection and summary of deregulated lncRNAs in cancer is essential to thoroughly understand the mechanisms and functions of lncRNAs. Here, we developed the Lnc2Cancer database, which contains 1057 manually curated associations between 531 lncRNAs and 86 human cancers. Each association includes lncRNA and cancer name, the lncRNA expression pattern, experimental techniques, a brief functional description, the original reference and additional annotation information. Lnc2Cancer provides a user-friendly interface to conveniently browse, retrieve and download data. Lnc2Cancer also offers a submission page for researchers to submit newly validated lncRNA-cancer associations. With the rapidly increasing interest in lncRNAs, Lnc2Cancer will significantly improve our understanding of lncRNA deregulation in cancer and has the potential to be a timely and valuable resource. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. 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...

  16. Trash or Treasure: extracellular microRNAs and cell-to-cell communication

    Directory of Open Access Journals (Sweden)

    Nobuyoshi eKosaka

    2013-09-01

    Full Text Available Circulating RNAs in human body fluids are promising candidates for diagnostic purposes. However, the biological significance of circulating RNAs remains elusive. Recently, small non-coding RNAs, microRNAs (miRNAs, were isolated from multiple human body fluids, and these circulating miRNAs have been implicated as novel disease biomarkers. Concurrently, miRNAs were also identified in the extracellular space associated with extracellular vesicles (EVs, which are small membrane vesicles secreted from various types of cells. The function of these secreted miRNAs has been revealed in several papers. Circulating miRNAs have been experimentally found to be associated with EVs, however, other types of extracellular miRNAs were also described. This review discusses studies related to extracellular miRNAs, including circulating miRNAs and secreted miRNAs, to highlight the importance of studying not only secreted miRNAs but also circulating miRNAs to determine the contribution of extracellular miRNAs especially in cancer development.

  17. Non-Coding RNAs and Endometrial Cancer

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    Cristina Vallone

    2018-03-01

    Full Text Available Non-coding RNAs (ncRNAs are involved in the regulation of cell metabolism and neoplastic transformation. Recent studies have tried to clarify the significance of these information carriers in the genesis and progression of various cancers and their use as biomarkers for the disease; possible targets for the inhibition of growth and invasion by the neoplastic cells have been suggested. The significance of ncRNAs in lung cancer, bladder cancer, kidney cancer, and melanoma has been amply investigated with important results. Recently, the role of long non-coding RNAs (lncRNAs has also been included in cancer studies. Studies on the relation between endometrial cancer (EC and ncRNAs, such as small ncRNAs or micro RNAs (miRNAs, transfer RNAs (tRNAs, ribosomal RNAs (rRNAs, antisense RNAs (asRNAs, small nuclear RNAs (snRNAs, Piwi-interacting RNAs (piRNAs, small nucleolar RNAs (snoRNAs, competing endogenous RNAs (ceRNAs, lncRNAs, and long intergenic ncRNAs (lincRNAs have been published. The recent literature produced in the last three years was extracted from PubMed by two independent readers, which was then selected for the possible relation between ncRNAs, oncogenesis in general, and EC in particular.

  18. 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.

  19. Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs.

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    Federica Collino

    Full Text Available BACKGROUND: Cell-derived microvesicles (MVs have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs and liver resident stem cells (HLSCs were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs. METHODOLOGY/PRINCIPAL FINDINGS: MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation. CONCLUSIONS: This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem

  20. C. elegans microRNAs.

    Science.gov (United States)

    Vella, Monica C; Slack, Frank J

    2005-09-21

    MicroRNAs (miRNAs) are small, non-coding regulatory RNAs found in many phyla that control such diverse events as development, metabolism, cell fate and cell death. They have also been implicated in human cancers. The C. elegans genome encodes hundreds of miRNAs, including the founding members of the miRNA family lin-4 and let-7. Despite the abundance of C. elegans miRNAs, few miRNA targets are known and little is known about the mechanism by which they function. However, C. elegans research continues to push the boundaries of discovery in this area. lin-4 and let-7 are the best understood miRNAs. They control the timing of adult cell fate determination in hypodermal cells by binding to partially complementary sites in the mRNA of key developmental regulators to repress protein expression. For example, lin-4 is predicted to bind to seven sites in the lin-14 3' untranslated region (UTR) to repress LIN-14, while let-7 is predicted to bind two let-7 complementary sites in the lin-41 3' UTR to down-regulate LIN-41. Two other miRNAs, lsy-6 and mir-273, control left-right asymmetry in neural development, and also target key developmental regulators for repression. Approximately one third of the C. elegans miRNAs are differentially expressed during development indicating a major role for miRNAs in C. elegans development. Given the remarkable conservation of developmental mechanism across phylogeny, many of the principles of miRNAs discovered in C. elegans are likely to be applicable to higher animals.

  1. An integrated expression atlas of miRNAs and their promoters in human and mouse

    DEFF Research Database (Denmark)

    de Rie, Derek; Abugessaisa, Imad; Alam, Tanvir

    2017-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libr...

  2. Small interfering RNAs from bidirectional transcripts of GhMML3_A12 regulate cotton fiber development.

    Science.gov (United States)

    Wan, Qun; Guan, Xueying; Yang, Nannan; Wu, Huaitong; Pan, Mengqiao; Liu, Bingliang; Fang, Lei; Yang, Shouping; Hu, Yan; Ye, Wenxue; Zhang, Hua; Ma, Peiyong; Chen, Jiedan; Wang, Qiong; Mei, Gaofu; Cai, Caiping; Yang, Donglei; Wang, Jiawei; Guo, Wangzhen; Zhang, Wenhua; Chen, Xiaoya; Zhang, Tianzhen

    2016-06-01

    Natural antisense transcripts (NATs) are commonly observed in eukaryotic genomes, but only a limited number of such genes have been identified as being involved in gene regulation in plants. In this research, we investigated the function of small RNA derived from a NAT in fiber cell development. Using a map-based cloning strategy for the first time in tetraploid cotton, we cloned a naked seed mutant gene (N1 ) encoding a MYBMIXTA-like transcription factor 3 (MML3)/GhMYB25-like in chromosome A12, GhMML3_A12, that is associated with fuzz fiber development. The extremely low expression of GhMML3_A12 in N1 is associated with NAT production, driven by its 3' antisense promoter, as indicated by the promoter-driven histochemical staining assay. In addition, small RNA deep sequencing analysis suggested that the bidirectional transcriptions of GhMML3_A12 form double-stranded RNAs and generate 21-22 nt small RNAs. Therefore, in a fiber-specific manner, small RNA derived from the GhMML3_A12 locus can mediate GhMML3_A12 mRNA self-cleavage and result in the production of naked seeds followed by lint fiber inhibition in N1 plants. The present research reports the first observation of gene-mediated NATs and siRNA directly controlling fiber development in cotton. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  3. The regulatory effect of miRNAs is a heritable genetic trait in humans

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    Geeleher Paul

    2012-08-01

    Full Text Available Abstract Background microRNAs (miRNAs have been shown to regulate the expression of a large number of genes and play key roles in many biological processes. Several previous studies have quantified the inhibitory effect of a miRNA indirectly by considering the expression levels of genes that are predicted to be targeted by the miRNA and this approach has been shown to be robust to the choice of prediction algorithm. Given a gene expression dataset, Cheng et al. defined the regulatory effect score (RE-score of a miRNA as the difference in the gene expression rank of targets of the miRNA compared to non-targeted genes. Results Using microarray data from parent-offspring trios from the International HapMap project, we show that the RE-score of most miRNAs is correlated between parents and offspring and, thus, inter-individual variation in RE-score has a genetic component in humans. Indeed, the mean RE-score across miRNAs is correlated between parents and offspring, suggesting genetic differences in the overall efficiency of the miRNA biogenesis pathway between individuals. To explore the genetics of this quantitative trait further, we carried out a genome-wide association study of the mean RE-score separately in two HapMap populations (CEU and YRI. No genome-wide significant associations were discovered; however, a SNP rs17409624, in an intron of DROSHA, was significantly associated with mean RE-score in the CEU population following permutation-based control for multiple testing based on all SNPs mapped to the canonical miRNA biogenesis pathway; of 244 individual miRNA RE-scores assessed in the CEU, 214 were associated (p p = 0.04 with mean RE-score in the YRI population. Interestingly, the same SNP was associated with 17 (8.5% of all expressed miRNA expression levels in the CEU. We also show here that the expression of the targets of most miRNAs is more highly correlated with global changes in miRNA regulatory effect than with the expression of

  4. Targeting MicroRNAs with Small Molecules a Novel Approach to Treating Breast Cancer

    Science.gov (United States)

    2011-10-01

    or deoxyribozyme, is a catalytic DNA that site-specifically cleaves the target RNA Watson – Crick base pairing to a complementary target sequence...RNA A-helix fold among the selected pre- miRNA targets. Furthermore, 3D characteristics including Watson - Crick base pairs and wobble base pairs...phosphorothioate backbone in addition to 2′-O-methoxyethyl AMOs are ASOs against miRNAs and therefore produce ASO–miRNA duplexes through Watson – Crick binding

  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. MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis.

    Science.gov (United States)

    Boštjančič, Emanuela; Zidar, Nina; Glavač, Damjan

    2012-10-15

    Cardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3'-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI. The protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed. Combing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.

  7. 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.

  8. Visualized and precise design of artificial small RNAs for regulating T7 RNA polymerase and enhancing recombinant protein folding in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yujia Zhao

    2016-12-01

    Full Text Available Small non-coding RNAs (sRNAs have received much attention in recent years due to their unique biological properties, which can efficiently and specifically tune target gene expressions in bacteria. Inspired by natural sRNAs, recent works have proposed the use of artificial sRNAs (asRNAs as genetic tools to regulate desired gene that has been applied in several fields, such as metabolic engineering and bacterial physiology studies. However, the rational design of asRNAs is still a challenge. In this study, we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs. T7 expression system was one of the most useful recombinant protein expression systems. However, it was deeply limited by the formation of inclusion body. To settle this problem, we designed a series of asRNAs to inhibit the T7 RNA polymerase (Gene1 expression to balance the rate between transcription and folding of recombinant protein. Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E. coli, the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.

  9. On revealing the gene targets of Ebola virus microRNAs involved in the human skin microbiome

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    Pei-Chun Hsu

    2018-01-01

    Full Text Available Ebola virus, a negative-sense single-stranded RNA virus, causes severe viral hemorrhagic fever and has a high mortality rate. Histopathological and immunopathological analyses of Ebola virus have revealed that histopathological changes in skin tissue are associated with various degrees of endothelial cell swelling and necrosis. The interactions of microbes within or on a host are a crucial for the skin immune shield. The discovery of microRNAs (miRNAs in Ebola virus implies that immune escape, endothelial cell rupture, and tissue dissolution during Ebola virus infection are a result of the effects of Ebola virus miRNAs. Keratinocytes obtained from normal skin can attach and spread through expression of the thrombospondin family of proteins, playing a role in initiation of cell-mediated immune responses in the skin. Several miRNAs have been shown to bind the 3′ untranslated region of thrombospondin mRNA, thereby controlling its stability and translational activity. In this study, we discovered short RNA sequences that may act as miRNAs from Propionibacterium acnes using a practical workflow of bioinformatics methods. Subsequently, we deciphered the common target gene. These RNA sequences tended to bind to the same thrombospondin protein, THSD4, emphasizing the potential importance of the synergistic binding of miRNAs from Ebola virus, Propionibacterium acnes, and humans to the target. These results provide important insights into the molecular mechanisms of thrombospondin proteins and miRNAs in Ebola virus infection.

  10. Involvement of miRNAs in the differentiation of human glioblastoma multiforme stem-like cells.

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    Beatriz Aldaz

    Full Text Available Glioblastoma multiforme (GBM-initiating cells (GICs represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.

  11. Non-Coding RNAs: Multi-Tasking Molecules in the Cell

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    Anita Quintal Gomes

    2013-07-01

    Full Text Available In the last years it has become increasingly clear that the mammalian transcriptome is highly complex and includes a large number of small non-coding RNAs (sncRNAs and long noncoding RNAs (lncRNAs. Here we review the biogenesis pathways of the three classes of sncRNAs, namely short interfering RNAs (siRNAs, microRNAs (miRNAs and PIWI-interacting RNAs (piRNAs. These ncRNAs have been extensively studied and are involved in pathways leading to specific gene silencing and the protection of genomes against virus and transposons, for example. Also, lncRNAs have emerged as pivotal molecules for the transcriptional and post-transcriptional regulation of gene expression which is supported by their tissue-specific expression patterns, subcellular distribution, and developmental regulation. Therefore, we also focus our attention on their role in differentiation and development. SncRNAs and lncRNAs play critical roles in defining DNA methylation patterns, as well as chromatin remodeling thus having a substantial effect in epigenetics. The identification of some overlaps in their biogenesis pathways and functional roles raises the hypothesis that these molecules play concerted functions in vivo, creating complex regulatory networks where cooperation with regulatory proteins is necessary. We also highlighted the implications of biogenesis and gene expression deregulation of sncRNAs and lncRNAs in human diseases like cancer.

  12. Ligand-targeted delivery of small interfering RNAs to malignant cells and tissues.

    Science.gov (United States)

    Thomas, Mini; Kularatne, Sumith A; Qi, Longwu; Kleindl, Paul; Leamon, Christopher P; Hansen, Michael J; Low, Philip S

    2009-09-01

    Potential clinical applications of small interfering RNA (siRNA) are hampered primarily by delivery issues. We have successfully addressed the delivery problems associated with off-site targeting of highly toxic chemotherapeutic agents by attaching the drugs to tumor-specific ligands that will carry the attached cargo into the desired cancer cell. Indeed, several such tumor-targeted drugs are currently undergoing human clinical trials. We now show that efficient targeting of siRNA to malignant cells and tissues can be achieved by covalent conjugation of small-molecular-weight, high-affinity ligands, such as folic acid and DUPA (2-[3-(1, 3-dicarboxy propyl)-ureido] pentanedioic acid), to siRNA. The former ligand binds a folate receptor that is overexpressed on a variety of cancers, whereas the latter ligand binds to prostate-specific membrane antigen that is overexpressed specifically on prostate cancers and the neovasculature of all solid tumors. Using these ligands, we show remarkable receptor-mediated targeting of siRNA to cancer tissues in vitro and in vivo.

  13. Primate-specific spliced PMCHL RNAs are non-protein coding in human and macaque tissues

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    Delerue-Audegond Audrey

    2008-12-01

    Full Text Available Abstract Background Brain-expressed genes that were created in primate lineage represent obvious candidates to investigate molecular mechanisms that contributed to neural reorganization and emergence of new behavioural functions in Homo sapiens. PMCHL1 arose from retroposition of a pro-melanin-concentrating hormone (PMCH antisense mRNA on the ancestral human chromosome 5p14 when platyrrhines and catarrhines diverged. Mutations before divergence of hylobatidae led to creation of new exons and finally PMCHL1 duplicated in an ancestor of hominids to generate PMCHL2 at the human chromosome 5q13. A complex pattern of spliced and unspliced PMCHL RNAs were found in human brain and testis. Results Several novel spliced PMCHL transcripts have been characterized in human testis and fetal brain, identifying an additional exon and novel splice sites. Sequencing of PMCHL genes in several non-human primates allowed to carry out phylogenetic analyses revealing that the initial retroposition event took place within an intron of the brain cadherin (CDH12 gene, soon after platyrrhine/catarrhine divergence, i.e. 30–35 Mya, and was concomitant with the insertion of an AluSg element. Sequence analysis of the spliced PMCHL transcripts identified only short ORFs of less than 300 bp, with low (VMCH-p8 and protein variants or no evolutionary conservation. Western blot analyses of human and macaque tissues expressing PMCHL RNA failed to reveal any protein corresponding to VMCH-p8 and protein variants encoded by spliced transcripts. Conclusion Our present results improve our knowledge of the gene structure and the evolutionary history of the primate-specific chimeric PMCHL genes. These genes produce multiple spliced transcripts, bearing short, non-conserved and apparently non-translated ORFs that may function as mRNA-like non-coding RNAs.

  14. Competition between virus-derived and endogenous small RNAs regulates gene expression in Caenorhabditis elegans.

    Science.gov (United States)

    Sarkies, Peter; Ashe, Alyson; Le Pen, Jérémie; McKie, Mikel A; Miska, Eric A

    2013-08-01

    Positive-strand RNA viruses encompass more than one-third of known virus genera and include many medically and agriculturally relevant human, animal, and plant pathogens. The nematode Caenorhabditis elegans and its natural pathogen, the positive-strand RNA virus Orsay, have recently emerged as a new animal model to understand the mechanisms and evolution of innate immune responses. In particular, the RNA interference (RNAi) pathway is required for C. elegans resistance to viral infection. Here we report the first genome-wide analyses of gene expression upon viral infection in C. elegans. Using the laboratory strain N2, we identify a novel C. elegans innate immune response specific to viral infection. A subset of these changes is driven by the RNAi response to the virus, which redirects the Argonaute protein RDE-1 from its endogenous small RNA cofactors, leading to loss of repression of endogenous RDE-1 targets. Additionally, we show that a C. elegans wild isolate, JU1580, has a distinct gene expression signature in response to viral infection. This is associated with a reduction in microRNA (miRNA) levels and an up-regulation of their target genes. Intriguingly, alterations in miRNA levels upon JU1580 infection are associated with a transformation of the antiviral transcriptional response into an antibacterial-like response. Together our data support a model whereby antiviral RNAi competes with endogenous small RNA pathways, causing widespread transcriptional changes. This provides an elegant mechanism for C. elegans to orchestrate its antiviral response, which may have significance for the relationship between small RNA pathways and immune regulation in other organisms.

  15. Expression of antisense small RNAs in response to stress in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Gómez Lozano, María; Marvig, Rasmus Lykke; Tulstrup, Monica Vera-Lise

    2014-01-01

    Background: RNA sequencing technologies reveal that bacteria express RNA molecules other than mRNA, rRNA or tRNA. During the last years genome-wide bacterial transcriptomes have been shown to comprise intergenic RNA, antisense RNA, and untranslated regions, all capable of performing diverse...... that the extent of overlap between the studies is very limited. Conclusions: RNA-seq experiments are revealing hundreds of novel transcripts in all bacterial genomes investigated. The comparison between independent studies that used RNA-seq to detect novel asRNAs in P. aeruginosa shows that the overlap between...

  16. Circular RNAs

    DEFF Research Database (Denmark)

    Han, Yi-Neng; Xia, Shengqiang; Zhang, Yuan-Yuan

    2017-01-01

    Circular RNAs (circRNAs) are a novel type of universal and diverse endogenous noncoding RNAs (ncRNAs) and they form a covalently closed continuous loop without 5' or 3' tails unlike linear RNAs. Most circRNAs are presented with characteristics of abundance, stability, conservatism, and often exhi...... and expression regulators, RBP sponges in cancer as well as current research methods of circRNAs, providing evidence for the significance of circRNAs in cancer diagnosis and clinical treatment....

  17. High-Throughput Sequencing of MicroRNAs in Adenovirus Type 3 Infected Human Laryngeal Epithelial Cells

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    Yuhua Qi

    2010-01-01

    Full Text Available Adenovirus infection can cause various illnesses depending on the infecting serotype, such as gastroenteritis, conjunctivitis, cystitis, and rash illness, but the infection mechanism is still unknown. MicroRNAs (miRNA have been reported to play essential roles in cell proliferation, cell differentiation, and pathogenesis of human diseases including viral infections. We analyzed the miRNA expression profiles from adenovirus type 3 (AD3 infected Human laryngeal epithelial (Hep2 cells using a SOLiD deep sequencing. 492 precursor miRNAs were identified in the AD3 infected Hep2 cells, and 540 precursor miRNAs were identified in the control. A total of 44 miRNAs demonstrated high expression and 36 miRNAs showed lower expression in the AD3 infected cells than control. The biogenesis of miRNAs has been analyzed, and some of the SOLiD results were confirmed by Quantitative PCR analysis. The present studies may provide a useful clue for the biological function research into AD3 infection.

  18. Proteomic screening of human targets of viral microRNAs reveals functions associated with immune evasion and angiogenesis.

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    Amelia M Gallaher

    Full Text Available Kaposi's sarcoma (KS is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV. The virus expresses unique microRNAs (miRNAs, but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1 following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α, and up-regulation of a protein involved in stimulating angiogenesis (HMOX1. This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.

  19. Mating of the stichotrichous ciliate Oxytricha trifallax induces production of a class of 27 nt small RNAs derived from the parental macronucleus.

    Directory of Open Access Journals (Sweden)

    Alan M Zahler

    Full Text Available Ciliated protozoans possess two types of nuclei; a transcriptionally silent micronucleus, which serves as the germ line nucleus, and a transcriptionally active macronucleus, which serves as the somatic nucleus. The macronucleus is derived from a new diploid micronucleus after mating, with epigenetic information contributed by the parental macronucleus serving to guide the formation of the new macronucleus. In the stichotrichous ciliate Oxytricha trifallax, the macronuclear DNA is highly processed to yield gene-sized nanochromosomes with telomeres at each end. Here we report that soon after mating of Oxytricha trifallax, abundant 27 nt small RNAs are produced that are not present prior to mating. We performed next generation sequencing of Oxytricha small RNAs from vegetative and mating cells. Using sequence comparisons between macronuclear and micronuclear versions of genes, we found that the 27 nt RNA class derives from the parental macronucleus, not the developing macronucleus. These small RNAs are produced equally from both strands of macronuclear nanochromosomes, but in a highly non-uniform distribution along the length of the nanochromosome, and with a particular depletion in the 30 nt telomere-proximal positions. This production of small RNAs from the parental macronucleus during macronuclear development stands in contrast to the mechanism of epigenetic control in the distantly related ciliate Tetrahymena. In that species, 28-29 nt scanRNAs are produced from the micronucleus and these micronuclear-derived RNAs serve as epigenetic controllers of macronuclear development. Unlike the Tetrahymena scanRNAs, the Oxytricha macronuclear-derived 27 mers are not modified by 2'O-methylation at their 3' ends. We propose models for the role of these "27macRNAs" in macronuclear development.

  20. Small RNAs and extracellular vesicles in filarial nematodes: From nematode development to diagnostics.

    Science.gov (United States)

    Quintana, J F; Babayan, S A; Buck, A H

    2017-02-01

    Parasitic nematodes have evolved sophisticated mechanisms to communicate with their hosts in order to survive and successfully establish an infection. The transfer of RNA within extracellular vesicles (EVs) has recently been described as a mechanism that could contribute to this communication in filarial nematodes. It has been shown that these EVs are loaded with several types of RNAs, including microRNAs, leading to the hypothesis that parasites could actively use these molecules to manipulate host gene expression and to the exciting prospect that these pathways could result in new diagnostic and therapeutic strategies. Here, we review the literature on the diverse RNAi pathways that operate in nematodes and more specifically our current knowledge of extracellular RNA (exRNA) and EVs derived from filarial nematodes in vitro and within their hosts. We further detail some of the issues and questions related to the capacity of RNA-mediated communication to function in parasite-host interactions and the ability of exRNA to enable us to distinguish and detect different nematode parasites in their hosts. © 2016 The Authors. Parasite Immunology published by John Wiley & Sons Ltd.

  1. Rootstock-to-scion transfer of transgene-derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry.

    Science.gov (United States)

    Zhao, Dongyan; Song, Guo-qing

    2014-12-01

    Small interfering RNAs (siRNAs) are silencing signals in plants. Virus-resistant transgenic rootstocks developed through siRNA-mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock-to-scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV-hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV-hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long-distance (1.2 m) transfer of PNRSV-hpRNA-derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for 'using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  2. Altered expression of long non-coding RNAs during genotoxic stress-induced cell death in human glioma cells.

    Science.gov (United States)

    Liu, Qian; Sun, Shanquan; Yu, Wei; Jiang, Jin; Zhuo, Fei; Qiu, Guoping; Xu, Shiye; Jiang, Xuli

    2015-04-01

    Long non-coding RNAs (lncRNAs), a recently discovered class of non-coding genes, are transcribed throughout the genome. Emerging evidence suggests that lncRNAs may be involved in modulating various aspects of tumor biology, including regulating gene activity in response to external stimuli or DNA damage. No data are available regarding the expression of lncRNAs during genotoxic stress-induced apoptosis and/or necrosis in human glioma cells. In this study, we detected a change in the expression of specific candidate lncRNAs (neat1, GAS5, TUG1, BC200, Malat1, MEG3, MIR155HG, PAR5, and ST7OT1) during DNA damage-induced apoptosis in human glioma cell lines (U251 and U87) using doxorubicin (DOX) and resveratrol (RES). We also detected the expression pattern of these lncRNAs in human glioma cell lines under necrosis induced using an increased dose of DOX. Our results reveal that the lncRNA expression patterns are distinct between genotoxic stress-induced apoptosis and necrosis in human glioma cells. The sets of lncRNA expressed during genotoxic stress-induced apoptosis were DNA-damaging agent-specific. Generally, MEG3 and ST7OT1 are up-regulated in both cell lines under apoptosis induced using both agents. The induction of GAS5 is only clearly detected during DOX-induced apoptosis, whereas the up-regulation of neat1 and MIR155HG is only found during RES-induced apoptosis in both cell lines. However, TUG1, BC200 and MIR155HG are down regulated when necrosis is induced using a high dose of DOX in both cell lines. In conclusion, our findings suggest that the distinct regulation of lncRNAs may possibly involve in the process of cellular defense against genotoxic agents.

  3. MSDD: a manually curated database of experimentally supported associations among miRNAs, SNPs and human diseases.

    Science.gov (United States)

    Yue, Ming; Zhou, Dianshuang; Zhi, Hui; Wang, Peng; Zhang, Yan; Gao, Yue; Guo, Maoni; Li, Xin; Wang, Yanxia; Zhang, Yunpeng; Ning, Shangwei; Li, Xia

    2018-01-04

    The MiRNA SNP Disease Database (MSDD, http://www.bio-bigdata.com/msdd/) is a manually curated database that provides comprehensive experimentally supported associations among microRNAs (miRNAs), single nucleotide polymorphisms (SNPs) and human diseases. SNPs in miRNA-related functional regions such as mature miRNAs, promoter regions, pri-miRNAs, pre-miRNAs and target gene 3'-UTRs, collectively called 'miRSNPs', represent a novel category of functional molecules. miRSNPs can lead to miRNA and its target gene dysregulation, and resulting in susceptibility to or onset of human diseases. A curated collection and summary of miRSNP-associated diseases is essential for a thorough understanding of the mechanisms and functions of miRSNPs. Here, we describe MSDD, which currently documents 525 associations among 182 human miRNAs, 197 SNPs, 153 genes and 164 human diseases through a review of more than 2000 published papers. Each association incorporates information on the miRNAs, SNPs, miRNA target genes and disease names, SNP locations and alleles, the miRNA dysfunctional pattern, experimental techniques, a brief functional description, the original reference and additional annotation. MSDD provides a user-friendly interface to conveniently browse, retrieve, download and submit novel data. MSDD will significantly improve our understanding of miRNA dysfunction in disease, and thus, MSDD has the potential to serve as a timely and valuable resource. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization

    International Nuclear Information System (INIS)

    Lawrence, J.B.; Singer, R.H.; Villnave, C.A.; Stein, J.L.; Stein, G.S.

    1988-01-01

    We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs

  5. 3' terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2013-09-21

    Post-transcriptional 3' end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3' RACE coupled with high-throughput sequencing to characterize the 3' terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. The 3' terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3' terminus of an in vitro transcribed MRP RNA control and the differing 3' terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). 3' RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3' terminal sequences of noncoding RNAs.

  6. 3′ terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing

    Science.gov (United States)

    2013-01-01

    Background Post-transcriptional 3′ end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3′ RACE coupled with high-throughput sequencing to characterize the 3′ terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. Results The 3′ terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3′ terminus of an in vitro transcribed MRP RNA control and the differing 3′ terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). Conclusions 3′ RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3′ terminal sequences of noncoding RNAs. PMID:24053768

  7. The PRC2-binding long non-coding RNAs in human and mouse genomes are associated with predictive sequence features

    Science.gov (United States)

    Tu, Shiqi; Yuan, Guo-Cheng; Shao, Zhen

    2017-01-01

    Recently, long non-coding RNAs (lncRNAs) have emerged as an important class of molecules involved in many cellular processes. One of their primary functions is to shape epigenetic landscape through interactions with chromatin modifying proteins. However, mechanisms contributing to the specificity of such interactions remain poorly understood. Here we took the human and mouse lncRNAs that were experimentally determined to have physical interactions with Polycomb repressive complex 2 (PRC2), and systematically investigated the sequence features of these lncRNAs by developing a new computational pipeline for sequences composition analysis, in which each sequence is considered as a series of transitions between adjacent nucleotides. Through that, PRC2-binding lncRNAs were found to be associated with a set of distinctive and evolutionarily conserved sequence features, which can be utilized to distinguish them from the others with considerable accuracy. We further identified fragments of PRC2-binding lncRNAs that are enriched with these sequence features, and found they show strong PRC2-binding signals and are more highly conserved across species than the other parts, implying their functional importance.

  8. The Effect of Botulinum Toxin Type A on Expression Profiling of Long Noncoding RNAs in Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Ying-ying Miao

    2017-01-01

    Full Text Available Objective. This study was aimed at analyzing the expressions of long noncoding RNAs (lncRNAs in Botulinum Toxin Type A (BoNTA treated human dermal fibroblasts (HDFs in vitro. Methods. We used RNA sequencing to characterize the lncRNAs and mRNAs transcriptome in the control and BoNTA treated group, in conjunction with application of GO (gene ontology analysis and KEGG (kyoto encyclopedia of genes and genomes analysis to delineate the alterations in gene expression. We also obtained quantitative real time polymerase chain reaction (qRT-PCR to confirm some differentially expressed genes. Results. Numerous differentially expressed genes were observed by microarrays between the two groups. qRT-PCR confirmed the changes of six lncRNAs (RP11-517C16.2-001, FR271872, LOC283352, RP11-401E9.3, FGFR3P, and XXbac-BPG16N22.5 and nine mRNAs (NOS2, C13orf15, FOS, FCN2, SPINT1, PLAC8, BIRC5, NOS2, and COL19A1. Farther studies indicated that the downregulating effect of BoNTA on the expression of FGFR3P was time-related and the dosage of BoNTA at a range from 2.5 U/106 cells to 7.5 U/106 cells increased the expression of FGFR3P and COL19A1 in HDFs as well. Conclusion. The expression profiling of lncRNAs was visibly changed in BoNTA treated HDFs. Further studies should focus on several lncRNAs to investigate their functions in BoNTA treated HDFs and the underlying mechanisms.

  9. HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

    Directory of Open Access Journals (Sweden)

    Valentina Vongrad

    Full Text Available MiRNAs and other small noncoding RNAs (sncRNAs are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM.The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP, which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

  10. The effects of potato virus Y-derived virus small interfering RNAs of three biologically distinct strains on potato (Solanum tuberosum) transcriptome.

    Science.gov (United States)

    Moyo, Lindani; Ramesh, Shunmugiah V; Kappagantu, Madhu; Mitter, Neena; Sathuvalli, Vidyasagar; Pappu, Hanu R

    2017-07-17

    Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome. The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR. The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3' UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic

  11. Regulation of coagulation factor XI expression by microRNAs in the human liver.

    Directory of Open Access Journals (Sweden)

    Salam Salloum-Asfar

    Full Text Available High levels of factor XI (FXI increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK demonstrated a direct interaction between miR-181a-5p and 3'untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.

  12. Long non-coding RNAs: Mechanism of action and functional utility

    OpenAIRE

    Bhat, Shakil Ahmad; Ahmad, Syed Mudasir; Mumtaz, Peerzada Tajamul; Malik, Abrar Ahad; Dar, Mashooq Ahmad; Urwat, Uneeb; Shah, Riaz Ahmad; Ganai, Nazir Ahmad

    2016-01-01

    Recent RNA sequencing studies have revealed that most of the human genome is transcribed, but very little of the total transcriptomes has the ability to encode proteins. Long non-coding RNAs (lncRNAs) are non-coding transcripts longer than 200 nucleotides. Members of the non-coding genome include microRNA (miRNA), small regulatory RNAs and other short RNAs. Most of long non-coding RNA (lncRNAs) are poorly annotated. Recent recognition about lncRNAs highlights their effects in many biological ...

  13. Panning for Long Noncoding RNAs

    Directory of Open Access Journals (Sweden)

    Li Yang

    2013-02-01

    Full Text Available The recent advent of high-throughput approaches has revealed widespread transcription of the human genome, leading to a new appreciation of transcription regulation, especially from noncoding regions. Distinct from most coding and small noncoding RNAs, long noncoding RNAs (lncRNAs are generally expressed at low levels, are less conserved and lack protein-coding capacity. These intrinsic features of lncRNAs have not only hampered their full annotation in the past several years, but have also generated controversy concerning whether many or most of these lncRNAs are simply the result of transcriptional noise. Here, we assess these intrinsic features that have challenged lncRNA discovery and further summarize recent progress in lncRNA discovery with integrated methodologies, from which new lessons and insights can be derived to achieve better characterization of lncRNA expression regulation. Full annotation of lncRNA repertoires and the implications of such annotation will provide a fundamental basis for comprehensive understanding of pervasive functions of lncRNAs in biological regulation.

  14. Survival and Diversity of Human Homologous Dietary MicroRNAs in Conventionally Cooked Top Sirloin and Dried Bovine Tissue Extracts.

    Directory of Open Access Journals (Sweden)

    Joseph T Dever

    Full Text Available Dietary microRNAs (miRNAs, notably those found in milk, are currently being investigated for their potential to elicit biological effects via canonical binding to human messenger RNA targets once ingested. Besides milk, beef and other bovine tissue-derived ingredients could also be a relevant source of potentially bioactive dietary miRNAs. In this study, we characterized the human homologous miRNA profiles in food-grade, bovine-sourced sirloin, heart and adrenal tissue (raw, cooked, and pasteurized, freeze-dried extracts via deep-sequencing and quantitative reverse transcription PCR (RT-qPCR. A total of 198 human homologous miRNAs were detected at 10 or more normalized reads in all replicates (n = 3 of at least one preparation method. Tissue origin rather than preparation method was the major differentiating factor of miRNA profiles, and adrenal-based miRNA profiles were the most distinct. The ten most prevalent miRNAs in each tissue represented 71-93% of the total normalized counts for all annotated miRNAs. In cooked sirloin, the most abundant miRNAs were miR-10b-5p, (48.8% of total annotated miRNA reads along with the muscle-specific miR-1 (24.1% and miR-206 (4.8%. In dried heart extracts, miR-1 (17.0%, miR-100-5p (16.1% and miR-99a-5p (11.0% gave the highest normalized read counts. In dried adrenal extracts, miR-10b-5p (71.2% was the most prominent followed by miR-143-3p (7.1% and 146b-5p (3.7%. Sequencing results for five detected and two undetected miRNAs were successfully validated by RT-qPCR. We conclude that edible, bovine tissues contain unique profiles of human homologous dietary miRNAs that survive heat-based preparation methods.

  15. 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.

  16. DES-ncRNA: A knowledgebase for exploring information about human micro and long noncoding RNAs based on literature-mining

    KAUST Repository

    Salhi, Adil

    2017-04-07

    Noncoding RNAs (ncRNAs), particularly microRNAs (miRNAs) and long ncRNAs (lncRNAs), are important players in diseases and emerge as novel drug targets. Thus, unraveling the relationships between ncRNAs and other biomedical entities in cells are critical for better understanding ncRNA roles that may eventually help develop their use in medicine. To support ncRNA research and facilitate retrieval of relevant information regarding miRNAs and lncRNAs from the plethora of published ncRNA-related research, we developed DES-ncRNA ( www.cbrc.kaust.edu.sa/des_ncrna ). DES-ncRNA is a knowledgebase containing text- and data-mined information from public scientific literature and other public resources. Exploration of mined information is enabled through terms and pairs of terms from 19 topic-specific dictionaries including, for example, antibiotics, toxins, drugs, enzymes, mutations, pathways, human genes and proteins, drug indications and side effects, mutations, diseases, etc. DES-ncRNA contains approximately 878,000 associations of terms from these dictionaries of which 36,222 (5,373) are with regards to miRNAs (lncRNAs). We provide several ways to explore information regarding ncRNAs to users including controlled generation of association networks as well as hypotheses generation. We show an example how DES-ncRNA can aid research on Alzheimer\\'s disease and suggest potential therapeutic role for Fasudil. DES-ncRNA is a powerful tool that can be used on its own or as a complement to the existing resources, to support research in human ncRNA. To our knowledge, this is the only knowledgebase dedicated to human miRNAs and lncRNAs derived primarily through literature-mining enabling exploration of a broad spectrum of associated biomedical entities, not paralleled by any other resource.

  17. DES-ncRNA: A knowledgebase for exploring information about human micro and long noncoding RNAs based on literature-mining

    KAUST Repository

    Salhi, Adil; Essack, Magbubah; Alam, Tanvir; Bajic, Vladan P.; Ma, Lina; Radovanovic, Aleksandar; Marchand, Benoit; Schmeier, Sebastian; Zhang, Zhang; Bajic, Vladimir B.

    2017-01-01

    Noncoding RNAs (ncRNAs), particularly microRNAs (miRNAs) and long ncRNAs (lncRNAs), are important players in diseases and emerge as novel drug targets. Thus, unraveling the relationships between ncRNAs and other biomedical entities in cells are critical for better understanding ncRNA roles that may eventually help develop their use in medicine. To support ncRNA research and facilitate retrieval of relevant information regarding miRNAs and lncRNAs from the plethora of published ncRNA-related research, we developed DES-ncRNA ( www.cbrc.kaust.edu.sa/des_ncrna ). DES-ncRNA is a knowledgebase containing text- and data-mined information from public scientific literature and other public resources. Exploration of mined information is enabled through terms and pairs of terms from 19 topic-specific dictionaries including, for example, antibiotics, toxins, drugs, enzymes, mutations, pathways, human genes and proteins, drug indications and side effects, mutations, diseases, etc. DES-ncRNA contains approximately 878,000 associations of terms from these dictionaries of which 36,222 (5,373) are with regards to miRNAs (lncRNAs). We provide several ways to explore information regarding ncRNAs to users including controlled generation of association networks as well as hypotheses generation. We show an example how DES-ncRNA can aid research on Alzheimer's disease and suggest potential therapeutic role for Fasudil. DES-ncRNA is a powerful tool that can be used on its own or as a complement to the existing resources, to support research in human ncRNA. To our knowledge, this is the only knowledgebase dedicated to human miRNAs and lncRNAs derived primarily through literature-mining enabling exploration of a broad spectrum of associated biomedical entities, not paralleled by any other resource.

  18. DES-ncRNA: A knowledgebase for exploring information about human micro and long noncoding RNAs based on literature-mining.

    Science.gov (United States)

    Salhi, Adil; Essack, Magbubah; Alam, Tanvir; Bajic, Vladan P; Ma, Lina; Radovanovic, Aleksandar; Marchand, Benoit; Schmeier, Sebastian; Zhang, Zhang; Bajic, Vladimir B

    2017-07-03

    Noncoding RNAs (ncRNAs), particularly microRNAs (miRNAs) and long ncRNAs (lncRNAs), are important players in diseases and emerge as novel drug targets. Thus, unraveling the relationships between ncRNAs and other biomedical entities in cells are critical for better understanding ncRNA roles that may eventually help develop their use in medicine. To support ncRNA research and facilitate retrieval of relevant information regarding miRNAs and lncRNAs from the plethora of published ncRNA-related research, we developed DES-ncRNA ( www.cbrc.kaust.edu.sa/des_ncrna ). DES-ncRNA is a knowledgebase containing text- and data-mined information from public scientific literature and other public resources. Exploration of mined information is enabled through terms and pairs of terms from 19 topic-specific dictionaries including, for example, antibiotics, toxins, drugs, enzymes, mutations, pathways, human genes and proteins, drug indications and side effects, mutations, diseases, etc. DES-ncRNA contains approximately 878,000 associations of terms from these dictionaries of which 36,222 (5,373) are with regards to miRNAs (lncRNAs). We provide several ways to explore information regarding ncRNAs to users including controlled generation of association networks as well as hypotheses generation. We show an example how DES-ncRNA can aid research on Alzheimer disease and suggest potential therapeutic role for Fasudil. DES-ncRNA is a powerful tool that can be used on its own or as a complement to the existing resources, to support research in human ncRNA. To our knowledge, this is the only knowledgebase dedicated to human miRNAs and lncRNAs derived primarily through literature-mining enabling exploration of a broad spectrum of associated biomedical entities, not paralleled by any other resource.

  19. Muscle specific microRNAs are regulated by endurance exercise in human skeletal muscle

    DEFF Research Database (Denmark)

    Nielsen, Søren; Scheele, Camilla; Yfanti, Christina

    2010-01-01

    Muscle specific miRNAs, myomiRs, have been shown to control muscle development in vitro and are differentially expressed at rest in diabetic skeletal muscle. Therefore, we investigated the expression of these myomiRs, including miR-1, miR-133a, miR-133b and miR-206 in muscle biopsies from vastus...... lateralis of healthy young males (n = 10) in relation to a hyperinsulinaemic–euglycaemic clamp as well as acute endurance exercise before and after 12 weeks of endurance training. The subjects increased their endurance capacity, VO2max (l min-1) by 17.4% (P improved insulin sensitivity by 19......, but their role in regulating human skeletal muscle adaptation remains unknown....

  20. Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions

    Science.gov (United States)

    Kuligina, Elena V.; Bariakin, Dmitry N.; Kozlov, Vadim V.; Richter, Vladimir A.; Semenov, Dmitry V.

    2017-01-01

    Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000g and 160,000g, and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC) patients. It was determined that 16,000g blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000g pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs' fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches. PMID:28127559

  1. Gene-chip studies of adipogenesis-regulated microRNAs in mouse primary adipocytes and human obesity

    Directory of Open Access Journals (Sweden)

    Gallagher Iain J

    2011-03-01

    Full Text Available Abstract Background Adipose tissue abundance relies partly on the factors that regulate adipogenesis, i.e. proliferation and differentiation of adipocytes. While components of the transcriptional program that initiates adipogenesis is well-known, the importance of microRNAs in adipogenesis is less well studied. We thus set out to investigate whether miRNAs would be actively modulated during adipogenesis and obesity. Methods Several models exist to study adipogenesis in vitro, of which the cell line 3T3-L1 is the most well known, albeit not the most physiologically appropriate. Thus, as an alternative, we produced EXIQON microarray of brown and white primary murine adipocytes (prior to and following differentiation to yield global profiles of miRNAs. Results We found 65 miRNAs regulated during in vitro adipogenesis in primary adipocytes. We evaluated the similarity of our responses to those found in non-primary cell models, through literature data-mining. When comparing primary adipocyte profiles, with those of cell lines reported in the literature, we found a high degree of difference in 'adipogenesis' regulated miRNAs suggesting that the model systems may not be accurately representing adipogenesis. The expression of 10 adipogenesis-regulated miRNAs were studied using real-time qPCR and then we selected 5 miRNAs, that showed robust expression, were profiled in subcutaneous adipose tissue obtained from 20 humans with a range of body mass indices (BMI, range = 21-48, and all samples have U133+2 Affymetrix profiles provided. Of the miRNAs tested, mir-21 was robustly expressed in human adipose tissue and positively correlated with BMI (R2 = 0.49, p Conclusion In conclusion, we provide a preliminary analysis of miRNAs associated with primary cell in vitro adipogenesis and demonstrate that the inflammation-associated miRNA, mir-21 is up-regulated in subcutaneous adipose tissue in human obesity. Further, we provide a novel transcriptomics database of

  2. Emerging RNA-based drugs: siRNAs, microRNAs and derivates.

    Science.gov (United States)

    Pereira, Tiago Campos; Lopes-Cendes, Iscia

    2012-09-01

    An emerging new category of therapeutic agents based on ribonucleic acid has emerged and shown very promising in vitro, animal and pre-clinical results, known as small interfering RNAs (siRNAs), microRNAs mimics (miRNA mimics) and their derivates. siRNAs are small RNA molecules that promote potent and specific silencing of mutant, exogenous or aberrant genes through a mechanism known as RNA interference. These agents have called special attention to medicine since they have been used to experimentally treat a series of neurological conditions with distinct etiologies such as prion, viral, bacterial, fungal, genetic disorders and others. siRNAs have also been tested in other scenarios such as: control of anxiety, alcohol consumption, drug-receptor blockage and inhibition of pain signaling. Although in a much earlier stage, miRNAs mimics, anti-miRs and small activating RNAs (saRNAs) also promise novel therapeutic approaches to control gene expression. In this review we intend to introduce clinicians and medical researchers to the most recent advances in the world of siRNA- and miRNA-mediated gene control, its history, applications in cells, animals and humans, delivery methods (an yet unsolved hurdle), current status and possible applications in future clinical practice.

  3. Unsuccessful detection of plant microRNAs in beer, extra virgin olive oil and human plasma after an acute ingestion of extra virgin olive oil

    Science.gov (United States)

    The recent description of the presence of exogenous plant microRNAs from rice in human plasma had profound implications for the interpretation of microRNAs function in human health. If validated, these results suggest that food should not be considered only as a macronutrient and micronutrient suppl...

  4. 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.

  5. MicroRNAs, Innate Immunity and Ventricular Rupture in Human Myocardial Infarction

    Directory of Open Access Journals (Sweden)

    Nina Zidar

    2011-01-01

    Full Text Available MicroRNAs are non-coding RNAs, functionioning as post-transcriptional regulators of gene expression. Some microRNAs have been demonstrated to play a role in regulation of innate immunity. After myocardial infarction (MI, innate immunity is activated leading to an acute inflammatory reaction. There is evidence that an intense inflammatory reaction might contribute to the development of ventricular rupture (VR after MI.

  6. MicroRNAs: A Puzzling Tool in Cancer Diagnostics and Therapy.

    Science.gov (United States)

    D'Angelo, Barbara; Benedetti, Elisabetta; Cimini, Annamaria; Giordano, Antonio

    2016-11-01

    MicroRNAs (miRNAs) constitute a dominating class of small RNAs that regulate diverse cellular functions. Due the pivotal role of miRNAs in biological processes, a deregulated miRNA expression is likely involved in human cancers. MicroRNAs possess tumor suppressor capability, as well as display oncogenic characteristics. Interestingly, miRNAs exist in various biological fluids as circulating entities. Changes in the profile of circulating miRNAs are indicative of pathophysiological conditions in human cancer. This concept has led to consider circulating miRNAs valid biomarkers in cancer diagnostics. Furthermore, current research promotes the use of miRNAs as a target in cancer therapy. However, miRNAs are an evolving research field. Although miRNAs have been demonstrated to be potentially valuable tools both in cancer diagnosis and treatment, a greater effort should be made to improve our understanding of miRNAs biology. This review describes the biology of microRNAs, emphasizing on the use of miRNAs in cancer diagnostics and therapy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  7. Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

    Science.gov (United States)

    Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

    2016-06-01

    Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia

    Science.gov (United States)

    Marcial-Quino, Jaime; Gómez-Manzo, Saúl; Fierro, Francisco; Vanoye-Carlo, America; Rufino-González, Yadira; Sierra-Palacios, Edgar; Castillo-Villanueva, Adriana; Castillo-Rodríguez, Rosa Angélica; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto; Reyes-Vivas, Horacio

    2016-01-01

    Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect—in a rapid, sensitive, specific, and reproducible way—the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia. PMID:27999395

  9. Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia

    Directory of Open Access Journals (Sweden)

    Jaime Marcial-Quino

    2016-12-01

    Full Text Available Stem-loop quantitative reverse transcription PCR (RT-qPCR is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL-based design to detect—in a rapid, sensitive, specific, and reproducible way—the small nucleolar RNA (snoRNA GlsR17 and its derived miRNA (miR2 of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi pathway in G. lamblia.

  10. 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.

  11. Web-based NGS data analysis using miRMaster: a large-scale meta-analysis of human miRNAs.

    Science.gov (United States)

    Fehlmann, Tobias; Backes, Christina; Kahraman, Mustafa; Haas, Jan; Ludwig, Nicole; Posch, Andreas E; Würstle, Maximilian L; Hübenthal, Matthias; Franke, Andre; Meder, Benjamin; Meese, Eckart; Keller, Andreas

    2017-09-06

    The analysis of small RNA NGS data together with the discovery of new small RNAs is among the foremost challenges in life science. For the analysis of raw high-throughput sequencing data we implemented the fast, accurate and comprehensive web-based tool miRMaster. Our toolbox provides a wide range of modules for quantification of miRNAs and other non-coding RNAs, discovering new miRNAs, isomiRs, mutations, exogenous RNAs and motifs. Use-cases comprising hundreds of samples are processed in less than 5 h with an accuracy of 99.4%. An integrative analysis of small RNAs from 1836 data sets (20 billion reads) indicated that context-specific miRNAs (e.g. miRNAs present only in one or few different tissues / cell types) still remain to be discovered while broadly expressed miRNAs appear to be largely known. In total, our analysis of known and novel miRNAs indicated nearly 22 000 candidates of precursors with one or two mature forms. Based on these, we designed a custom microarray comprising 11 872 potential mature miRNAs to assess the quality of our prediction. MiRMaster is a convenient-to-use tool for the comprehensive and fast analysis of miRNA NGS data. In addition, our predicted miRNA candidates provided as custom array will allow researchers to perform in depth validation of candidates interesting to them. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Small RNA profiling reveals important roles for miRNAs in Arabidopsis response to Bacillus velezensis FZB42.

    Science.gov (United States)

    Xie, Shanshan; Jiang, Haiyang; Xu, Zhilan; Xu, Qianqian; Cheng, Beijiu

    2017-09-20

    Bacillus velezensis FZB42 (previously classified as Bacillus amyloliquefaciens FZB42) has been confirmed to successfully colonize plant roots and enhance defense response against pathogen infection. This study indicated that FZB42 inoculation enhanced Arabidopsis defense response against Pseudomonas syringae DC3000 through inducing the expression of PR1, PDF1.2 and stomata closure. To further clarify the induced defense response at miRNA level, sRNA libraries from Arabidopsis roots inoculated with FZB42 and control were constructed and sequenced. The reads of 21nt and 24nt in length were the most abundant groups in FZB42-treated library and control library, respectively. 234 known miRNAs and 16 novel miRNAs were identified. Among them, 11 known miRNAs and 4 novel miRNAs were differentially expressed after FZB42 inoculation. Moreover cis-elements (TC-rich repeats, TCA-element and CGTCA-motif) associated with plant defense were also found in the promoters of these miRNAs. Additionally, 141 mRNAs were predicted as potential targets of these differentially expressed miRNAs. GO annotations of the target genes indicated their potential roles in polyamine biosynthetic process and intracellular protein transport biological process, which may contribute to increased defense response. Our findings indicated that Bacillus velezensis FZB42 inoculation altered the expression of Arabidopsis miRNAs and their target genes, which were associated with defense response. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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    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. MicroRNAs in large herpesvirus DNA genomes: recent advances.

    Science.gov (United States)

    Sorel, Océane; Dewals, Benjamin G

    2016-08-01

    MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that regulate gene expression. They alter mRNA translation through base-pair complementarity, leading to regulation of genes during both physiological and pathological processes. Viruses have evolved mechanisms to take advantage of the host cells to multiply and/or persist over the lifetime of the host. Herpesviridae are a large family of double-stranded DNA viruses that are associated with a number of important diseases, including lymphoproliferative diseases. Herpesviruses establish lifelong latent infections through modulation of the interface between the virus and its host. A number of reports have identified miRNAs in a very large number of human and animal herpesviruses suggesting that these short non-coding transcripts could play essential roles in herpesvirus biology. This review will specifically focus on the recent advances on the functions of herpesvirus miRNAs in infection and pathogenesis.

  15. Annotating Diseases Using Human Phenotype Ontology Improves Prediction of Disease-Associated Long Non-coding RNAs.

    Science.gov (United States)

    Le, Duc-Hau; Dao, Lan T M

    2018-05-23

    Recently, many long non-coding RNAs (lncRNAs) have been identified and their biological function has been characterized; however, our understanding of their underlying molecular mechanisms related to disease is still limited. To overcome the limitation in experimentally identifying disease-lncRNA associations, computational methods have been proposed as a powerful tool to predict such associations. These methods are usually based on the similarities between diseases or lncRNAs since it was reported that similar diseases are associated with functionally similar lncRNAs. Therefore, prediction performance is highly dependent on how well the similarities can be captured. Previous studies have calculated the similarity between two diseases by mapping exactly each disease to a single Disease Ontology (DO) term, and then use a semantic similarity measure to calculate the similarity between them. However, the problem of this approach is that a disease can be described by more than one DO terms. Until now, there is no annotation database of DO terms for diseases except for genes. In contrast, Human Phenotype Ontology (HPO) is designed to fully annotate human disease phenotypes. Therefore, in this study, we constructed disease similarity networks/matrices using HPO instead of DO. Then, we used these networks/matrices as inputs of two representative machine learning-based and network-based ranking algorithms, that is, regularized least square and heterogeneous graph-based inference, respectively. The results showed that the prediction performance of the two algorithms on HPO-based is better than that on DO-based networks/matrices. In addition, our method can predict 11 novel cancer-associated lncRNAs, which are supported by literature evidence. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Unsuccessful Detection of Plant MicroRNAs in Beer, Extra Virgin Olive Oil and Human Plasma After an Acute Ingestion of Extra Virgin Olive Oil.

    Science.gov (United States)

    Micó, Victor; Martín, Roberto; Lasunción, Miguel A; Ordovás, Jose M; Daimiel, Lidia

    2016-03-01

    The recent description of the presence of exogenous plant microRNAs from rice in human plasma had profound implications for the interpretation of microRNAs function in human health. If validated, these results suggest that food should not be considered only as a macronutrient and micronutrient supplier but it could also be a way of genomic interchange between kingdoms. Subsequently, several studies have tried to replicate these results in rice and other plant foods and most of them have failed to find plant microRNAs in human plasma. In this scenario, we aimed to detect plant microRNAs in beer and extra virgin olive oil (EVOO)--two plant-derived liquid products frequently consumed in Spain--as well as in human plasma after an acute ingestion of EVOO. Our hypothesis was that microRNAs present in beer and EVOO raw material could survive manufacturing processes, be part of these liquid products, be absorbed by human gut and circulate in human plasma. To test this hypothesis, we first optimized the microRNA extraction protocol to extract microRNAs from beer and EVOO, and then tried to detect microRNAs in those samples and in plasma samples of healthy volunteers after an acute ingestion of EVOO.

  17. Efficient inhibition of the formation of joint adhesions by ERK2 small interfering RNAs

    International Nuclear Information System (INIS)

    Li, Fengfeng; Ruan, Hongjiang; Fan, Cunyi; Zeng, Bingfang; Wang, Chunyang; Wang, Xiang

    2010-01-01

    Transforming growth factor-β1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which extracellular signal-regulated kinase (ERK)2 is considered to be crucial. Based on these theories, we examined the effects of a lentivirus-mediated small interfering RNA (siRNA) targeting ERK2 on the suppression of joint adhesion formation in vivo. The effects were assessed in vivo from different aspects including the adhesion score, histology and joint contracture angle. We found that the adhesions in the ERK2 siRNA group became soft and weak, and were easily stretched. Accordingly, the flexion contracture angles in the ERK2 siRNA group were also reduced (P < 0.05 compared with the control group). The animals appeared healthy, with no signs of impaired wound healing. In conclusion, local delivery of a lentivirus-mediated siRNA targeting ERK2 can ameliorate joint adhesion formation effectively and safely.

  18. Antifibrotic effects of Smad4 small interfering RNAs in injured skeletal muscle after acute contusion.

    Science.gov (United States)

    Li, H; Chen, J; Chen, S; Zhang, Q; Chen, S

    2011-10-01

    Muscle injuries are common musculoskeletal problems encountered in sports medicine clinics. In this study, we examined the effect of lentivirus-mediated small interfering RNA (siRNA) targeting Smad4 on the suppression of the fibrosis in injured skeletal muscles. We found that Smad4-siRNA could efficiently knock down the expression of Smad4 in the C2C12 myoblast cells and in the contunded mice gastrocnemius muscle. The expression of mRNA level of Smad4 decreased to 11% and 49% compared to the control group, respectively, and the expression of protein level decreased to 13% and 57% respectively. Moreover, the lentivirus-mediated siRNA was stably transfected only into the skeletal muscle and not into the liver of the animals. In contunded mice gastrocnemius, the collagenous and vimentin-positive area in the Smad4 siRNA group reduced to 36% and 37% compared to the control group, respectively. Furthermore, compared to the scrambled Smad4 siRNA-injected mice and PBS control-injected mice, the muscle function of the mice injected with lentivirus-mediated Smad4 siRNA improved in terms of both fast-twitch and tetanic strength (P<0.05). The results suggest that the gene therapy of inhibiting Smad4 by lentivirus-mediated siRNA could be a useful approach to prevent scar tissue formation and improve the function of injured skeletal muscle. © Georg Thieme Verlag KG Stuttgart · New York.

  19. A Computational Approach for Predicting Role of Human MicroRNAs in MERS-CoV Genome

    Directory of Open Access Journals (Sweden)

    Md Mahmudul Hasan

    2014-01-01

    Full Text Available The new epidemic Middle East Respiratory Syndrome (MERS is caused by a type of human coronavirus called MERS-CoV which has global fatality rate of about 30%. We are investigating potential antiviral therapeutics against MERS-CoV by using host microRNAs (miRNAs which may downregulate viral gene expression to quell viral replication. We computationally predicted potential 13 cellular miRNAs from 11 potential hairpin sequences of MERS-CoV genome. Our study provided an interesting hypothesis that those miRNAs, that is, hsa-miR-628-5p, hsa-miR-6804-3p, hsa-miR-4289, hsa-miR-208a-3p, hsa-miR-510-3p, hsa-miR-18a-3p, hsa-miR-329-3p, hsa-miR-548ax, hsa-miR-3934-5p, hsa-miR-4474-5p, hsa-miR-7974, hsa-miR-6865-5p, and hsa-miR-342-3p, would be antiviral therapeutics against MERS-CoV infection.

  20. Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

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    Desy S. Lee

    2015-09-01

    Full Text Available Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs. We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo, to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

  1. MiR-107 and MiR-185 can induce cell cycle arrest in human non small cell lung cancer cell lines.

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    Yukari Takahashi

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are short single stranded noncoding RNAs that suppress gene expression through either translational repression or degradation of target mRNAs. The annealing between messenger RNAs and 5' seed region of miRNAs is believed to be essential for the specific suppression of target gene expression. One miRNA can have several hundred different targets in a cell. Rapidly accumulating evidence suggests that many miRNAs are involved in cell cycle regulation and consequentially play critical roles in carcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Introduction of synthetic miR-107 or miR-185 suppressed growth of the human non-small cell lung cancer cell lines. Flow cytometry analysis revealed these miRNAs induce a G1 cell cycle arrest in H1299 cells and the suppression of cell cycle progression is stronger than that by Let-7 miRNA. By the gene expression analyses with oligonucleotide microarrays, we find hundreds of genes are affected by transfection of these miRNAs. Using miRNA-target prediction analyses and the array data, we listed up a set of likely targets of miR-107 and miR-185 for G1 cell cycle arrest and validate a subset of them using real-time RT-PCR and immunoblotting for CDK6. CONCLUSIONS/SIGNIFICANCE: We identified new cell cycle regulating miRNAs, miR-107 and miR-185, localized in frequently altered chromosomal regions in human lung cancers. Especially for miR-107, a large number of down-regulated genes are annotated with the gene ontology term 'cell cycle'. Our results suggest that these miRNAs may contribute to regulate cell cycle in human malignant tumors.

  2. Human embryos secrete microRNAs into culture media--a potential biomarker for implantation.

    Science.gov (United States)

    Rosenbluth, Evan M; Shelton, Dawne N; Wells, Lindsay M; Sparks, Amy E T; Van Voorhis, Bradley J

    2014-05-01

    To determine whether human blastocysts secrete microRNA (miRNAs) into culture media and whether these reflect embryonic ploidy status and can predict in vitro fertilization (IVF) outcomes. Experimental study of human embryos and IVF culture media. Academic IVF program. 91 donated, cryopreserved embryos that developed into 28 tested blastocysts, from 13 couples who had previously completed IVF cycles. None. Relative miRNA expression in IVF culture media. Blastocysts were assessed by chromosomal comparative genomic hybridization analysis, and the culture media from 55 single-embryo transfer cycles was tested for miRNA expression using an array-based quantitative real-time polymerase chain reaction analysis. The expression of the identified miRNA was correlated with pregnancy outcomes. Ten miRNA were identified in the culture media; two were specific to spent media (miR-191 and miR-372), and one was only present in media before the embryos had been cultured (miR-645). MicroRNA-191 was more highly concentrated in media from aneuploid embryos, and miR-191, miR-372, and miR-645 were more highly concentrated in media from failed IVF/non-intracytoplasmic sperm injection cycles. Additionally, miRNA were found to be more highly concentrated in ICSI and day-5 media samples when compared with regularly inseminated and day-4 samples, respectively. MicroRNA can be detected in IVF culture media. Some of these miRNA are differentially expressed according to the fertilization method, chromosomal status, and pregnancy outcome, which makes them potential biomarkers for predicting IVF success. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. The Role of MicroRNAs in Bovine Infection and Immunity

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    Nathan eLawless

    2014-11-01

    Full Text Available MicroRNAs (miRNAs are a class of small, non-coding RNAs that are recognised as critical regulators of immune gene expression during infection. Many immunologically significant human miRNAs have been found to be conserved in agriculturally important species, including cattle. Discovering how bovine miRNAs mediate the immune defence during infection is critical to understanding the aetiology of the most prevalent bovine diseases. Here, we review current knowledge of miRNAs in the bovine genome, and discuss the advances in understanding of miRNAs as regulators of immune cell function, and bovine immune response activation, regulation, and resolution. Finally, we consider the future perspectives on miRNAs in bovine viral disease, their role as potential biomarkers and in therapy.

  4. 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

  5. Repertoire of noncoding RNAs in corpus luteum of early pregnancy in buffalo (Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    A. Jerome

    2017-09-01

    Full Text Available Aim: The present study was designed to identify other noncoding RNAs (ncRNAs in the corpus luteum (CL during early pregnancy in buffalo. Materials and Methods: For this study, CL (n=2 from two buffalo gravid uteri, obtained from the slaughter house, was transported to laboratory after snap freezing in liquid nitrogen (-196°C. The stage of pregnancy was determined by measuring the crown-rump region of the fetus. This was followed by isolation of RNA and deep sequencing. Post-deep sequencing, the obtained reads were checked and aligned against various ncRNA databases (GtRNA, RFAM, and deep guide. Various parameters, namely, frequency of specific ncRNAs, length, mismatch, and genomic location target in several model species were deciphered. Results: Frequency of piwi-interacting RNAs (piwi-RNAs, having target location in rodents and human genomes, were significantly higher compared to other piwi-RNAs and ncRNAs. Ribosomal RNAs (rRNAs deduced had nucleotides (nts ranging from 17 to 50 nts, but the occurrence of small length rRNAs was more than lengthier fragments. The target on 16S rRNA species confirms the conservation of 16S rRNA across species. With respect to transfer RNA (tRNA, the abundantly occurring tRNAs were unique with no duplication. Small nucleolar RNAs (snoRNAs, identified in this study, showed a strong tendency for coding box C/D snoRNAs in comparison to H/ACA snoRNAs. Regulatory and evolutionary implications of these identified ncRNAs are yet to be delineated in many species, including buffaloes. Conclusion: This is the first report of identification of other ncRNAs in CL of early pregnancy in buffalo.

  6. MicroRNAs Change the Landscape of Cancer Resistance.

    Science.gov (United States)

    Zhu, Jun; Zhu, Wei; Wu, Wei

    2018-01-01

    One of the major challenges in the cancer treatment is the development of drug resistance. It represents a major obstacle to curing cancer with constrained efficacy of both conventional chemotherapy and targeted therapies, even recent immune checkpoint blockade therapy. Deciphering the mechanisms of resistance is critical to further understanding the multifactorial pathways involved, and developing more specific targeted treatments. To date, numerous studies have reported the potential role of microRNAs (miRNAs) in the resistance to various cancer treatments. MicroRNAs are a family of small noncoding RNAs that regulate gene expression by sequence-specific targeting of mRNAs causing translational repression or mRNA degradation. More than 1200 validated human miRNAs have been identified in human genome. While one miRNA can regulate hundreds of targets, a single target can also be affected by multiple miRNAs. Evidence suggests that dysregulation of specific miRNAs may be involved in the acquisition of resistance, thereby modulating the sensitivity of cancer cells to treatment. Therefore, manipulation of miRNAs may be an attractive strategy for more effective individualized therapies through reprograming resistant network in cancer cells.

  7. Profiling of circulating microRNAs after a bout of acute resistance exercise in humans.

    Directory of Open Access Journals (Sweden)

    Shuji Sawada

    Full Text Available Recent studies have revealed a new aspect of physiological regulation in which microRNAs (miRNAs play fundamental roles in diverse biological and pathological processes. Furthermore, it was recently discovered that miRNAs are stably secreted into blood and that circulating miRNAs may play important roles in cell-cell communication. Here, we examined whether the circulating miRNA profile is affected by acute resistance exercise. Twelve males performed a resistance exercise session (bench press and leg press, consisting of five sets of 10 repetitions at 70% of maximum strength, with a 1 min rest between sets. Blood samples were taken before exercise, and at 0 and 60 min, 1 day, and 3 days after exercise. The circulating miRNA profile was determined by microarray analysis. Quantitative real-time PCR confirmed that the miR-149* level increased three days after resistance exercise. In contrast, the miR-146a and miR-221 levels decreased three days after resistance exercise. Our findings suggest that circulating miRNA levels change in response to acute resistance exercise, and miRNAs may play important roles in resistance-exercise-induced adaptation.

  8. The role of microRNAs in stemness of cancer stem cells

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    Seyed Mohammad Ali Hosseini Rad

    2013-12-01

    Full Text Available Cancer is one of the most important diseases of humans, for which no cure has been found so far. Understanding the causes of cancer can pave the way for its treatment. Alteration in genetic elements such as oncogenes and tumor suppressor genes results in cancer. The most recent theory for the origin of cancer has been provided by cancer stem cells (CSCs. Tumor-initiating cells (T-ICs or CSCs are a small population isolated from tumors and hematologic malignancies. Since CSCs are similar to embryonic stem cells (ESCs in many aspects (such as pluripotency and self-renewal, recognizing the signaling pathways through which ESCs maintain their stemness can also help identify CSC signaling. One component of these signaling pathways is non-coding RNAs (ncRNAs. ncRNAs are classified in two groups: microRNAs (miRNAs and long non-coding RNAs (lncRNAs. miRNAs undergo altered expression in cancer. In this regard, they are classified as Onco-miRNAs or tumor suppressor miRNAs. Some miRNAs play similar roles in ESCs and CSCs, such as let-7 and miR-302. This review focuses on the miRNAs involved in stemness of ESCs and CSCs by presenting a summary of the role of miRNAs in other tumor cells.

  9. Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular.

    Science.gov (United States)

    Izuogu, Osagie G; Alhasan, Abd A; Mellough, Carla; Collin, Joseph; Gallon, Richard; Hyslop, Jonathon; Mastrorosa, Francesco K; Ehrmann, Ingrid; Lako, Majlinda; Elliott, David J; Santibanez-Koref, Mauro; Jackson, Michael S

    2018-04-20

    Circular RNAs (circRNAs) are predominantly derived from protein coding genes, and some can act as microRNA sponges or transcriptional regulators. Changes in circRNA levels have been identified during human development which may be functionally important, but lineage-specific analyses are currently lacking. To address this, we performed RNAseq analysis of human embryonic stem (ES) cells differentiated for 90 days towards 3D laminated retina. A transcriptome-wide increase in circRNA expression, size, and exon count was observed, with circRNA levels reaching a plateau by day 45. Parallel statistical analyses, controlling for sample and locus specific effects, identified 239 circRNAs with expression changes distinct from the transcriptome-wide pattern, but these all also increased in abundance over time. Surprisingly, circRNAs derived from long non-coding RNAs (lncRNAs) were found to account for a significantly larger proportion of transcripts from their loci of origin than circRNAs from coding genes. The most abundant, circRMST:E12-E6, showed a > 100X increase during differentiation accompanied by an isoform switch, and accounts for > 99% of RMST transcripts in many adult tissues. The second most abundant, circFIRRE:E10-E5, accounts for > 98% of FIRRE transcripts in differentiating human ES cells, and is one of 39 FIRRE circRNAs, many of which include multiple unannotated exons. Our results suggest that during human ES cell differentiation, changes in circRNA levels are primarily globally controlled. They also suggest that RMST and FIRRE, genes with established roles in neurogenesis and topological organisation of chromosomal domains respectively, are processed as circular lncRNAs with only minor linear species.

  10. Hallmarks of Human Small Antral Follicle Development

    DEFF Research Database (Denmark)

    Kristensen, Stine G; Mamsen, Linn S; Jeppesen, Janni V

    2018-01-01

    Regulation of human ovarian steroidogenesis differs from other species and precise knowledge on how human small antral follicles (hSAF) develop and acquire competence for continued growth and steroid output is still incomplete. The present study has characterized almost 1,000 normal hSAF collected...... increased steroid output profoundly. Furthermore, the highly significant association between FSHR and AR mRNA gene expression enforces important functions of androgens in follicular development. Collectively, these data reintroduce the understanding of the follicular phase as two parted in which regulation...

  11. Downregulation of 14q32 microRNAs in primary human desmoplastic medulloblastoma.

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    Danielle Ribeiro Lucon

    2013-09-01

    Full Text Available Medulloblastoma (MB is one of the most common pediatric cancers, likely originating from abnormal development of cerebellar progenitor neurons. MicroRNA (miRNA has been shown to play an important role in the development of the central nervous system. Microarray analysis was used to investigate miRNA expression in desmoplastic MB from patients diagnosed at a young age (1 or 2 years old. Normal fetal or newborn cerebellum was used as control. A total of 84 differentially expressed miRNAs (64 downregulated and 20 upregulated were found. Most downregulated miRNAs (32/64 were found to belong to the cluster of miRNAs at the 14q32 locus, suggesting that this miRNA locus is regulated as a module in MB. Possible mechanisms of 14q32 miRNAs downregulation were investigated by the analysis of publicly available gene expression data sets. First, expression of estrogen-related receptor γ (ESRRG, a reported positive transcriptional regulator of some 14q32 miRNAs, was found downregulated in desmoplastic MB. Second, expression of the parentally imprinted gene MEG3 was lower in MB in comparison to normal cerebellum, suggesting a possible epigenetic silencing of the 14q32 locus. miR-129-5p (11p11.2/7q32.1, miR-206 (6p12.2, and miR-323-3p (14q32.2, were chosen for functional studies in DAOY cells. Overexpression of miR-129-5p using mimics decreased DAOY proliferation. No effect was found with miR-206 or miR-323 mimics.

  12. Small-bodied humans from Palau, Micronesia.

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    Lee R Berger

    Full Text Available UNLABELLED: Newly discovered fossil assemblages of small bodied Homo sapiens from Palau, Micronesia possess characters thought to be taxonomically primitive for the genus Homo. BACKGROUND: Recent surface collection and test excavation in limestone caves in the rock islands of Palau, Micronesia, has produced a sizeable sample of human skeletal remains dating roughly between 940-2890 cal ybp. PRINCIPLE FINDINGS: Preliminary analysis indicates that this material is important for two reasons. First, individuals from the older time horizons are small in body size even relative to "pygmoid" populations from Southeast Asia and Indonesia, and thus may represent a marked case of human insular dwarfism. Second, while possessing a number of derived features that align them with Homo sapiens, the human remains from Palau also exhibit several skeletal traits that are considered to be primitive for the genus Homo. SIGNIFICANCE: These features may be previously unrecognized developmental correlates of small body size and, if so, they may have important implications for interpreting the taxonomic affinities of fossil specimens of Homo.

  13. Ambient temperature regulates the expression of a small set of sRNAs influencing plant development through NF-YA2 and YUC2.

    Science.gov (United States)

    Gyula, Péter; Baksa, Ivett; Tóth, Tamás; Mohorianu, Irina; Dalmay, Tamás; Szittya, György

    2018-06-01

    Plants substantially alter their developmental program upon changes in the ambient temperature. The 21-24 nt small RNAs (sRNAs) are important gene expression regulators, which play a major role in development and adaptation. However, little is known about how the different sRNA classes respond to changes in the ambient temperature. We profiled the sRNA populations in four different tissues of Arabidopsis thaliana plants grown at 15, 21 and 27 °C. We found that only a small fraction (0.6%) of the sRNA loci are ambient temperature-controlled. We identified thermoresponsive miRNAs and identified their target genes using degradome libraries. We verified that the target of the thermoregulated miR169, NF-YA2, is also ambient temperature-regulated. NF-YA2, as the component of the conserved transcriptional regulator NF-Y complex, binds the promoter of the flowering time regulator FT and the auxin biosynthesis gene YUC2. Other differentially expressed loci include thermoresponsive phased siRNA loci that target various auxin pathway genes and tRNA fragments. Furthermore, a temperature dependent 24-nt heterochromatic siRNA locus in the promoter of YUC2 may contribute to the epigenetic regulation of auxin homeostasis. This holistic approach facilitated a better understanding of the role of different sRNA classes in ambient temperature adaptation of plants. This article is protected by copyright. All rights reserved.

  14. Digital Genome-Wide ncRNA Expression, Including SnoRNAs, across 11 Human Tissues Using PolyA-Neutral Amplification

    Science.gov (United States)

    Castle, John C.; Armour, Christopher D.; Löwer, Martin; Haynor, David; Biery, Matthew; Bouzek, Heather; Chen, Ronghua; Jackson, Stuart; Johnson, Jason M.; Rohl, Carol A.; Raymond, Christopher K.

    2010-01-01

    Non-coding RNAs (ncRNAs) are an essential class of molecular species that have been difficult to monitor on high throughput platforms due to frequent lack of polyadenylation. Using a polyadenylation-neutral amplification protocol and next-generation sequencing, we explore ncRNA expression in eleven human tissues. ncRNAs 7SL, U2, 7SK, and HBII-52 are expressed at levels far exceeding mRNAs. C/D and H/ACA box snoRNAs are associated with rRNA methylation and pseudouridylation, respectively: spleen expresses both, hypothalamus expresses mainly C/D box snoRNAs, and testes show enriched expression of both H/ACA box snoRNAs and RNA telomerase TERC. Within the snoRNA 14q cluster, 14q(I-6) is expressed at much higher levels than other cluster members. More reads align to mitochondrial than nuclear tRNAs. Many lincRNAs are actively transcribed, particularly those overlapping known ncRNAs. Within the Prader-Willi syndrome loci, the snoRNA HBII-85 (group I) cluster is highly expressed in hypothalamus, greater than in other tissues and greater than group II or III. Additionally, within the disease locus we find novel transcription across a 400,000 nt span in ovaries. This genome-wide polyA-neutral expression compendium demonstrates the richness of ncRNA expression, their high expression patterns, their function-specific expression patterns, and is publicly available. PMID:20668672

  15. Digital genome-wide ncRNA expression, including SnoRNAs, across 11 human tissues using polyA-neutral amplification.

    Directory of Open Access Journals (Sweden)

    John C Castle

    Full Text Available Non-coding RNAs (ncRNAs are an essential class of molecular species that have been difficult to monitor on high throughput platforms due to frequent lack of polyadenylation. Using a polyadenylation-neutral amplification protocol and next-generation sequencing, we explore ncRNA expression in eleven human tissues. ncRNAs 7SL, U2, 7SK, and HBII-52 are expressed at levels far exceeding mRNAs. C/D and H/ACA box snoRNAs are associated with rRNA methylation and pseudouridylation, respectively: spleen expresses both, hypothalamus expresses mainly C/D box snoRNAs, and testes show enriched expression of both H/ACA box snoRNAs and RNA telomerase TERC. Within the snoRNA 14q cluster, 14q(I-6 is expressed at much higher levels than other cluster members. More reads align to mitochondrial than nuclear tRNAs. Many lincRNAs are actively transcribed, particularly those overlapping known ncRNAs. Within the Prader-Willi syndrome loci, the snoRNA HBII-85 (group I cluster is highly expressed in hypothalamus, greater than in other tissues and greater than group II or III. Additionally, within the disease locus we find novel transcription across a 400,000 nt span in ovaries. This genome-wide polyA-neutral expression compendium demonstrates the richness of ncRNA expression, their high expression patterns, their function-specific expression patterns, and is publicly available.

  16. A deletion of the HBII-85 class of small nucleolar RNAs (snoRNAs) is associated with hyperphagia, obesity and hypogonadism

    NARCIS (Netherlands)

    de Smith, Adam J.; Purmann, Carolin; Walters, Robin G.; Ellis, Richard J.; Holder, Susan E.; Van Haelst, Mieke M.; Brady, Angela F.; Fairbrother, Una L.; Dattani, Mehul; Keogh, Julia M.; Henning, Elana; Yeo, Giles S. H.; O'Rahilly, Stephen; Froguel, Philippe; Farooqi, I. Sadaf; Blakemore, Alexandra I. F.

    2009-01-01

    Genetic studies in patients with severe early-onset obesity have provided insights into the molecular and physiological pathways that regulate body weight in humans. We report a 19-year-old male with hyperphagia and severe obesity, mild learning difficulties and hypogonadism, in whom diagnostic

  17. Network of microRNAs-mRNAs Interactions in Pancreatic Cancer

    Science.gov (United States)

    Naderi, Elnaz; Mostafaei, Mehdi; Pourshams, Akram

    2014-01-01

    Background. MicroRNAs are small RNA molecules that regulate the expression of certain genes through interaction with mRNA targets and are mainly involved in human cancer. This study was conducted to make the network of miRNAs-mRNAs interactions in pancreatic cancer as the fourth leading cause of cancer death. Methods. 56 miRNAs that were exclusively expressed and 1176 genes that were downregulated or silenced in pancreas cancer were extracted from beforehand investigations. MiRNA–mRNA interactions data analysis and related networks were explored using MAGIA tool and Cytoscape 3 software. Functional annotations of candidate genes in pancreatic cancer were identified by DAVID annotation tool. Results. This network is made of 217 nodes for mRNA, 15 nodes for miRNA, and 241 edges that show 241 regulations between 15 miRNAs and 217 target genes. The miR-24 was the most significantly powerful miRNA that regulated series of important genes. ACVR2B, GFRA1, and MTHFR were significant target genes were that downregulated. Conclusion. Although the collected previous data seems to be a treasure trove, there was no study simultaneous to analysis of miRNAs and mRNAs interaction. Network of miRNA-mRNA interactions will help to corroborate experimental remarks and could be used to refine miRNA target predictions for developing new therapeutic approaches. PMID:24895587

  18. Network of microRNAs-mRNAs Interactions in Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Elnaz Naderi

    2014-01-01

    Full Text Available Background. MicroRNAs are small RNA molecules that regulate the expression of certain genes through interaction with mRNA targets and are mainly involved in human cancer. This study was conducted to make the network of miRNAs-mRNAs interactions in pancreatic cancer as the fourth leading cause of cancer death. Methods. 56 miRNAs that were exclusively expressed and 1176 genes that were downregulated or silenced in pancreas cancer were extracted from beforehand investigations. MiRNA–mRNA interactions data analysis and related networks were explored using MAGIA tool and Cytoscape 3 software. Functional annotations of candidate genes in pancreatic cancer were identified by DAVID annotation tool. Results. This network is made of 217 nodes for mRNA, 15 nodes for miRNA, and 241 edges that show 241 regulations between 15 miRNAs and 217 target genes. The miR-24 was the most significantly powerful miRNA that regulated series of important genes. ACVR2B, GFRA1, and MTHFR were significant target genes were that downregulated. Conclusion. Although the collected previous data seems to be a treasure trove, there was no study simultaneous to analysis of miRNAs and mRNAs interaction. Network of miRNA-mRNA interactions will help to corroborate experimental remarks and could be used to refine miRNA target predictions for developing new therapeutic approaches.

  19. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism

    NARCIS (Netherlands)

    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

  20. MicroRNAs in Coronary Heart Disease: Ready to Enter the Clinical Arena?

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    Elena Cavarretta

    2016-01-01

    Full Text Available Coronary artery disease (CAD and its complication remain the leading cause of mortality in industrialized countries despite great advances in terms of diagnosis, prognosis, and treatment options. MicroRNAs (miRNAs, small noncoding RNAs, act as posttranscriptional gene expression modulators and have been implicated as key regulators in several physiological and pathological processes linked to CAD. Circulating miRNAs have been evaluated as promising novel biomarkers of CAD, acute coronary syndromes, and acute myocardial infarction, with prognostic implications. Several challenges related to technical aspects, miRNAs normalization, drugs interaction, and quality reporting of statistical multivariable analysis of the miRNAs observational studies remain unresolved. MicroRNA-based therapies in cardiovascular diseases are not ready yet for human trials but definitely appealing. Through this review we will provide clinicians with a concise overview of the pros and cons of microRNAs.

  1. Implications of microRNAs in Colorectal Cancer Development, Diagnosis, Prognosis and Therapeutics

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    Haiyan eZhai

    2011-11-01

    Full Text Available MicroRNAs (miRNAs are a class of non-coding small RNAs with critical regulatory functions as post-transcriptional regulators. Due to the fundamental importance and broad impact of miRNAs on multiple genes and pathways, dysregulated miRNAs have been associated with human diseases, including cancer. Colorectal cancer (CRC is among the most deadly diseases, and miRNAs offer a new frontier for target discovery and novel biomarkers for both diagnosis and prognosis. In this review, we summarize the recent advancement of miRNA research in CRC, in particular, the roles of miRNAs in colorectal cancer stem cells, EMT, chemoresistance, therapeutics, diagnosis and prognosis.

  2. Retrotransposons and non-protein coding RNAs

    DEFF Research Database (Denmark)

    Mourier, Tobias; Willerslev, Eske

    2009-01-01

    does not merely represent spurious transcription. We review examples of functional RNAs transcribed from retrotransposons, and address the collection of non-protein coding RNAs derived from transposable element sequences, including numerous human microRNAs and the neuronal BC RNAs. Finally, we review...

  3. Lnc2Meth: a manually curated database of regulatory relationships between long non-coding RNAs and DNA methylation associated with human disease.

    Science.gov (United States)

    Zhi, Hui; Li, Xin; Wang, Peng; Gao, Yue; Gao, Baoqing; Zhou, Dianshuang; Zhang, Yan; Guo, Maoni; Yue, Ming; Shen, Weitao; Ning, Shangwei; Jin, Lianhong; Li, Xia

    2018-01-04

    Lnc2Meth (http://www.bio-bigdata.com/Lnc2Meth/), an interactive resource to identify regulatory relationships between human long non-coding RNAs (lncRNAs) and DNA methylation, is not only a manually curated collection and annotation of experimentally supported lncRNAs-DNA methylation associations but also a platform that effectively integrates tools for calculating and identifying the differentially methylated lncRNAs and protein-coding genes (PCGs) in diverse human diseases. The resource provides: (i) advanced search possibilities, e.g. retrieval of the database by searching the lncRNA symbol of interest, DNA methylation patterns, regulatory mechanisms and disease types; (ii) abundant computationally calculated DNA methylation array profiles for the lncRNAs and PCGs; (iii) the prognostic values for each hit transcript calculated from the patients clinical data; (iv) a genome browser to display the DNA methylation landscape of the lncRNA transcripts for a specific type of disease; (v) tools to re-annotate probes to lncRNA loci and identify the differential methylation patterns for lncRNAs and PCGs with user-supplied external datasets; (vi) an R package (LncDM) to complete the differentially methylated lncRNAs identification and visualization with local computers. Lnc2Meth provides a timely and valuable resource that can be applied to significantly expand our understanding of the regulatory relationships between lncRNAs and DNA methylation in various human diseases. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. RsmV a small non-coding regulatory RNA in Pseudomonas aeruginosa that sequesters RsmA and RsmF from target mRNAs.

    Science.gov (United States)

    Janssen, Kayley H; Diaz, Manisha R; Gode, Cindy J; Wolfgang, Matthew C; Yahr, Timothy L

    2018-06-04

    The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression. Acute virulence is associated with twitching and swimming motility, expression of a type III secretion system (T3SS), and the absence of alginate, Psl, or Pel polysaccharide production. Traits associated with chronic infection include growth as a biofilm, reduced motility, and expression of a type VI secretion system (T6SS). The Rsm post-transcriptional regulatory system plays important roles in the inverse control of phenotypes associated with acute and chronic virulence. RsmA and RsmF are RNA-binding proteins that interact with target mRNAs to control gene expression at the post-transcriptional level. Previous work found that RsmA activity is controlled by at least three small, non-coding regulatory RNAs (RsmW, RsmY, and RsmZ). In this study, we took an in-silico approach to identify additional sRNAs that might function in the sequestration of RsmA and/or RsmF and identified RsmV, a 192 nt transcript with four predicted RsmA/RsmF consensus binding sites. RsmV is capable of sequestering RsmA and RsmF in vivo to activate translation of tssA1 , a component of the T6SS, and to inhibit T3SS gene expression. Each of the predicted RsmA/RsmF consensus binding sites contribute to RsmV activity. Electrophoretic mobility shifts assays show that RsmF binds RsmV with >10-fold higher affinity than RsmY and RsmZ. Gene expression studies revealed that the temporal expression pattern of RsmV differs from RsmW, RsmY, and RsmZ. These findings suggest that each sRNA may play distinct roles in controlling RsmA and RsmF activity. IMPORTANCE The CsrA/RsmA family of RNA-binding proteins play important roles in post-transcriptional control of gene expression. The activity of CsrA/RsmA proteins is controlled by small non-coding RNAs that function as decoys to sequester CsrA/RsmA from target mRNAs. Pseudomonas aeruginosa has two Csr

  5. Human Reliability Analysis for Small Modular Reactors

    Energy Technology Data Exchange (ETDEWEB)

    Ronald L. Boring; David I. Gertman

    2012-06-01

    Because no human reliability analysis (HRA) method was specifically developed for small modular reactors (SMRs), the application of any current HRA method to SMRs represents tradeoffs. A first- generation HRA method like THERP provides clearly defined activity types, but these activity types do not map to the human-system interface or concept of operations confronting SMR operators. A second- generation HRA method like ATHEANA is flexible enough to be used for SMR applications, but there is currently insufficient guidance for the analyst, requiring considerably more first-of-a-kind analyses and extensive SMR expertise in order to complete a quality HRA. Although no current HRA method is optimized to SMRs, it is possible to use existing HRA methods to identify errors, incorporate them as human failure events in the probabilistic risk assessment (PRA), and quantify them. In this paper, we provided preliminary guidance to assist the human reliability analyst and reviewer in understanding how to apply current HRA methods to the domain of SMRs. While it is possible to perform a satisfactory HRA using existing HRA methods, ultimately it is desirable to formally incorporate SMR considerations into the methods. This may require the development of new HRA methods. More practicably, existing methods need to be adapted to incorporate SMRs. Such adaptations may take the form of guidance on the complex mapping between conventional light water reactors and small modular reactors. While many behaviors and activities are shared between current plants and SMRs, the methods must adapt if they are to perform a valid and accurate analysis of plant personnel performance in SMRs.

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

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    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

  7. MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells.

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    Hisataka Ogawa

    Full Text Available Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

  8. Comparing artificial neural networks, general linear models and support vector machines in building predictive models for small interfering RNAs.

    Directory of Open Access Journals (Sweden)

    Kyle A McQuisten

    2009-10-01

    Full Text Available Exogenous short interfering RNAs (siRNAs induce a gene knockdown effect in cells by interacting with naturally occurring RNA processing machinery. However not all siRNAs induce this effect equally. Several heterogeneous kinds of machine learning techniques and feature sets have been applied to modeling siRNAs and their abilities to induce knockdown. There is some growing agreement to which techniques produce maximally predictive models and yet there is little consensus for methods to compare among predictive models. Also, there are few comparative studies that address what the effect of choosing learning technique, feature set or cross validation approach has on finding and discriminating among predictive models.Three learning techniques were used to develop predictive models for effective siRNA sequences including Artificial Neural Networks (ANNs, General Linear Models (GLMs and Support Vector Machines (SVMs. Five feature mapping methods were also used to generate models of siRNA activities. The 2 factors of learning technique and feature mapping were evaluated by complete 3x5 factorial ANOVA. Overall, both learning techniques and feature mapping contributed significantly to the observed variance in predictive models, but to differing degrees for precision and accuracy as well as across different kinds and levels of model cross-validation.The methods presented here provide a robust statistical framework to compare among models developed under distinct learning techniques and feature sets for siRNAs. Further comparisons among current or future modeling approaches should apply these or other suitable statistically equivalent methods to critically evaluate the performance of proposed models. ANN and GLM techniques tend to be more sensitive to the inclusion of noisy features, but the SVM technique is more robust under large numbers of features for measures of model precision and accuracy. Features found to result in maximally predictive models are

  9. The Binding Sites of miR-619-5p in the mRNAs of Human and Orthologous Genes.

    Science.gov (United States)

    Atambayeva, Shara; Niyazova, Raigul; Ivashchenko, Anatoliy; Pyrkova, Anna; Pinsky, Ilya; Akimniyazova, Aigul; Labeit, Siegfried

    2017-06-01

    Normally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs. This significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications. The search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. For the binding sites of the miR-619-5p the hybridization free energy of the bonds was equal to 100% of the maximum potential free energy. The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3'UTR (214 sites), CDS (3 sites), and 5'UTR (4 sites). The mRNAs of CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites, and the mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these miR-619-5p binding sites are located in the 3'UTRs. The miR-619-5p binding site in the 5'UTR of mRNA of human USP29 gene is found in the mRNAs of orthologous genes of primates. Binding sites of miR-619-5p in the coding regions of mRNAs of C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is present in the orthologous proteins. Binding sites of miR-619-5p in the mRNAs of transcription factor genes ZNF429 and ZNF429 encode the AHACNP oligopeptide in another reading frame. Binding sites of miR-619-5p in the 3'UTRs of all human target genes are also present in the 3'UTRs of orthologous genes of mammals. The completely complementary binding sites for miR-619-5p are conservative in the orthologous mammalian genes. The majority of miR-619-5p binding sites are located in the 3'UTRs but some genes have miRNA binding sites in the 5'UTRs of mRNAs. Several genes have binding sites for miRNAs in the CDSs that are read in different open reading frames. Identical nucleotide sequences of binding sites encode different amino acids in different proteins. The binding sites of miR-619-5p

  10. Circular RNAs and systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lian-Ju; Huang, Qing; Pan, Hai-Feng; Ye, Dong-Qing, E-mail: ydqahmu@gmail.com

    2016-08-15

    Circular RNAs (circRNAs) are a large class of noncoding RNAs that form covalently closed RNA circles. The discovery of circRNAs discloses a new layer of gene regulation occurred post-transcriptionally. Identification of endogenous circRNAs benefits from the advance in high-throughput RNA sequencing and remains challenging. Many studies probing into the mechanisms of circRNAs formation occurred cotranscriptionally or posttranscriptionally emerge and conclude that canonical splicing mechanism, sequence properties, and certain regulatory factors are at play in the process. Although our knowledge on functions of circRNAs is rather limited, a few circRNAs are shown to sponge miRNA and regulate gene transcription. The clearest case is one circRNA CDR1as that serves as sponge of miR-7. Researches on circRNAs in human diseases such as cancers highlight the function and physical relevance of circRNAs. Given the implication of miRNAs in the initiation and progression of systemic lupus erythematosus (SLE) and the roles of circRNAs in sponging miRNA and gene regulation, it is appealing to speculate that circRNAs may associate with SLE and may be potential therapeutic targets for treatment of SLE. Future studies should attach more importance to the relationship between circRNAs and SLE. This review will concern identification, biogenesis, and function of circRNAs, introduce reports exploring the association of circRNAs with human diseases, and conjecture the potential roles of circRNAs in SLE. - Highlights: • Studies have discovered thousands of circRNAs and interpreted their biogenesis. • Cytoplasmic circRNAs sponge miRNA and nuclear circRNAs modulate gene transcription. • Aberrant expression of circRNAs has been observed in various cancers. • CircRNAs may partake in the pathogenesis of systemic lupus erythematosus.

  11. Circular RNAs and systemic lupus erythematosus

    International Nuclear Information System (INIS)

    Li, Lian-Ju; Huang, Qing; Pan, Hai-Feng; Ye, Dong-Qing

    2016-01-01

    Circular RNAs (circRNAs) are a large class of noncoding RNAs that form covalently closed RNA circles. The discovery of circRNAs discloses a new layer of gene regulation occurred post-transcriptionally. Identification of endogenous circRNAs benefits from the advance in high-throughput RNA sequencing and remains challenging. Many studies probing into the mechanisms of circRNAs formation occurred cotranscriptionally or posttranscriptionally emerge and conclude that canonical splicing mechanism, sequence properties, and certain regulatory factors are at play in the process. Although our knowledge on functions of circRNAs is rather limited, a few circRNAs are shown to sponge miRNA and regulate gene transcription. The clearest case is one circRNA CDR1as that serves as sponge of miR-7. Researches on circRNAs in human diseases such as cancers highlight the function and physical relevance of circRNAs. Given the implication of miRNAs in the initiation and progression of systemic lupus erythematosus (SLE) and the roles of circRNAs in sponging miRNA and gene regulation, it is appealing to speculate that circRNAs may associate with SLE and may be potential therapeutic targets for treatment of SLE. Future studies should attach more importance to the relationship between circRNAs and SLE. This review will concern identification, biogenesis, and function of circRNAs, introduce reports exploring the association of circRNAs with human diseases, and conjecture the potential roles of circRNAs in SLE. - Highlights: • Studies have discovered thousands of circRNAs and interpreted their biogenesis. • Cytoplasmic circRNAs sponge miRNA and nuclear circRNAs modulate gene transcription. • Aberrant expression of circRNAs has been observed in various cancers. • CircRNAs may partake in the pathogenesis of systemic lupus erythematosus.

  12. microRNAs and the mammary gland: a new understanding of gene expression

    Directory of Open Access Journals (Sweden)

    Isabel Gigli

    2013-01-01

    Full Text Available MicroRNAs (miRNAs have been identified in cells as well as in exosomes in biological fluids such as milk. In mammary gland, most of the miRNAs studied have functions related to immunity and show alterations in their pattern of expression during lactation. In mastitis, the inflammatory response caused by Streptococcus uberis alters the expression of miRNAs that may regulate the innate immune system. These small RNAs are stable at room temperature and are resistant to repeated freeze/thaw cycles, acidic conditions and degradation by RNAse, making them resistant to industrial procedures. These properties mean that miRNAs could have multiple applications in veterinary medicine and biotechnology. Indeed, lactoglobulin-free milk has been produced in transgenic cows expressing specific miRNAs. Although plant and animal miRNAs have undergone independent evolutionary adaptation recent studies have demonstrated a cross-kingdom passage in which rice miRNA was isolated from human serum. This finding raises questions about the possible effect that miRNAs present in foods consumed by humans could have on human gene regulation. Further studies are needed before applying miRNA biotechnology to the milk industry. New discoveries and a greater knowledge of gene expression will lead to a better understanding of the role of miRNAs in physiology, nutrition and evolution.

  13. Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants

    KAUST Repository

    Khraiwesh, Basel

    2012-02-01

    Small, non-coding RNAs are a distinct class of regulatory RNAs in plants and animals that control a variety of biological processes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved through a series of pathways. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs control the expression of cognate target genes by binding to reverse complementary sequences, resulting in cleavage or translational inhibition of the target RNAs. siRNAs have a similar structure, function, and biogenesis as miRNAs but are derived from long double-stranded RNAs and can often direct DNA methylation at target sequences. Besides their roles in growth and development and maintenance of genome integrity, small RNAs are also important components in plant stress responses. One way in which plants respond to environmental stress is by modifying their gene expression through the activity of small RNAs. Thus, understanding how small RNAs regulate gene expression will enable researchers to explore the role of small RNAs in biotic and abiotic stress responses. This review focuses on the regulatory roles of plant small RNAs in the adaptive response to stresses. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress. © 2011 Elsevier B.V.

  14. Cloning, characterization and expression analysis of porcine microRNAs

    Directory of Open Access Journals (Sweden)

    Desilva Udaya

    2009-02-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are small ~22-nt regulatory RNAs that can silence target genes, by blocking their protein production or degrading the mRNAs. Pig is an important animal in the agriculture industry because of its utility in the meat production. Besides, pig has tremendous biomedical importance as a model organism because of its closer proximity to humans than the mouse model. Several hundreds of miRNAs have been identified from mammals, humans, mice and rats, but little is known about the miRNA component in the pig genome. Here, we adopted an experimental approach to identify conserved and unique miRNAs and characterize their expression patterns in diverse tissues of pig. Results By sequencing a small RNA library generated using pooled RNA from the pig heart, liver and thymus; we identified a total of 120 conserved miRNA homologs in pig. Expression analysis of conserved miRNAs in 14 different tissue types revealed heart-specific expression of miR-499 and miR-208 and liver-specific expression of miR-122. Additionally, miR-1 and miR-133 in the heart, miR-181a and miR-142-3p in the thymus, miR-194 in the liver, and miR-143 in the stomach showed the highest levels of expression. miR-22, miR-26b, miR-29c and miR-30c showed ubiquitous expression in diverse tissues. The expression patterns of pig-specific miRNAs also varied among the tissues examined. Conclusion Identification of 120 miRNAs and determination of the spatial expression patterns of a sub-set of these in the pig is a valuable resource for molecular biologists, breeders, and biomedical investigators interested in post-transcriptional gene regulation in pig and in related mammals, including humans.

  15. MicroRNAs define distinct human neuroblastoma cell phenotypes and regulate their differentiation and tumorigenicity

    International Nuclear Information System (INIS)

    Samaraweera, Leleesha; Grandinetti, Kathryn B; Huang, Ruojun; Spengler, Barbara A; Ross, Robert A

    2014-01-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties. A miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation. Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD. Thus, miRNAs define distinct NB cell phenotypes

  16. Biophysical and biological contributions of polyamine-coated carbon nanotubes and bidimensional buckypapers in the delivery of miRNAs to human cells

    Directory of Open Access Journals (Sweden)

    Celluzzi A

    2017-12-01

    Full Text Available Antonella Celluzzi,1,* Alessandro Paolini,1,* Valentina D’Oria,1 Roberta Risoluti,2 Stefano Materazzi,2 Marco Pezzullo,1 Stefano Casciardi,3 Simona Sennato,4 Federico Bordi,4 Andrea Masotti1 1Bambino Gesù Children’s Hospital, IRCCS, Research Laboratories, 2Department of Chemistry, Sapienza University of Rome, 3Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, National Institution for Insurance Against Accidents at Work (INAIL Research, Monte Porzio Catone, 4CNR-ISC UOS Roma, Department of Physics, Sapienza University of Rome, Roma, Italy *These authors contributed equally to this work Abstract: Recent findings in nanomedicine have revealed that carbon nanotubes (CNTs can be used as potential drug carriers, therapeutic agents and diagnostics tools. Moreover, due to their ability to cross cellular membranes, their nanosize dimension, high surface area and relatively good biocompatibility, CNTs have also been employed as a novel gene delivery vector system. In our previous work, we functionalized CNTs with two polyamine polymers, polyethyleneimine (PEI and polyamidoamine dendrimer (PAMAM. These compounds have low cytotoxicity, ability to conjugate microRNAs (such as miR-503 and, at the same time, transfect efficiently endothelial cells. The parameters contributing to the good efficiency of transfection that we observed were not investigated in detail. In fact, the diameter and length of CNTs are important parameters to be taken into account when evaluating the effects on drug delivery efficiency. In order to investigate the biophysical and biological contributions of polymer-coated CNTs in delivery of miRNAs to human cells, we decided to investigate three different preparations, characterized by different dimensions and aspect ratios. In particular, we took into account very small CNTs, a suspension of CNTs starting from the commercial product and a 2D material based on CNTs (ie, buckypapers [BPs] to examine the

  17. The Potential of MicroRNAs as Prostate Cancer Biomarkers

    NARCIS (Netherlands)

    L. Fabris (Linda); Y. Ceder (Yvonne); A.M. Chinnaiyan (Arul); G.W. Jenster (Guido); K.D. Sorensen (Karina D.); S.A. Tomlins (Scott A); T. Visakorpi (Tapio); G.A. Calin (George)

    2016-01-01

    textabstractContext: Short noncoding RNAs known as microRNAs (miRNAs) control protein expression through the degradation of RNA or the inhibition of protein translation. The miRNAs influence a wide range of biologic processes and are often deregulated in cancer. This family of small RNAs constitutes

  18. Titanium and Zirconium Levels Are Associated with Changes in MicroRNAs Expression: Results from a Human Cross-Sectional Study on Obese Population

    Science.gov (United States)

    Dioni, Laura; Angelici, Laura; Vigna, Luisella; Farronato, Giampietro; Pesatori, Angela Cecilia; Bollati, Valentina

    2016-01-01

    Objectives In this study on 90 individuals we aimed at evaluating the microRNAs (miRNAs) expression profile associated with personal levels of Titanium (Ti) and Zirconium (Zr) traced in hair samples. Ti and Zr materials are broadly used for dental implants but the biological reactions triggered by a long term presence of these materials in the oral cavity still need to be assessed. MiRNAs are mechanisms that need to be investigated as they play a fundamental role in the control of gene expression following external stimuli and contribute to a wide range of pathophysiological processes. Methods Using the TaqMan® Low-Density Array, we assessed the expression levels of 377 human miRNAs in peripheral blood of 90 subjects. Hair samples were analyzed for Ti and Zr content using Inductively Coupled Plasma-Mass Spectrometry. We performed multivariable regression analysis to investigate the effects of Ti and Zr exposure on miRNA expression levels. We used the Ingenuity Pathway Analysis (IPA) software to explore the functional role of the investigated miRNAs and the related target genes. Results Seven miRNAs (miR-99b, miR-142-5p, miR-152, miR-193a-5p, miR-323-3p, miR-335, miR-494) resulted specifically associated with Zr levels. The functional target analysis showed that miRNAs are involved in mechanisms such as inflammation, skeletal and connective tissue disorders. Conclusions Our data suggest that Zr is more bioactive than Ti and show that miRNAs are relevant molecular mechanisms sensitive to Zr exposure. PMID:27611787

  19. Role of microRNAs in the age-related changes in skeletal muscle and diet or exercise interventions to promote healthy aging in humans.

    Science.gov (United States)

    McGregor, Robin A; Poppitt, Sally D; Cameron-Smith, David

    2014-09-01

    Progressive age-related changes in skeletal muscle mass and composition, underpin decreases in muscle function, which can inturn lead to impaired mobility and quality of life in older adults. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression in skeletal muscle and are associated with aging. Accumulating evidence suggests that miRNAs play an important role in the age-related changes in skeletal muscle mass, composition and function. At the cellular level, miRNAs have been demonstrated to regulate muscle cell proliferation and differentiation. Furthermore, miRNAs are involved in the transitioning of muscle stem cells from a quiescent, to either an activated or senescence state. Evidence from animal and human studies has shown miRNAs are modulated in muscle atrophy and hypertrophy. In addition, miRNAs have been implicated in changes in muscle fiber composition, fat infiltration and insulin resistance. Both exercise and dietary interventions can combat age-related changes in muscle mass, composition and function, which may be mediated by miRNA modulation in skeletal muscle. Circulating miRNA species derived from myogenic cell populations represent potential biomarkers of aging muscle and the molecular responses to exercise or diet interventions, but larger validation studies are required. In future therapeutic approaches targeting miRNAs, either through exercise, diet or drugs may be able to slow down or prevent the age-related changes in skeletal muscle mass, composition, function, hence help maintain mobility and quality of life in old age. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Synaptic vesicles isolated from the electric organ of Torpedo californica and from the central nervous system of Mus musculus contain small ribonucleic acids (sRNAs

    Directory of Open Access Journals (Sweden)

    Huinan Li

    2017-06-01

    Full Text Available Synaptic vesicles (SVs are presynaptic organelles that load and release small molecule neurotransmitters at chemical synapses. In addition to classic neurotransmitters, we have demonstrated that SVs isolated from the Peripheral Nervous Systems (PNS of the electric organ of Torpedo californica, a model cholinergic synapse, and SVs isolated from the Central Nervous System (CNS of Mus musculus (mouse contain small ribonucleic acids (sRNAs; ≤50 nucleotides (Scientific Reports, 5:1–14(14918 Li et al. (2015 [1]. Our previous publication provided the five most abundant sequences associated with the T. californica SVs, and the ten most abundant sequences associated with the mouse SVs, representing 59% and 39% of the total sRNA reads sequenced, respectively. We provide here a full repository of the SV sRNAs sequenced from T. californica and the mouse deposited in the NCBI as biosamples. Three data studies are included: SVs isolated from the electric organ of T. californica using standard techniques, SVs isolated from the electric organ of T. californica using standard techniques with an additional affinity purification step, and finally, SVs isolated from the CNS of mouse. The three biosamples are available at https://www.ncbi.nlm.nih.gov/biosample/ SRS1523467, SRS1523466, and SRS1523472 respectively.

  1. Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients

    Directory of Open Access Journals (Sweden)

    Chencheng Yao

    2017-12-01

    Full Text Available Human spermatogenesis includes three main stages, namely, the mitosis of spermatogonia, meiosis of spermatocytes, and spermiogenesis of spermatids, which are precisely regulated by epigenetic and genetic factors. Abnormality of epigenetic and genetic factors can result in aberrant spermatogenesis and eventual male infertility. However, epigenetic regulators in controlling each stage of normal and abnormal human spermatogenesis remain unknown. Here, we have revealed for the first time the distinct microRNA profiles in human spermatogonia, pachytene spermatocytes, and round spermatids between obstructive azoospermia (OA patients and non-obstructive azoospermia (NOA patients. Human spermatogonia, pachytene spermatocytes, and round spermatids from OA patients and NOA patients were isolated using STA-PUT velocity sedimentation and identified by numerous hallmarks for these cells. RNA deep sequencing showed that 396 microRNAs were differentially expressed in human spermatogonia between OA patients and NOA patients and 395 differentially expressed microRNAs were found in human pachytene spermatocytes between OA patients and NOA patients. Moreover, 378 microRNAs were differentially expressed in human round spermatids between OA patients and NOA patients. The differential expression of numerous microRNAs identified by RNA deep sequencing was verified by real-time PCR. Moreover, a number of novel targeting genes for microRNAs were predicted using various kinds of software and further verified by real-time PCR. This study thus sheds novel insights into epigenetic regulation of human normal spermatogenesis and the etiology of azoospermia, and it could offer new targets for molecular therapy to treat male infertility.

  2. Phytohormones and microRNAs as sensors and regulators of leaf senescence: assigning macro roles to small molecules.

    Science.gov (United States)

    Sarwat, Maryam; Naqvi, Afsar Raza; Ahmad, Parvaiz; Ashraf, Muhammad; Akram, Nudrat Aisha

    2013-12-01

    Ageing or senescence is an intricate and highly synchronized developmental phase in the life of plant parts including leaf. Senescence not only means death of a plant part, but during this process, different macromolecules undergo degradation and the resulting components are transported to other parts of the plant. During the period from when a leaf is young and green to the stage when it senesces, a multitude of factors such as hormones, environmental factors and senescence associated genes (SAGs) are involved. Plant hormones including salicylic acid, abscisic acid, jasmonic acid and ethylene advance leaf senescence, whereas others like cytokinins, gibberellins, and auxins delay this process. The environmental factors which generally affect plant development and growth, can hasten senescence, the examples being nutrient dearth, water stress, pathogen attack, radiations, high temperature and light intensity, waterlogging, and air, water or soil contamination. Other important influences include carbohydrate accumulation and high carbon/nitrogen level. To date, although several genes involved in this complex process have been identified, still not much information exists in the literature on the signalling mechanism of leaf senescence. Now, the Arabidopsis mutants have paved our way and opened new vistas to elucidate the signalling mechanism of leaf senescence for which various mutants are being utilized. Recent studies demonstrating the role of microRNAs in leaf senescence have reinforced our knowledge of this intricate process. This review provides a comprehensive and critical analysis of the information gained particularly on the roles of several plant growth regulators and microRNAs in regulation of leaf senescence. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. MicroRNAs as tumour suppressors in canine and human melanoma cells and as a prognostic factor in canine melanomas.

    Science.gov (United States)

    Noguchi, S; Mori, T; Hoshino, Y; Yamada, N; Maruo, K; Akao, Y

    2013-06-01

    Malignant melanoma (MM) is one of the most aggressive cancers in dogs and in humans. However, the molecular mechanisms of its development and progression remain unclear. Presently, we examined the expression profile of microRNAs (miRs) in canine oral MM tissues and paired normal oral mucosa tissues by using the microRNA-microarray assay and quantitative RT-PCR. Importantly, a decreased expression of miR-203 was significantly associated with a shorter survival time. Also, miR-203 and -205 were markedly down-regulated in canine and human MM cell lines tested. Furthermore, the ectopic expression of miR-205 had a significant inhibitory effect on the cell growth of canine and human melanoma cells tested by targeting erbb3. Our data suggest that miR-203 is a new prognostic factor in canine oral MMs and that miR-205 functions as a tumour suppressor by targeting erbb3 in both canine and human MM cells. © 2011 John Wiley & Sons Ltd.

  4. Tumor suppressor microRNAs are downregulated in myelodysplastic syndrome with spliceosome mutations

    DEFF Research Database (Denmark)

    Aslan, Derya; Garde, Christian; Nygaard, Mette Katrine

    2016-01-01

    Spliceosome mutations are frequently observed in patients with myelodysplastic syndromes (MDS). However, it is largely unknown how these mutations contribute to the disease. MicroRNAs (miRNAs) are small noncoding RNAs, which have been implicated in most human cancers due to their role in post...... the most downregulated miRNAs were several tumor-suppressor miRNAs, including several let-7 family members, miR-423, and miR-103a. Finally, we observed that the predicted targets of the most downregulated miRNAs were involved in apoptosis, hematopoiesis, and acute myeloid leukemia among other cancer......- and metabolic pathways. Our data indicate that spliceosome mutations may play an important role in MDS pathophysiology by affecting the expression of tumor suppressor miRNA genes involved in the development and progression of MDS....

  5. MetastamiRs: Non-Coding MicroRNAs Driving Cancer Invasion and Metastasis

    Directory of Open Access Journals (Sweden)

    Sergio Rodriguez-Cuevas

    2012-01-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs of ~22 nucleotides that function as negative regulators of gene expression by either inhibiting translation or inducing deadenylation-dependent degradation of target transcripts. Notably, deregulation of miRNAs expression is associated with the initiation and progression of human cancers where they act as oncogenes or tumor suppressors contributing to tumorigenesis. Abnormal miRNA expression may provide potential diagnostic and prognostic tumor biomarkers and new therapeutic targets in cancer. Recently, several miRNAs have been shown to initiate invasion and metastasis by targeting multiple proteins that are major players in these cellular events, thus they have been denominated as metastamiRs. Here, we present a review of the current knowledge of miRNAs in cancer with a special focus on metastamiRs. In addition we discuss their potential use as novel specific markers for cancer progression.

  6. Long Non-Coding RNAs Embedded in the Rb and p53 Pathways

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, Murugan; Jones, Matthew F.; Lal, Ashish, E-mail: ashish.lal@nih.gov [Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-12-04

    In recent years, long non-coding RNAs (lncRNAs) have gained significant attention as a novel class of gene regulators. Although a small number of lncRNAs have been shown to regulate gene expression through diverse mechanisms including transcriptional regulation, mRNA splicing and translation, the physiological function and mechanism of action of the vast majority are not known. Profiling studies in cell lines and tumor samples have suggested a potential role of lncRNAs in cancer. Indeed, distinct lncRNAs have been shown to be embedded in the p53 and Rb networks, two of the major tumor suppressor pathways that control cell cycle progression and survival. Given the fact that inactivation of Rb and p53 is a hallmark of human cancer, in this review we discuss recent evidence on the function of lncRNAs in the Rb and p53 signaling pathways.

  7. Long Non-Coding RNAs Embedded in the Rb and p53 Pathways

    International Nuclear Information System (INIS)

    Subramanian, Murugan; Jones, Matthew F.; Lal, Ashish

    2013-01-01

    In recent years, long non-coding RNAs (lncRNAs) have gained significant attention as a novel class of gene regulators. Although a small number of lncRNAs have been shown to regulate gene expression through diverse mechanisms including transcriptional regulation, mRNA splicing and translation, the physiological function and mechanism of action of the vast majority are not known. Profiling studies in cell lines and tumor samples have suggested a potential role of lncRNAs in cancer. Indeed, distinct lncRNAs have been shown to be embedded in the p53 and Rb networks, two of the major tumor suppressor pathways that control cell cycle progression and survival. Given the fact that inactivation of Rb and p53 is a hallmark of human cancer, in this review we discuss recent evidence on the function of lncRNAs in the Rb and p53 signaling pathways

  8. A global downregulation of microRNAs occurs in human quiescent satellite cells during myogenesis

    NARCIS (Netherlands)

    Koning, Merel; Werker, Paul M N; van Luyn, Marja J A; Krenning, Guido; Harmsen, Martin C

    2012-01-01

    During myogenesis, human satellite cells differentiate and form multinucleated myotubes, while a fraction of the human satellite cells enter quiescence. These quiescent satellite cells are able to activate, proliferate and contribute to muscle regeneration. Post-transcriptional regulation of

  9. Novel classes of non-coding RNAs and cancer

    Directory of Open Access Journals (Sweden)

    Sana Jiri

    2012-05-01

    Full Text Available Abstract For the many years, the central dogma of molecular biology has been that RNA functions mainly as an informational intermediate between a DNA sequence and its encoded protein. But one of the great surprises of modern biology was the discovery that protein-coding genes represent less than 2% of the total genome sequence, and subsequently the fact that at least 90% of the human genome is actively transcribed. Thus, the human transcriptome was found to be more complex than a collection of protein-coding genes and their splice variants. Although initially argued to be spurious transcriptional noise or accumulated evolutionary debris arising from the early assembly of genes and/or the insertion of mobile genetic elements, recent evidence suggests that the non-coding RNAs (ncRNAs may play major biological roles in cellular development, physiology and pathologies. NcRNAs could be grouped into two major classes based on the transcript size; small ncRNAs and long ncRNAs. Each of these classes can be further divided, whereas novel subclasses are still being discovered and characterized. Although, in the last years, small ncRNAs called microRNAs were studied most frequently with more than ten thousand hits at PubMed database, recently, evidence has begun to accumulate describing the molecular mechanisms by which a wide range of novel RNA species function, providing insight into their functional roles in cellular biology and in human disease. In this review, we summarize newly discovered classes of ncRNAs, and highlight their functioning in cancer biology and potential usage as biomarkers or therapeutic targets.

  10. BioM2MetDisease: a manually curated database for associations between microRNAs, metabolites, small molecules and metabolic diseases.

    Science.gov (United States)

    Xu, Yanjun; Yang, Haixiu; Wu, Tan; Dong, Qun; Sun, Zeguo; Shang, Desi; Li, Feng; Xu, Yingqi; Su, Fei; Liu, Siyao; Zhang, Yunpeng; Li, Xia

    2017-01-01

    BioM2MetDisease is a manually curated database that aims to provide a comprehensive and experimentally supported resource of associations between metabolic diseases and various biomolecules. Recently, metabolic diseases such as diabetes have become one of the leading threats to people’s health. Metabolic disease associated with alterations of multiple types of biomolecules such as miRNAs and metabolites. An integrated and high-quality data source that collection of metabolic disease associated biomolecules is essential for exploring the underlying molecular mechanisms and discovering novel therapeutics. Here, we developed the BioM2MetDisease database, which currently documents 2681 entries of relationships between 1147 biomolecules (miRNAs, metabolites and small molecules/drugs) and 78 metabolic diseases across 14 species. Each entry includes biomolecule category, species, biomolecule name, disease name, dysregulation pattern, experimental technique, a brief description of metabolic disease-biomolecule relationships, the reference, additional annotation information etc. BioM2MetDisease provides a user-friendly interface to explore and retrieve all data conveniently. A submission page was also offered for researchers to submit new associations between biomolecules and metabolic diseases. BioM2MetDisease provides a comprehensive resource for studying biology molecules act in metabolic diseases, and it is helpful for understanding the molecular mechanisms and developing novel therapeutics for metabolic diseases. http://www.bio-bigdata.com/BioM2MetDisease/. © The Author(s) 2017. Published by Oxford University Press.

  11. Genome-wide analysis of EgEVE_1, a transcriptionally active endogenous viral element associated to small RNAs in Eucalyptus genomes

    Directory of Open Access Journals (Sweden)

    Helena Sanches Marcon

    2017-02-01

    Full Text Available Abstract Endogenous viral elements (EVEs are the result of heritable horizontal gene transfer from viruses to hosts. In the last years, several EVE integration events were reported in plants by the exponential availability of sequenced genomes. Eucalyptus grandis is a forest tree species with a sequenced genome that is poorly studied in terms of evolution and mobile genetic elements composition. Here we report the characterization of E. grandis endogenous viral element 1 (EgEVE_1, a transcriptionally active EVE with a size of 5,664 bp. Phylogenetic analysis and genomic distribution demonstrated that EgEVE_1 is a newly described member of the Caulimoviridae family, distinct from the recently characterized plant Florendoviruses. Genomic distribution of EgEVE_1 and Florendovirus is also distinct. EgEVE_1 qPCR quantification in Eucalyptus urophylla suggests that this genome has more EgEVE_1 copies than E. grandis. EgEVE_1 transcriptional activity was demonstrated by RT-qPCR in five Eucalyptus species and one intrageneric hybrid. We also identified that Eucalyptus EVEs can generate small RNAs (sRNAs,that might be involved in de novo DNA methylation and virus resistance. Our data suggest that EVE families in Eucalyptus have distinct properties, and we provide the first comparative analysis of EVEs in Eucalyptus genomes.

  12. Small RNAs were involved in homozygous state-associated silencing of a marker gene (Neomycin phosphotransferase II: nptII) in transgenic tomato plants.

    Science.gov (United States)

    Deng, Lei; Pan, Yu; Chen, Xuqing; Chen, Guoping; Hu, Zongli

    2013-07-01

    Homozygous state-associated co-suppression is not a very common phenomenon. In our experiments, two transgenic plants 3A29 and 1195A were constructed by being transformed with the constructs pBIN-353A and pBIN119A containing nptII gene as a marker respectively. The homozygous progeny from these two independent transgenic lines 3A29 and 1195A, displayed kanamycin-sensitivity and produced a short main root without any lateral roots as untransformed control (wild-type) seedlings when germinated on kanamycin media. For the seedlings derived from putative hemizygous plants, the percentage of the seedlings showing normal growth on kanamycin media was about 50% and lower than the expected percentage (75%). Southern analysis of the genomic DNA confirmed that the homozygous and hemizygous plants derived from the same lines contained the same multiple nptII transgenes, which were located on the same site of chromosome. Northern analysis suggested that the marker nptII gene was expressed in the primary and the hemizygous transformants, but it was silenced in the homozygous transgenic plants. Further Northern analysis indicated that antisense and sense small nptII-derived RNAs were present in the transgenic plants and the blotting signal of nptII-derived small RNA was much higher in the homozygous transgenic plants than that of hemizygous transgenic plants. Additionally, read-through transcripts from the TRAMP gene to the nptII gene were detected. These results suggest that the read-through transcripts may be involved in homozygous state-associated silencing of the nptII transgene in transgenic tomato plants and a certain threshold level of the nptII-derived small RNAs is required for the homozygous state-associated co-suppression of the nptII transgene. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  13. All Small Nuclear RNAs (snRNAs) of the [U4/U6.U5] Tri-snRNP Localize to Nucleoli; Identification of the Nucleolar Localization Element of U6 snRNA

    Science.gov (United States)

    Gerbi, Susan A.; Lange, Thilo Sascha

    2002-01-01

    Previously, we showed that spliceosomal U6 small nuclear RNA (snRNA) transiently passes through the nucleolus. Herein, we report that all individual snRNAs of the [U4/U6.U5] tri-snRNP localize to nucleoli, demonstrated by fluorescence microscopy of nucleolar preparations after injection of fluorescein-labeled snRNA into Xenopus oocyte nuclei. Nucleolar localization of U6 is independent from [U4/U6] snRNP formation since sites of direct interaction of U6 snRNA with U4 snRNA are not nucleolar localization elements. Among all regions in U6, the only one required for nucleolar localization is its 3′ end, which associates with the La protein and subsequently during maturation of U6 is bound by Lsm proteins. This 3′-nucleolar localization element of U6 is both essential and sufficient for nucleolar localization and also required for localization to Cajal bodies. Conversion of the 3′ hydroxyl of U6 snRNA to a 3′ phosphate prevents association with the La protein but does not affect U6 localization to nucleoli or Cajal bodies. PMID:12221120

  14. miRNAs and other non-coding RNAs in posttraumatic stress disorder: A systematic review of clinical and animal studies.

    Science.gov (United States)

    Schmidt, Ulrike; Keck, Martin E; Buell, Dominik R

    2015-06-01

    In the last couple of years, non-coding (nc) RNAs like micro-RNAs (miRNAs), small interference RNAs (siRNAs) and long ncRNAs (lncRNAs) have emerged as promising candidates for biomarkers and drug-targets in a variety of psychiatric disorders. In contrast to reports on ncRNAs in affective disorders, schizophrenia and anxiety disorders, manuscripts on ncRNAs in posttraumatic stress disorder (PTSD) and associated animal models are scarce. Aiming to stimulate ncRNA research in PTSD and to identify the hitherto most promising ncRNA candidates and associated pathways for psychotrauma research, we conducted the first review on ncRNAs in PTSD. We aimed to identify studies reporting on the expression, function and regulation of ncRNAs in PTSD patients and in animals exhibiting a PTSD-like syndrome. Following the PRISMA guidelines for systematic reviews, we systematically screened the PubMed database for clinical and animal studies on ncRNAs in PTSD, animal models for PTSD and animal models employing a classical fear conditioning paradigm. Using 112 different combinations of search terms, we retrieved 523 articles of which we finally included and evaluated three clinical and 12 animal studies. In addition, using the web-based tool DIANA miRPath v2.0, we searched for molecular pathways shared by the predicted targets of the here-evaluated miRNA candidates. Our findings suggest that mir-132, which has been found to be regulated in three of the here included studies, as well as miRNAs with an already established role in Alzheimer's disease (AD) seem to be particularly promising candidates for future miRNA studies in PTSD. These results are limited by the low number of human trials and by the heterogeneity of included animal studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. 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.

  16. Rifampin modulation of xeno- and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes.

    Science.gov (United States)

    Gufford, Brandon T; Robarge, Jason D; Eadon, Michael T; Gao, Hongyu; Lin, Hai; Liu, Yunlong; Desta, Zeruesenay; Skaar, Todd C

    2018-04-01

    Rifampin is a pleiotropic inducer of multiple drug metabolizing enzymes and transporters. This work utilized a global approach to evaluate rifampin effects on conjugating enzyme gene expression with relevance to human xeno- and endo-biotic metabolism. Primary human hepatocytes from 7 subjects were treated with rifampin (10 μmol/L, 24 hours). Standard methods for RNA-seq library construction, EZBead preparation, and NextGen sequencing were used to measure UDP-glucuronosyl transferase UGT, sulfonyltransferase SULT, N acetyltransferase NAT, and glutathione-S-transferase GST mRNA expression compared to vehicle control (0.01% MeOH). Rifampin-induced (>1.25-fold) mRNA expression of 13 clinically important phase II drug metabolizing genes and repressed (>1.25-fold) the expression of 3 genes ( P  accounting for simultaneous induction of both CYP3A4 and UGT1A4 predicted a ~10-fold decrease in parent midazolam exposure with only a ~2-fold decrease in midazolam N-glucuronide metabolite exposure. These data reveal differential effects of rifampin on the human conjugating enzyme transcriptome and potential associations with miRNAs that form the basis for future mechanistic studies to elucidate the interplay of conjugating enzyme regulatory elements.

  17. Noncanonical microRNAs and endogenous siRNAs in lytic infection of murine gammaherpesvirus.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available MicroRNA (miRNA and endogenous small interfering RNA (endo-siRNA are two essential classes of small noncoding RNAs (sncRNAs in eukaryotes. The class of miRNA is diverse and there exist noncanonical miRNAs that bypass the canonical miRNA biogenesis pathway. In order to identify noncanonical miRNAs and endo-siRNAs responding to virus infection and study their potential function, we sequenced small-RNA species from cells lytically infected with murine gammaherpesvirus 68 (MHV68. In addition to three novel canonical miRNAs in mouse, two antisense miRNAs in virus and 25 novel noncanonical miRNAs, including miRNAs derived from transfer RNAs, small nucleolar RNAs and introns, in the host were identified. These noncanonical miRNAs exhibited features distinct from that of canonical miRNAs in lengths of hairpins, base pairings and first nucleotide preference. Many of the novel miRNAs are conserved in mammals. Besides several known murine endo-siRNAs detected by the sequencing profiling, a novel locus in the mouse genome was identified to produce endo-siRNAs. This novel endo-siRNA locus is comprised of two tandem inverted B4 short interspersed nuclear elements (SINEs. Unexpectedly, the SINE-derived endo-siRNAs were found in a variety of sequencing data and virus-infected cells. Moreover, a murine miRNA was up-regulated more than 35 fold in infected than in mock-treated cells. The putative targets of the viral and the up-regulated murine miRNAs were potentially involved in processes of gene transcription and protein phosphorylation, and localized to membranes, suggesting their potential role in manipulating the host basal immune system during lytic infection. Our results extended the number of noncanonical miRNAs in mammals and shed new light on their potential functions of lytic infection of MHV68.

  18. 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.

  19. Diversity of human small intestinal Streptococcus and Veillonella populations

    NARCIS (Netherlands)

    van den Bogert, Bartholomeus; Erkus, Oylum; Boekhorst, Jos; de Goffau, Marcus; Smid, Eddy J.; Zoetendal, Erwin G.; Kleerebezem, Michiel

    Molecular and cultivation approaches were employed to study the phylogenetic richness and temporal dynamics of Streptococcus and Veillonella populations in the small intestine. Microbial profiling of human small intestinal samples collected from four ileostomy subjects at four time points displayed

  20. Circulating microRNAs as Potential Biomarkers of Infectious Disease

    Science.gov (United States)

    Correia, Carolina N.; Nalpas, Nicolas C.; McLoughlin, Kirsten E.; Browne, John A.; Gordon, Stephen V.; MacHugh, David E.; Shaughnessy, Ronan G.

    2017-01-01

    microRNAs (miRNAs) are a class of small non-coding endogenous RNA molecules that regulate a wide range of biological processes by post-transcriptionally regulating gene expression. Thousands of these molecules have been discovered to date, and multiple miRNAs have been shown to coordinately fine-tune cellular processes key to organismal development, homeostasis, neurobiology, immunobiology, and control of infection. The fundamental regulatory role of miRNAs in a variety of biological processes suggests that differential expression of these transcripts may be exploited as a novel source of molecular biomarkers for many different disease pathologies or abnormalities. This has been emphasized by the recent discovery of remarkably stable miRNAs in mammalian biofluids, which may originate from intracellular processes elsewhere in the body. The potential of circulating miRNAs as biomarkers of disease has mainly been demonstrated for various types of cancer. More recently, however, attention has focused on the use of circulating miRNAs as diagnostic/prognostic biomarkers of infectious disease; for example, human tuberculosis caused by infection with Mycobacterium tuberculosis, sepsis caused by multiple infectious agents, and viral hepatitis. Here, we review these developments and discuss prospects and challenges for translating circulating miRNA into novel diagnostics for infectious disease. PMID:28261201

  1. Global Intersection of Long Non-Coding RNAs with Processed and Unprocessed Pseudogenes in the Human Genome

    Directory of Open Access Journals (Sweden)

    Michael John Milligan

    2016-03-01

    Full Text Available Pseudogenes are abundant in the human genome and had long been thought of purely as nonfunctional gene fossils. Recent observations point to a role for pseudogenes in regulating genes transcriptionally and post-transcriptionally in human cells. To computationally interrogate the network space of integrated pseudogene and long non-coding RNA regulation in the human transcriptome, we developed and implemented an algorithm to identify all long non-coding RNA (lncRNA transcripts that overlap the genomic spans, and specifically the exons, of any human pseudogenes in either sense or antisense orientation. As inputs to our algorithm, we imported three public repositories of pseudogenes: GENCODE v17 (processed and unprocessed, Ensembl 72; Retroposed Pseudogenes V5 (processed only and Yale Pseudo60 (processed and unprocessed, Ensembl 60; two public lncRNA catalogs: Broad Institute, GENCODE v17; NCBI annotated piRNAs; and NHGRI clinical variants. The data sets were retrieved from the UCSC Genome Database using the UCSC Table Browser. We identified 2277 loci containing exon-to-exon overlaps between pseudogenes, both processed and unprocessed, and long non-coding RNA genes. Of these loci we identified 1167 with Genbank EST and full-length cDNA support providing direct evidence of transcription on one or both strands with exon-to-exon overlaps. The analysis converged on 313 pseudogene-lncRNA exon-to-exon overlaps that were bidirectionally supported by both full-length cDNAs and ESTs. In the process of identifying transcribed pseudogenes, we generated a comprehensive, positionally non-redundant encyclopedia of human pseudogenes, drawing upon multiple, and formerly disparate public pseudogene repositories. Collectively, these observations suggest that pseudogenes are pervasively transcribed on both strands and are common drivers of gene regulation.

  2. Identification and analysis of miRNAs in human breast cancer and teratoma samples using deep sequencing

    DEFF Research Database (Denmark)

    Nygaard, Sanne; Jacobsen, Anders; Lindow, Morten

    2009-01-01

    ABSTRACT: BACKGROUND: MiRNAs play important roles in cellular control and in various disease states such as cancers, where they may serve as markers or possibly even therapeutics. Identifying the whole repertoire of miRNAs and understanding their expression patterns is therefore an important goal...

  3. Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

    International Nuclear Information System (INIS)

    Kwon, ChangHyuk; Tak, Hyosun; Rho, Mina; Chang, Hae Ryung; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Lee, Eun Kyung; Nam, Seungyoon

    2014-01-01

    Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies

  4. Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, ChangHyuk, E-mail: netbuyer@hanmail.net [Cancer Genomics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Tak, Hyosun, E-mail: chuberry@naver.com [Department of Biochemistry, College of Medicine, Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Rho, Mina, E-mail: minarho@hanyang.ac.kr [Department of Computer Science, Hanyang University, Seoul 133-791 (Korea, Republic of); Chang, Hae Ryung, E-mail: heyhae@ncc.re.kr [New Experimental Therapeutics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, Yon Hui, E-mail: yhkim@ncc.re.kr [New Experimental Therapeutics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, Kyung Tae, E-mail: bioktkim@ncc.re.kr [Molecular Epidemiology Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Balch, Curt, E-mail: curt.balch@gmail.com [Medical Sciences Program, Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Bloomington, IN 47405 (United States); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Nam, Seungyoon, E-mail: seungyoon.nam@ncc.re.kr [Cancer Genomics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of)

    2014-03-28

    Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies.

  5. let-7 miRNAs Can Act through Notch to Regulate Human Gliogenesis

    Directory of Open Access Journals (Sweden)

    M. Patterson

    2014-11-01

    Full Text Available It is clear that neural differentiation from human pluripotent stem cells generates cells that are developmentally immature. Here, we show that the let-7 plays a functional role in the developmental decision making of human neural progenitors, controlling whether these cells make neurons or glia. Through gain- and loss-of-function studies on both tissue and pluripotent derived cells, our data show that let-7 specifically regulates decision making in this context by regulation of a key chromatin-associated protein, HMGA2. Furthermore, we provide evidence that the let-7/HMGA2 circuit acts on HES5, a NOTCH effector and well-established node that regulates fate decisions in the nervous system. These data link the let-7 circuit to NOTCH signaling and suggest that this interaction serves to regulate human developmental progression.

  6. The 3' region of Human Papillomavirus type 16 early mRNAs decrease expression

    DEFF Research Database (Denmark)

    Vinther, J.; Rosenstierne, M.W.; Kristiansen, Karen

    2005-01-01

    Background: High risk human papillomavirus (HR-HPV) infects mucosal surfaces and HR-HPV infection is required for development of cervical cancer. Accordingly, enforced expression of the early HR-HPV proteins can induce immortalisation of human cells. In most cervical cancers and cervical cancer...... cell lines the HR-HPV double stranded DNA genome has been integrated into the host cell genome. Methods: We have used a retroviral GUS reporter system to generate pools of stably transfected HaCaT and SiHa cells. The HPV-16 early sequences that are deleted upon integration of the HPV-16 genome...

  7. MicroRNAs: From Female Fertility, Germ Cells, and Stem Cells to Cancer in Humans

    Czech Academy of Sciences Publication Activity Database

    Virant-Klun, I.; Stahlberg, A.; Kubista, Mikael; Skutella, T.

    2016-01-01

    Roč. 2016, č. 2016 (2016), č. článku 3984937. E-ISSN 1687-9678 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : PREMATURE OVARIAN FAILURE * CUMULUS-OOCYTE COMPLEX * HUMAN GRANULOSA-CELLS Subject RIV: EB - Genetics ; Molecular Biology

  8. Challenges and Opportunities of MicroRNAs in Lymphomas

    Directory of Open Access Journals (Sweden)

    Giacoma De Tullio

    2014-09-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that control the expression of many target messenger RNAs (mRNAs involved in normal cell functions (differentiation, proliferation and apoptosis. Consequently their aberrant expression and/or functions are related to pathogenesis of many human diseases including cancers. Haematopoiesis is a highly regulated process controlled by a complex network of molecular mechanisms that simultaneously regulate commitment, differentiation, proliferation, and apoptosis of hematopoietic stem cells (HSC. Alterations on this network could affect the normal haematopoiesis, leading to the development of haematological malignancies such as lymphomas. The incidence of lymphomas is rising and a significant proportion of patients are refractory to standard therapies. Accurate diagnosis, prognosis and therapy still require additional markers to be used for diagnostic and prognostic purpose and evaluation of clinical outcome. The dysregulated expression or function of miRNAs in various types of lymphomas has been associated with lymphoma pathogenesis. Indeed, many recent findings suggest that almost all lymphomas seem to have a distinct and specific miRNA profile and some miRNAs are related to therapy resistance or have a distinct kinetics during therapy. MiRNAs are easily detectable in fresh or paraffin-embedded diagnostic tissue and serum where they are highly stable and quantifiable within the diagnostic laboratory at each consultation. Accordingly they could be specific biomarkers for lymphoma diagnosis, as well as useful for evaluating prognosis or disease response to the therapy, especially for evaluation of early relapse detection and for greatly assisting clinical decisions making. Here we summarize the current knowledge on the role of miRNAs in normal and aberrant lymphopoiesis in order to highlight their clinical value as specific diagnosis and prognosis markers of lymphoid malignancies or for prediction of therapy

  9. Targeting Non-Coding RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting.

    Science.gov (United States)

    Basak, Jolly; Nithin, Chandran

    2015-01-01

    Non-coding RNAs (ncRNAs) have emerged as versatile master regulator of biological functions in recent years. MicroRNAs (miRNAs) are small endogenous ncRNAs of 18-24 nucleotides in length that originates from long self-complementary precursors. Besides their direct involvement in developmental processes, plant miRNAs play key roles in gene regulatory networks and varied biological processes. Alternatively, long ncRNAs (lncRNAs) are a large and diverse class of transcribed ncRNAs whose length exceed that of 200 nucleotides. Plant lncRNAs are transcribed by different RNA polymerases, showing diverse structural features. Plant lncRNAs also are important regulators of gene expression in diverse biological processes. There has been a breakthrough in the technology of genome editing, the CRISPR-Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9) technology, in the last decade. CRISPR loci are transcribed into ncRNA and eventually form a functional complex with Cas9 and further guide the complex to cleave complementary invading DNA. The CRISPR-Cas technology has been successfully applied in model plants such as Arabidopsis and tobacco and important crops like wheat, maize, and rice. However, all these studies are focused on protein coding genes. Information about targeting non-coding genes is scarce. Hitherto, the CRISPR-Cas technology has been exclusively used in vertebrate systems to engineer miRNA/lncRNAs, but it is still relatively unexplored in plants. While briefing miRNAs, lncRNAs and applications of the CRISPR-Cas technology in human and animals, this review essentially elaborates several strategies to overcome the challenges of applying the CRISPR-Cas technology in editing ncRNAs in plants and the future perspective of this field.

  10. Targeting non-coding RNAs in Plants with the CRISPR-Cas technology is a challenge yet worth accepting

    Directory of Open Access Journals (Sweden)

    Jolly eBasak

    2015-11-01

    Full Text Available Non-coding RNAs (ncRNAs have emerged as versatile master regulator of biological functions in recent years. MicroRNAs (miRNAs are small endogenous ncRNAs of 18-24 nucleotides in length that originates from long self-complementary precursors. Besides their direct involvement in developmental processes, plant miRNAs play key roles in gene regulatory networks and varied biological processes. Alternatively, long ncRNAs (lncRNAs are a large and diverse class of transcribed ncRNAs whose length exceed that of 200 nucleotides. Plant lncRNAs are transcribed by different RNA polymerases, showing diverse structural features. Plant lncRNAs also are important regulators of gene expression in diverse biological processes. There has been a breakthrough in the technology of genome editing, the CRISPR-Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 technology, in the last decade. CRISPR loci are transcribed into ncRNA and eventually form a functional complex with Cas9 and further guide the complex to cleave complementary invading DNA. The CRISPR-Cas technology has been successfully applied in model plants such as Arabidopsis and tobacco and important crops like wheat, maize and rice. However, all these studies are focused on protein coding genes. Information about targeting non-coding genes is scarce. Hitherto, the CRISPR-Cas technology has been exclusively used in vertebrate systems to engineer miRNA/lncRNAs, but it is still relatively unexplored in plants. While briefing miRNAs, lncRNAs and applications of the CRISPR-Cas technology in human and animals, this review essentially elaborates several strategies to overcome the challenges of applying the CRISPR-Cas technology in editing ncRNAs in plants and the future perspective of this field.

  11. Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

    Directory of Open Access Journals (Sweden)

    Valentina Iadevaia

    2015-09-01

    Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

  12. Antiviral activity of Small interfering RNAs: Specificity testing using heterologous virus reveals interferon-related effects overlooked by conventional mismatch controls

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Lorenzen, Niels; Pedersen, Finn Skou

    2006-01-01

    to the viral glycoprotein gene of the target-virus efficiently inhibited viral multiplication in infected cell cultures, while two of three corresponding mismatched siRNAs did not have this effect. This suggested specific interference, but similar results were obtained when the same siRNAs were tested against...... a heterologous virus. Further analyses revealed that the siRNAs induced a non-target-specific anti-viral effect correlating with upregulation of the interferon induced Mx gene....

  13. Regulatory RNAs in the Less Studied Streptococcal Species: from Nomenclature to Identification

    Directory of Open Access Journals (Sweden)

    Mohamed Amine Zorgani

    2016-07-01

    Full Text Available Streptococcal species are Gram-positive bacteria involved in severe and invasive diseases in humans and animals. Although this group includes different pathogenic species involved in life-threatening infections for humans, it also includes beneficial species, such as Streptococcus thermophilus, which is used in yogurt production. In bacteria virulence factors are controlled by various regulatory networks including regulatory RNAs. For clearness and to develop logical thinking, we start this review with a revision of regulatory RNAs nomenclature. Previous reviews are mostly dealing with Streptococcus pyogenes and Streptococcus pneumoniae regulatory RNAs. We especially focused our analysis on regulatory RNAs in Streptococcus agalactiae, Streptococcus mutans, Streptococcus thermophilus and other less studied Streptococcus species. Although S. agalactiae RNome remains largely unknown, sRNAs (small RNAs are supposed to mediate regulation during environmental adaptation and host infection. In the case of S. mutans, sRNAs are suggested to be involved in competence regulation, carbohydrate metabolism and Toxin-Antitoxin systems. A new category of miRNA-size small RNAs (msRNAs was also identified for the first time in this species. The analysis of S. thermophilus sRNome shows that many sRNAs are associated to the bacterial immune system known as CRISPR-Cas system. Only few of the other different Streptococcus species have been the subject of studies pointed toward the characterization of regulatory RNAs. Finally, understanding bacterial sRNome can constitute one step forward to the elaboration of new strategies in therapy such as substitution of antibiotics in the management of S. agalactiae neonatal infections, prevention of S. mutans dental caries or use of S. thermophilus CRISPR-Cas system in genome editing applications.

  14. Two insulin-like growth factor I messenger RNAs are expressed in human liver

    International Nuclear Information System (INIS)

    Rotwein, P.

    1986-01-01

    Through use of a synthetic radiolabelled oligonucleotide probe, human insulin-like growth factor I (IGF-I) cDNA clones were isolated from a liver library. Two types of cDNAs were defined by restriction enzyme analysis and DNA sequencing. Both encode IGF-I precursors of either 195 or 153 amino acids. The two predicted protein precursors are identical from their amino terminus to a lysine residue 16 codons beyond the IGF-I sequence, and then they diverge. Both cDNAs predict additional unique carboxyl-terminal extension peptides. Since there is only one IGF-I gene in the human genome, the finding of two different cDNAs suggests that alternative RNA processing plays a role in IGF-I gene expression. The functions of the different extension peptides remain to be elucidates

  15. Reconstituted influenza virus envelopes as an efficient carrier system for cellular delivery of small-interfering RNAs

    NARCIS (Netherlands)

    de Jonge, J; Holtrop, M; Wilschut, J; Huckriede, A

    Application of RNA interference for in vivo evaluation of gene function or for therapeutic interventions has been hampered by a lack of suitable delivery methods for small interfering RNA ( siRNA). Here, we present reconstituted viral envelopes (virosomes) derived from influenza virus as suitable

  16. MicroRNAs let-7b/i suppress human glioma cell invasion and migration by targeting IKBKE directly

    International Nuclear Information System (INIS)

    Tian, Yuan; Hao, Shaobo; Ye, Minhua; Zhang, Anling; Nan, Yang; Wang, Guangxiu; Jia, Zhifan; Yu, Kai; Guo, Lianmei; Pu, Peiyu; Huang, Qiang; Zhong, Yue

    2015-01-01

    We demonstrated that IKBKE is overexpressed in human gliomas and that the downregulation of IKBKE markedly inhibits the proliferative and invasive abilities of glioma cells, which is consistent with the results reported by several different research groups. Therefore, IKBKE represents a promising therapeutic target for the treatment of glioma. In the present study, we verified that the microRNAs let-7b and let-7i target IKBKE through luciferase assays and found that let-7b/i mimics can knock down IKBKE and upregulate E-cadherin through western blot analysis. Moreover, the expression levels of let-7b/i were significantly lower in glioma cell lines than that in normal brain tissues, as determined by quantitative real-time PCR. Furthermore, let-7b/i inhibit the invasion and migration of glioma cells, as determined through wound healing and Transwell assays. The above-mentioned data suggest that let-7b/i inhibit the invasive ability of glioma cells by directly downregulating IKBKE and indirectly upregulating E-cadherin. - Highlights: • Let-7b and let-7i are downregulated in glioma cell lines. • IKBKE is a target gene of let-7b/i. • Let-7b/i inhibit the invasion and migration of glioma cells. • Let-7b/i upregulate E-cadherin by downregulating IKBKE

  17. MicroRNAs let-7b/i suppress human glioma cell invasion and migration by targeting IKBKE directly

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Yuan; Hao, Shaobo [Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052 (China); Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin 300052 (China); Key Laboratory of Neurotrauma, Variation and Regeneration, Ministry of Education and Tianjin Municipal Government (China); Ye, Minhua [Department of Neurosurgery, Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province 330006 (China); Zhang, Anling [Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin 300052 (China); Key Laboratory of Neurotrauma, Variation and Regeneration, Ministry of Education and Tianjin Municipal Government (China); Nan, Yang [Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052 (China); Wang, Guangxiu; Jia, Zhifan [Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin 300052 (China); Key Laboratory of Neurotrauma, Variation and Regeneration, Ministry of Education and Tianjin Municipal Government (China); Yu, Kai; Guo, Lianmei [Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052 (China); Pu, Peiyu [Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin 300052 (China); Key Laboratory of Neurotrauma, Variation and Regeneration, Ministry of Education and Tianjin Municipal Government (China); Huang, Qiang, E-mail: huangqiang209@163.com [Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052 (China); Zhong, Yue, E-mail: zhongyue2457@sina.com [Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052 (China)

    2015-03-06

    We demonstrated that IKBKE is overexpressed in human gliomas and that the downregulation of IKBKE markedly inhibits the proliferative and invasive abilities of glioma cells, which is consistent with the results reported by several different research groups. Therefore, IKBKE represents a promising therapeutic target for the treatment of glioma. In the present study, we verified that the microRNAs let-7b and let-7i target IKBKE through luciferase assays and found that let-7b/i mimics can knock down IKBKE and upregulate E-cadherin through western blot analysis. Moreover, the expression levels of let-7b/i were significantly lower in glioma cell lines than that in normal brain tissues, as determined by quantitative real-time PCR. Furthermore, let-7b/i inhibit the invasion and migration of glioma cells, as determined through wound healing and Transwell assays. The above-mentioned data suggest that let-7b/i inhibit the invasive ability of glioma cells by directly downregulating IKBKE and indirectly upregulating E-cadherin. - Highlights: • Let-7b and let-7i are downregulated in glioma cell lines. • IKBKE is a target gene of let-7b/i. • Let-7b/i inhibit the invasion and migration of glioma cells. • Let-7b/i upregulate E-cadherin by downregulating IKBKE.

  18. Dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding RNAs and fitness in human cells.

    Directory of Open Access Journals (Sweden)

    Claudia V Filomatori

    2017-03-01

    Full Text Available The Flavivirus genus includes a large number of medically relevant pathogens that cycle between humans and arthropods. This host alternation imposes a selective pressure on the viral population. Here, we found that dengue virus, the most important viral human pathogen transmitted by insects, evolved a mechanism to differentially regulate the production of viral non-coding RNAs in mosquitos and humans, with a significant impact on viral fitness in each host. Flavivirus infections accumulate non-coding RNAs derived from the viral 3'UTRs (known as sfRNAs, relevant in viral pathogenesis and immune evasion. We found that dengue virus host adaptation leads to the accumulation of different species of sfRNAs in vertebrate and invertebrate cells. This process does not depend on differences in the host machinery; but it was found to be dependent on the selection of specific mutations in the viral 3'UTR. Dissecting the viral population and studying phenotypes of cloned variants, the molecular determinants for the switch in the sfRNA pattern during host change were mapped to a single RNA structure. Point mutations selected in mosquito cells were sufficient to change the pattern of sfRNAs, induce higher type I interferon responses and reduce viral fitness in human cells, explaining the rapid clearance of certain viral variants after host change. In addition, using epidemic and pre-epidemic Zika viruses, similar patterns of sfRNAs were observed in mosquito and human infected cells, but they were different from those observed during dengue virus infections, indicating that distinct selective pressures act on the 3'UTR of these closely related viruses. In summary, we present a novel mechanism by which dengue virus evolved an RNA structure that is under strong selective pressure in the two hosts, as regulator of non-coding RNA accumulation and viral fitness. This work provides new ideas about the impact of host adaptation on the variability and evolution of

  19. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Sujun [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China); Southern Medical University, Guangzhou, Guangdong 510515 (China); Wu, Binwen, E-mail: wubinwengd@aliyun.com [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China); Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China)

    2014-02-14

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2{sup ∗}, -193b and -193a, and inversely inhibit miR-31 and -9{sup ∗}. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.

  20. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    International Nuclear Information System (INIS)

    Huang, Sujun; Wu, Binwen; Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia

    2014-01-01

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2 ∗ , -193b and -193a, and inversely inhibit miR-31 and -9 ∗ . Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC

  1. Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites

    International Nuclear Information System (INIS)

    Bodaghi, Sohrab; Jia Rong; Zheng Zhiming

    2009-01-01

    Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection

  2. Forging our understanding of lncRNAs in the brain.

    Science.gov (United States)

    Andersen, Rebecca E; Lim, Daniel A

    2018-01-01

    During both development and adulthood, the human brain expresses many thousands of long noncoding RNAs (lncRNAs), and aberrant lncRNA expression has been associated with a wide range of neurological diseases. Although the biological significance of most lncRNAs remains to be discovered, it is now clear that certain lncRNAs carry out important functions in neurodevelopment, neural cell function, and perhaps even diseases of the human brain. Given the relatively inclusive definition of lncRNAs-transcripts longer than 200 nucleotides with essentially no protein coding potential-this class of noncoding transcript is both large and very diverse. Furthermore, emerging data indicate that lncRNA genes can act via multiple, non-mutually exclusive molecular mechanisms, and specific functions are difficult to predict from lncRNA expression or sequence alone. Thus, the different experimental approaches used to explore the role of a lncRNA might each shed light upon distinct facets of its overall molecular mechanism, and combining multiple approaches may be necessary to fully illuminate the function of any particular lncRNA. To understand how lncRNAs affect brain development and neurological disease, in vivo studies of lncRNA function are required. Thus, in this review, we focus our discussion upon a small set of neural lncRNAs that have been experimentally manipulated in mice. Together, these examples illustrate how studies of individual lncRNAs using multiple experimental approaches can help reveal the richness and complexity of lncRNA function in both neurodevelopment and diseases of the brain.

  3. Structural dissection of human metapneumovirus phosphoprotein using small angle x-ray scattering.

    Science.gov (United States)

    Renner, Max; Paesen, Guido C; Grison, Claire M; Granier, Sébastien; Grimes, Jonathan M; Leyrat, Cédric

    2017-11-01

    The phosphoprotein (P) is the main and essential cofactor of the RNA polymerase (L) of non-segmented, negative-strand RNA viruses. P positions the viral polymerase onto its nucleoprotein-RNA template and acts as a chaperone of the nucleoprotein (N), thereby preventing nonspecific encapsidation of cellular RNAs. The phosphoprotein of human metapneumovirus (HMPV) forms homotetramers composed of a stable oligomerization domain (P core ) flanked by large intrinsically disordered regions (IDRs). Here we combined x-ray crystallography of P core with small angle x-ray scattering (SAXS)-based ensemble modeling of the full-length P protein and several of its fragments to provide a structural description of P that captures its dynamic character, and highlights the presence of varyingly stable structural elements within the IDRs. We discuss the implications of the structural properties of HMPV P for the assembly and functioning of the viral transcription/replication machinery.

  4. 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

  5. Identification of latent tuberculosis infection-related microRNAs in human U937 macrophages expressing Mycobacterium tuberculosis Hsp16.3.

    Science.gov (United States)

    Meng, Qing-Lin; Liu, Fei; Yang, Xing-Yuan; Liu, Xiao-Mei; Zhang, Xia; Zhang, Chun; Zhang, Zong-De

    2014-02-12

    Latent tuberculosis infection (LTBI) relies on a homeostasis of macrophages and Mycobacterium tuberculosis (Mtb). The small heat shock protein, Mtb Hsp16.3 (also known as latency-associated antigen), plays an important role in Mtb persistence within macrophages. However, the mechanism of LTBI remains elusive. The aim of this study was to delineate LTBI-related miRNA expression in U937 macrophages expressing Mtb Hsp16.3 protein. U937 macrophages were infected with an integrase-deficient Lentivirus vector to transiently express Mtb Hsp16.3, and green fluorescent protein (GFP) as a control. We used a microRNA (miRNA) microarray chip containing more than 1000 probes to identify the significant differentially expressed miRNAs in the infected U937 cells, and employed real-time quantitative polymerase chain reaction (qRT-PCR) for validation. Furthermore, we confirmed these candidate LTBI-related miRNAs in peripheral blood mononuclear cells from subjects with LTBI and in healthy control individuals. Functional annotation prediction of miRNA target genes and pathway enrichment analyses were used to explore the putative links between these miRNAs and LTBI. Analysis of the miRNA expression profile identified 149 miRNAs that were differentially expressed in U937 macrophages expressing Mtb Hsp16.3 compared with the control expressing GFP. The expression level of seven miRNAs (miR-424-5p, miR-493-5p, miR-296-5p, miR-27b-3p, miR-377-5p, miR-3680-5p, miR-191-5p) were validated by qRT-PCR. The expression level of four miRNAs (miR-424-5p, miR-27b-3p, miR-377-5p, miR-3680-5p) in the peripheral blood mononuclear cells samples from LTBI and healthy participants reflected the altered patterns observed in the microarray profile. The bioinformatic analyses suggest that the miRNAs may regulate Mtb latent infection by affecting the development of macrophage cells. The results suggest that miRNA expression may play a considerable role in the pathogenesis of LTBI, and this would increase our

  6. Novel meiotic miRNAs and indications for a role of phasiRNAs in meiosis

    Science.gov (United States)

    Small RNAs (sRNA) add additional layers to the regulation of gene expression, with siRNAs directing gene silencing at the DNA level by RdDM (RNA-directed DNA methylation), and miRNAs directing post-transcriptional regulation of specific target genes, mostly by mRNA cleavage. We used manually isolate...

  7. Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

    Science.gov (United States)

    Furuse, Yuki; Finethy, Ryan; Saka, Hector A; Xet-Mull, Ana M; Sisk, Dana M; Smith, Kristen L Jurcic; Lee, Sunhee; Coers, Jörn; Valdivia, Raphael H; Tobin, David M; Cullen, Bryan R

    2014-01-01

    MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

  8. Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

    Directory of Open Access Journals (Sweden)

    Yuki Furuse

    Full Text Available MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

  9. MicroRNAs associated with exercise and diet: a systematic review.

    Science.gov (United States)

    Flowers, Elena; Won, Gloria Y; Fukuoka, Yoshimi

    2015-01-01

    MicroRNAs are posttranscriptional regulators of gene expression. MicroRNAs reflect individual biologic adaptation to exposures in the environment. As such, measurement of circulating microRNAs presents an opportunity to evaluate biologic changes associated with behavioral interventions (i.e., exercise, diet) for weight loss. The aim of this study was to perform a systematic review of the literature to summarize what is known about circulating microRNAs associated with exercise, diet, and weight loss. We performed a systematic review of three scientific databases. We included studies reporting on circulating microRNAs associated with exercise, diet, and weight loss in humans. Of 1,219 studies identified in our comprehensive database search, 14 were selected for inclusion. Twelve reported on microRNAs associated with exercise, and two reported on microRNAs associated with diet and weight loss. The majority of studies used a quasiexperimental, cross-sectional design. There were numerous differences in the type and intensity of exercise and dietary interventions, the biologic source of microRNAs, and the methodological approaches used quantitate microRNAs. Data from several studies support an association between circulating microRNAs and exercise. The evidence for an association between circulating microRNAs and diet is weaker because of a small number of studies. Additional research is needed to validate previous observations using methodologically rigorous approaches to microRNA quantitation to determine the specific circulating microRNA signatures associated with behavioral approaches to weight loss. Future directions include longitudinal studies to determine if circulating microRNAs are predictive of response to behavioral interventions. Copyright © 2015 the American Physiological Society.

  10. miRNAs in Tuberculosis: New Avenues for Diagnosis and Host-Directed Therapy

    Directory of Open Access Journals (Sweden)

    Naveed Sabir

    2018-03-01

    Full Text Available Tuberculosis (TB is one of the most fatal infectious diseases and a leading cause of mortality, with 95% of these deaths occurring in developing countries. The causative agent, Mycobacterium tuberculosis (Mtb, has a well-established ability to circumvent the host’s immune system for its intracellular survival. microRNAs (miRNAs are small, non-coding RNAs having an important function at the post-transcriptional level and are involved in shaping immunity by regulating the repertoire of genes expressed in immune cells. It has been established in recent studies that the innate immune response against TB is significantly regulated by miRNAs. Moreover, differential expression of miRNA in Mtb infection can reflect the disease progression and may help distinguish between active and latent TB infection (LTBI. These findings encouraged the application of miRNAs as potential biomarkers. Similarly, active participation of miRNAs in modulation of autophagy and apoptosis responses against Mtb opens an exciting avenue for the exploitation of miRNAs as host directed therapy (HDT against TB. Nanoparticles mediated delivery of miRNAs to treat various diseases has been reported and this technology has a great potential to be used in TB. In reality, this exploitation of miRNAs as biomarkers and in HDT is still in its infancy stage, and more studies using animal models mimicking human TB are advocated to assess the role of miRNAs as biomarkers and therapeutic targets. In this review, we attempt to summarize the recent advancements in the role of miRNAs in TB as immune modulator, miRNAs’ capability to distinguish between active and latent TB and, finally, usage of miRNAs as therapeutic targets against TB.

  11. Research advances in microRNAs in regulating hepatitis C virus replication and antiviral therapy

    Directory of Open Access Journals (Sweden)

    CUI Xianghua

    2015-05-01

    Full Text Available Hepatitis C virus (HCV infection is one of the most common causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. MicroRNAs (miRNAs, a class of small, non-coding RNA, are involved in a variety of physiological and pathological processes in human bodies. The mechanism by which miRNAs regulate HCV replication is described, and the effects of liver-specific microRNA-122 antagonists on hepatitis C antiviral therapy are discussed. Our study indicates that miRNAs play an important regulatory role in HCV expression. Targeting miRNAs may be a potential therapeutic approach for treating HCV infection, but further studies are still in need.

  12. Long non-coding RNAs: Mechanism of action and functional utility

    Directory of Open Access Journals (Sweden)

    Shakil Ahmad Bhat

    2016-10-01

    Full Text Available Recent RNA sequencing studies have revealed that most of the human genome is transcribed, but very little of the total transcriptomes has the ability to encode proteins. Long non-coding RNAs (lncRNAs are non-coding transcripts longer than 200 nucleotides. Members of the non-coding genome include microRNA (miRNA, small regulatory RNAs and other short RNAs. Most of long non-coding RNA (lncRNAs are poorly annotated. Recent recognition about lncRNAs highlights their effects in many biological and pathological processes. LncRNAs are dysfunctional in a variety of human diseases varying from cancerous to non-cancerous diseases. Characterization of these lncRNA genes and their modes of action may allow their use for diagnosis, monitoring of progression and targeted therapies in various diseases. In this review, we summarize the functional perspectives as well as the mechanism of action of lncRNAs. Keywords: LncRNA, X-chromosome inactivation, Genome imprinting, Transcription regulation, Cancer, Immunity

  13. Genomic Organization of Zebrafish microRNAs

    Directory of Open Access Journals (Sweden)

    Paydar Ima

    2008-05-01

    Full Text Available Abstract Background microRNAs (miRNAs are small (~22 nt non-coding RNAs that regulate cell movement, specification, and development. Expression of miRNAs is highly regulated, both spatially and temporally. Based on direct cloning, sequence conservation, and predicted secondary structures, a large number of miRNAs have been identified in higher eukaryotic genomes but whether these RNAs are simply a subset of a much larger number of noncoding RNA families is unknown. This is especially true in zebrafish where genome sequencing and annotation is not yet complete. Results We analyzed the zebrafish genome to identify the number and location of proven and predicted miRNAs resulting in the identification of 35 new miRNAs. We then grouped all 415 zebrafish miRNAs into families based on seed sequence identity as a means to identify possible functional redundancy. Based on genomic location and expression analysis, we also identified those miRNAs that are likely to be encoded as part of polycistronic transcripts. Lastly, as a resource, we compiled existing zebrafish miRNA expression data and, where possible, listed all experimentally proven mRNA targets. Conclusion Current analysis indicates the zebrafish genome encodes 415 miRNAs which can be grouped into 44 families. The largest of these families (the miR-430 family contains 72 members largely clustered in two main locations along chromosome 4. Thus far, most zebrafish miRNAs exhibit tissue specific patterns of expression.

  14. Association of RNAs with Bacillus subtilis Hfq.

    Directory of Open Access Journals (Sweden)

    Michael Dambach

    Full Text Available The prevalence and characteristics of small regulatory RNAs (sRNAs have not been well characterized for Bacillus subtilis, an important model system for Gram-positive bacteria. However, B. subtilis was recently found to synthesize many candidate sRNAs during stationary phase. In the current study, we performed deep sequencing on Hfq-associated RNAs and found that a small subset of sRNAs associates with Hfq, an enigmatic RNA-binding protein that stabilizes sRNAs in Gram-negatives, but whose role is largely unknown in Gram-positive bacteria. We also found that Hfq associated with antisense RNAs, antitoxin transcripts, and many mRNA leaders. Several new candidate sRNAs and mRNA leader regions were also discovered by this analysis. Additionally, mRNA fragments overlapping with start or stop codons associated with Hfq, while, in contrast, relatively few full-length mRNAs were recovered. Deletion of hfq reduced the intracellular abundance of several representative sRNAs, suggesting that B. subtilis Hfq-sRNA interactions may be functionally significant in vivo. In general, we anticipate this catalog of Hfq-associated RNAs to serve as a resource in the functional characterization of Hfq in B. subtilis.

  15. Organization Context and Human Resource Management in the Small Firm

    NARCIS (Netherlands)

    J.M.P. de Kok (Jan); L.M. Uhlaner (Lorraine)

    2001-01-01

    textabstractThis paper examines the relationship between organization contextual variables and human resource management (HRM) practices in small firms. The proposed model is based on an integration of theoretical perspectives, including the resource-based approach, institutional theory, transaction

  16. A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: Comparative expression profiles of mRNAs, microRNAs and proteins among different feeders and conditioned media

    Directory of Open Access Journals (Sweden)

    Chou Chi-Hsien

    2010-10-01

    Full Text Available Abstract Background Human embryonic stem (hES cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast (MEF feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency. The hES-T3 cell line with normal female karyotype was previously used to differentiate into autogeneic fibroblast-like cells (T3HDF as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF for 14 passages. Results A feeder-free culture on Matrigel in hES medium conditioned by the autogeneic feeder cells (T3HDF was established to maintain the undifferentiated growth of hES-T3 cells (T3/CMHDF for 8 passages in this investigation. The gene expression profiles of mRNAs, microRNAs and proteins between the undifferentiated T3/HDF and T3/CMHDF cells were shown to be very similar, and their expression profiles were also found to be similar to those of T3/MEF and T3/CMMEF cells grown on MEF feeder and feeder-free Matrigel in MEF-conditioned medium, respectively. The undifferentiated state of T3/HDF and T3/CMHDF as well as T3/MEF andT3/CMMEF cells was evidenced by the very high expression levels of "stemness" genes and low expression levels of differentiation markers of ectoderm, mesoderm and endoderm in addition to the strong staining of OCT4 and NANOG. Conclusion The T3HDF feeder and T3HDF-conditioned medium were able to support the undifferentiated growth of hES cells, and they would be useful for drug development and toxicity testing in addition to the reduced risks of xenogeneic pathogens when used for medical applications such as cell therapies.

  17. Moderating effect of innovation on human capital and small firm ...

    African Journals Online (AJOL)

    This study evaluate the importance of human capital on the performance of small firms in the construction sector in developing countries. Primary data was obtained from 255 small contractors in the construction sector in the state of Kelantan, Malaysia. Analysis of the study was carried out through descriptive and multiple ...

  18. RNA-Seq of human neurons derived from iPS cells reveals candidate long non-coding RNAs involved in neurogenesis and neuropsychiatric disorders.

    Directory of Open Access Journals (Sweden)

    Mingyan Lin

    Full Text Available Genome-wide expression analysis using next generation sequencing (RNA-Seq provides an opportunity for in-depth molecular profiling of fundamental biological processes, such as cellular differentiation and malignant transformation. Differentiating human neurons derived from induced pluripotent stem cells (iPSCs provide an ideal system for RNA-Seq since defective neurogenesis caused by abnormalities in transcription factors, DNA methylation, and chromatin modifiers lie at the heart of some neuropsychiatric disorders. As a preliminary step towards applying next generation sequencing using neurons derived from patient-specific iPSCs, we have carried out an RNA-Seq analysis on control human neurons. Dramatic changes in the expression of coding genes, long non-coding RNAs (lncRNAs, pseudogenes, and splice isoforms were seen during the transition from pluripotent stem cells to early differentiating neurons. A number of genes that undergo radical changes in expression during this transition include candidates for schizophrenia (SZ, bipolar disorder (BD and autism spectrum disorders (ASD that function as transcription factors and chromatin modifiers, such as POU3F2 and ZNF804A, and genes coding for cell adhesion proteins implicated in these conditions including NRXN1 and NLGN1. In addition, a number of novel lncRNAs were found to undergo dramatic changes in expression, one of which is HOTAIRM1, a regulator of several HOXA genes during myelopoiesis. The increase we observed in differentiating neurons suggests a role in neurogenesis as well. Finally, several lncRNAs that map near SNPs associated with SZ in genome wide association studies also increase during neuronal differentiation, suggesting that these novel transcripts may be abnormally regulated in a subgroup of patients.

  19. On-the-fly selection of cell-specific enhancers, genes, miRNAs and proteins across the human body using SlideBase

    DEFF Research Database (Denmark)

    Ienasescu, Hans-Ioan; Li, Kang; Andersson, Robin

    2016-01-01

    Genomics consortia have produced large datasets profiling the expression of genes, micro-RNAs, enhancers and more across human tissues or cells. There is a need for intuitive tools to select subsets of such data that is the most relevant for specific studies. To this end, we present Slide...... for individual cell types/tissues, producing sets of genes, enhancers etc. which satisfy these constraints. Changes in slider settings result in simultaneous changes in the selected sets, updated in real time. SlideBase is linked to major databases from genomics consortia, including FANTOM, GTEx, The Human...

  20. High-Throughput Sequencing of Small RNA Transcriptomes in Maize Kernel Identifies miRNAs Involved in Embryo and Endosperm Development.

    Science.gov (United States)

    Xing, Lijuan; Zhu, Ming; Zhang, Min; Li, Wenzong; Jiang, Haiyang; Zou, Junjie; Wang, Lei; Xu, Miaoyun

    2017-12-14

    Maize kernel development is a complex biological process that involves the temporal and spatial expression of many genes and fine gene regulation at a transcriptional and post-transcriptional level, and microRNAs (miRNAs) play vital roles during this process. To gain insight into miRNA-mediated regulation of maize kernel development, a deep-sequencing technique was used to investigate the dynamic expression of miRNAs in the embryo and endosperm at three developmental stages in B73. By miRNA transcriptomic analysis, we characterized 132 known miRNAs and six novel miRNAs in developing maize kernel, among which, 15 and 14 miRNAs were commonly differentially expressed between the embryo and endosperm at 9 days after pollination (DAP), 15 DAP and 20 DAP respectively. Conserved miRNA families such as miR159, miR160, miR166, miR390, miR319, miR528 and miR529 were highly expressed in developing embryos; miR164, miR171, miR393 and miR2118 were highly expressed in developing endosperm. Genes targeted by those highly expressed miRNAs were found to be largely related to a regulation category, including the transcription, macromolecule biosynthetic and metabolic process in the embryo as well as the vitamin biosynthetic and metabolic process in the endosperm. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that these miRNAs displayed a negative correlation with the levels of their corresponding target genes. Importantly, our findings revealed that members of the miR169 family were highly and dynamically expressed in the developing kernel, which will help to exploit new players functioning in maize kernel development.

  1. 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

  2. MicroRNAs in Breast Cancer: One More Turn in Regulation.

    Science.gov (United States)

    Eroles, Pilar; Asensio, Pilar E; Tormo, Eduardo; Martin, Eduardo T; Pineda, Begoña; Merlo, Begoña P; Espin, Estefanía; Armas, Estefanía E; Lluch, Ana; Hernández, Ana L

    2016-01-01

    MicroRNAs (miRNAs) are small non-coding RNA molecules that critically regulate the expression of genes. MiRNAs are involved in physiological cellular processes; however, their deregulation has been associated with several pathologies, including cancer. In human breast cancer, differently expressed levels of miRNAs have been identified from those in normal breast tissues. Moreover, several miRNAs have been correlated with pathological phenotype, cancer subtype and therapy response in breast cancer. The resistance to therapy is increasingly a problem in patient management, and miRNAs are emerging as novel therapeutic targets and potential predictive biomarkers for treatment. This review provides an overview of the current situation of miRNAs in breast cancer, focusing on their involvement in resistance and the circulating miRNA. The mechanisms of therapeutic resistance regulated by miRNAs, such as the regulation of receptors, the modification of enzymes of drug metabolism, the inhibition of cell cycle control or pro-apoptotic proteins, the alteration of histone activity and the regulation of DNA repair machinery among others, are discussed for breast cancer clinical subtypes. Additionally, in this review, we summarize the recent knowledge that has established miRNA detection in peripheral body fluids as a suitable biomarker. We review the detection of miRNA in liquid biopsies and its implications for the diagnosis and monitoring of breast cancer. This new generation of cancer biomarkers may lead to a significant improvement in patient management.

  3. MicroRNAs in the host response to viral infections of veterinary importance

    Directory of Open Access Journals (Sweden)

    Mohamed Samir Ahmed

    2016-10-01

    Full Text Available The discovery of small regulatory non-coding RNAs has been an exciting advance in the field of genomics. MicroRNAs (miRNAs are endogenous RNA molecules, approximately 22 nucleotides in length that regulate gene expression, mostly at the post-transcriptional level. MiRNA profiling technologies have made it possible to identify and quantify novel miRNAs and to study their regulation and potential roles in disease pathogenesis. Although miRNAs have been extensively investigated in viral infections of humans, their implications in viral diseases affecting animals of veterinary importance are much less understood. The number of annotated miRNAs in different animal species is growing continuously, and novel roles in regulating host-pathogen interactions are being discovered, for instance miRNA-mediated augmentation of viral transcription and replication. In this review, we present an overview of synthesis and function of miRNAs and an update on the current state of research on host-encoded miRNAs in the genesis of viral infectious diseases in their natural animal host as well as in selected in vivo and in vitro laboratory models.

  4. A Regulatory MDM4 Genetic Variant Locating in the Binding Sequence of Multiple MicroRNAs Contributes to Susceptibility of Small Cell Lung Cancer.

    Directory of Open Access Journals (Sweden)

    Feng Gao

    Full Text Available A functional rs4245739 A>C single nucleotide polymorphism (SNP locating in the MDM43'-untranslated (3'-UTR region creates a miR-191-5p or miR-887-3p targeting sites. This change results in decreased expression of oncogene MDM4. Therefore, we examined the association between this SNP and small cell lung cancer (SCLC risk as well as its regulatory function in SCLC cells. Genotypes were determined in two independent case-control sets consisted of 520SCLC cases and 1040 controls from two regions of China. Odds ratios (ORs and 95% confidence intervals (CIs were estimated by logistic regression. The impact of the rs4245739 SNP on miR-191-5p/miR-887-3p mediated MDM4 expression regulation was investigated using luciferase reporter gene assays. We found that the MDM4 rs4245739AC and CC genotypes were significantly associated with decreased SCLC susceptibility compared with the AA genotype in both case-control sets (Shandong set: OR = 0.53, 95% CI = 0.32-0.89, P = 0.014; Jiangsu set: OR = 0.47, 95% CI = 0.26-0.879, P = 0.017. Stratified analyses indicated that there was a significantly multiplicative interaction between rs4245739 and smoking (Pinteractioin = 0.048. After co-tranfection of miRNAs and different allelic-MDM4 reporter constructs into SCLC cells, we found that the both miR-191-5p and miR-887-3p can lead to significantly decreased MDM4 expression activities in the construct with C-allelic 3'-UTR but not A-allelic 3'-UTR, suggesting a consistent genotype-phenotype correlation. Our data illuminate that the MDM4rs4245739SNP contributes to SCLC risk and support the notion that gene 3'-UTR genetic variants, impacting miRNA-binding, might modify SCLC susceptibility.

  5. Gene function analysis by artificial microRNAs in Physcomitrella patens.

    KAUST Repository

    Khraiwesh, Basel; Fattash, Isam; Arif, Muhammad Asif; Frank, Wolfgang

    2011-01-01

    MicroRNAs (miRNAs) are ~21 nt long small RNAs transcribed from endogenous MIR genes which form precursor RNAs with a characteristic hairpin structure. miRNAs control the expression of cognate target genes by binding to reverse complementary

  6. Annotation of mammalian primary microRNAs

    Directory of Open Access Journals (Sweden)

    Enright Anton J

    2008-11-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are important regulators of gene expression and have been implicated in development, differentiation and pathogenesis. Hundreds of miRNAs have been discovered in mammalian genomes. Approximately 50% of mammalian miRNAs are expressed from introns of protein-coding genes; the primary transcript (pri-miRNA is therefore assumed to be the host transcript. However, very little is known about the structure of pri-miRNAs expressed from intergenic regions. Here we annotate transcript boundaries of miRNAs in human, mouse and rat genomes using various transcription features. The 5' end of the pri-miRNA is predicted from transcription start sites, CpG islands and 5' CAGE tags mapped in the upstream flanking region surrounding the precursor miRNA (pre-miRNA. The 3' end of the pri-miRNA is predicted based on the mapping of polyA signals, and supported by cDNA/EST and ditags data. The predicted pri-miRNAs are also analyzed for promoter and insulator-associated regulatory regions. Results We define sets of conserved and non-conserved human, mouse and rat pre-miRNAs using bidirectional BLAST and synteny analysis. Transcription features in their flanking regions are used to demarcate the 5' and 3' boundaries of the pri-miRNAs. The lengths and boundaries of primary transcripts are highly conserved between orthologous miRNAs. A significant fraction of pri-miRNAs have lengths between 1 and 10 kb, with very few introns. We annotate a total of 59 pri-miRNA structures, which include 82 pre-miRNAs. 36 pri-miRNAs are conserved in all 3 species. In total, 18 of the confidently annotated transcripts express more than one pre-miRNA. The upstream regions of 54% of the predicted pri-miRNAs are found to be associated with promoter and insulator regulatory sequences. Conclusion Little is known about the primary transcripts of intergenic miRNAs. Using comparative data, we are able to identify the boundaries of a significant proportion of

  7. The Impact of MicroRNAs on Brain Aging and Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Stephan P. Persengiev

    2012-01-01

    Full Text Available The molecular instructions that govern gene expression regulation are encoded in the genome and ultimately determine the morphology and functional specifications of the human brain. As a consequence, changes in gene expression levels might be directly related to the functional decline associated with brain aging. Small noncoding RNAs, including miRNAs, comprise a group of regulatory molecules that modulate the expression of hundred of genes which play important roles in brain metabolism. Recent comparative studies in humans and nonhuman primates revealed that miRNAs regulate multiple pathways and interconnected signaling cascades that are the basis for the cognitive decline and neurodegenerative disorders during aging. Identifying the roles of miRNAs and their target genes in model organisms combined with system-level studies of the brain would provide more comprehensive understanding of the molecular basis of brain deterioration during the aging process.

  8. Noncoding RNAs in Cancer Medicine

    Directory of Open Access Journals (Sweden)

    Laura Cerchia

    2006-01-01

    Full Text Available Several signalling proteins involved in cell growth and differentiation represent attractive candidate targets for cancer diagnosis and/or therapy since they can act as oncogenes. Because of their high specificity and low immunogeneicity, using artificial small noncoding RNA (ncRNAs as therapeutics has recently become a highly promising and rapidly expanding field of interest. Indeed, ncRNAs may either interfere with RNA transcription, stability, translation or directly hamper the function of the targets by binding to their surface. The recent finding that the expression of several genes is under the control of small single-stranded regulatory RNAs, including miRNAs, makes these genes as appropriate targets for ncRNA gene silencing. Furthermore, another class of small ncRNA, aptamers, act as high-affinity ligands and potential antagonists of disease-associated proteins. We will review here the recent and innovative methods that have been developed and the possible applications of ncRNAs as inhibitors or tracers in cancer medicine.

  9. Small RNA Sequencing Reveals Dlk1-Dio3 Locus-Embedded MicroRNAs as Major Drivers of Ground-State Pluripotency.

    Science.gov (United States)

    Moradi, Sharif; Sharifi-Zarchi, Ali; Ahmadi, Amirhossein; Mollamohammadi, Sepideh; Stubenvoll, Alexander; Günther, Stefan; Salekdeh, Ghasem Hosseini; Asgari, Sassan; Braun, Thomas; Baharvand, Hossein

    2017-12-12

    Ground-state pluripotency is a cell state in which pluripotency is established and maintained through efficient repression of endogenous differentiation pathways. Self-renewal and pluripotency of embryonic stem cells (ESCs) are influenced by ESC-associated microRNAs (miRNAs). Here, we provide a comprehensive assessment of the "miRNome" of ESCs cultured under conditions favoring ground-state pluripotency. We found that ground-state ESCs express a distinct set of miRNAs compared with ESCs grown in serum. Interestingly, most "ground-state miRNAs" are encoded by an imprinted region on chromosome 12 within the Dlk1-Dio3 locus. Functional analysis revealed that ground-state miRNAs embedded in the Dlk1-Dio3 locus (miR-541-5p, miR-410-3p, and miR-381-3p) promoted pluripotency via inhibition of multi-lineage differentiation and stimulation of self-renewal. Overall, our results demonstrate that ground-state pluripotency is associated with a unique miRNA signature, which supports ground-state self-renewal by suppressing differentiation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  10. MicroRNAs in Breastmilk and the Lactating Breast: Potential Immunoprotectors and Developmental Regulators for the Infant and the Mother

    Directory of Open Access Journals (Sweden)

    Mohammed Alsaweed

    2015-10-01

    Full Text Available Human milk (HM is the optimal source of nutrition, protection and developmental programming for infants. It is species-specific and consists of various bioactive components, including microRNAs, small non-coding RNAs regulating gene expression at the post-transcriptional level. microRNAs are both intra- and extra-cellular and are present in body fluids of humans and animals. Of these body fluids, HM appears to be one of the richest sources of microRNA, which are highly conserved in its different fractions, with milk cells containing more microRNAs than milk lipids, followed by skim milk. Potential effects of exogenous food-derived microRNAs on gene expression have been demonstrated, together with the stability of milk-derived microRNAs in the gastrointestinal tract. Taken together, these strongly support the notion that milk microRNAs enter the systemic circulation of the HM fed infant and exert tissue-specific immunoprotective and developmental functions. This has initiated intensive research on the origin, fate and functional significance of milk microRNAs. Importantly, recent studies have provided evidence of endogenous synthesis of HM microRNA within the human lactating mammary epithelium. These findings will now form the basis for investigations of the role of microRNA in the epigenetic control of normal and aberrant mammary development, and particularly lactation performance.

  11. miRNAs in brain development

    International Nuclear Information System (INIS)

    Petri, Rebecca; Malmevik, Josephine; Fasching, Liana; Åkerblom, Malin; Jakobsson, Johan

    2014-01-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. In the brain, a large number of miRNAs are expressed and there is a growing body of evidence demonstrating that miRNAs are essential for brain development and neuronal function. Conditional knockout studies of the core components in the miRNA biogenesis pathway, such as Dicer and DGCR8, have demonstrated a crucial role for miRNAs during the development of the central nervous system. Furthermore, mice deleted for specific miRNAs and miRNA-clusters demonstrate diverse functional roles for different miRNAs during the development of different brain structures. miRNAs have been proposed to regulate cellular functions such as differentiation, proliferation and fate-determination of neural progenitors. In this review we summarise the findings from recent studies that highlight the importance of miRNAs in brain development with a focus on the mouse model. We also discuss the technical limitations of current miRNA studies that still limit our understanding of this family of non-coding RNAs and propose the use of novel and refined technologies that are needed in order to fully determine the impact of specific miRNAs in brain development. - Highlights: • miRNAs are essential for brain development and neuronal function. • KO of Dicer is embryonically lethal. • Conditional Dicer KO results in defective proliferation or increased apoptosis. • KO of individual miRNAs or miRNA families is necessary to determine function

  12. Locking-to-unlocking system is an efficient strategy to design DNA/silver nanoclusters (AgNCs) probe for human miRNAs

    DEFF Research Database (Denmark)