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Sample records for interfering rnas designed

  1. Design of functional small interfering RNAs targeting amyotrophic lateral sclerosis-associated mutant alleles

    Institute of Scientific and Technical Information of China (English)

    GENG Chang-ming; DING Hong-liu

    2011-01-01

    Background RNA interference (RNAi) is a potential cure for amyotrophic lateral sclerosis (ALS) caused by dominant,gain-of-function superoxide dismutase 1 (SOD1) mutations. The success of such therapy relies on the functional small interfering RNAs (siRNAs) that can effectively deliver RNAi. This study aimed to design the functional siRNAs targeting ALS-associated mutant alleles.Methods A modified dual luciferase system containing human SOD1 mRNA target was established to quantify siRNA efficacy. Coupled with validated siRNAs identified in the literature, we analyzed the rationale of siRNA design and subsequently developed an asymmetry rule-based strategy for designing siRNA. We then further tested the effectiveness of this design strategy in converting a naturally symmetric siRNA into functional siRNAs with favorable asymmetry for gene silencing of SOD1 alleles.Results The efficacies of siRNAs could vary tremendously by one base-pair position change. Functional siRNAs could target the whole span of SOD1 mRNA coding sequence as well as non-coding region. While there is no distinguishable pattern of the distribution of nucleobases in these validated siRNAs, the high percent of GC count at the last two positions of siRNAs (P18 and P19) indicated a strong effect of asymmetry rule. Introducing a mismatch at position 1 of the 5' of antisense strand of siRNA successfully converted the inactive siRNA into functional siRNAs that silence SOD1 with desired efficacy.Conclusions Asymmetry rule-based strategy that incorporates a mismatch into siRNA most consistently enhances RNAi efficacy and guarantees producing functional siRNAs that successfully silence ALS-associated SOD1 mutant alleles regardless target positions. This strategy could also be useful to design siRNAs for silencing other disease-associated dominant, gain-of-function mutant genes.

  2. Interfering Satellite RNAs of Bamboo mosaic virus

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    Kuan-Yu Lin

    2017-05-01

    Full Text Available Satellite RNAs (satRNAs are sub-viral agents that may interact with their cognate helper virus (HV and host plant synergistically and/or antagonistically. SatRNAs totally depend on the HV for replication, so satRNAs and HV usually evolve similar secondary or tertiary RNA structures that are recognized by a replication complex, although satRNAs and HV do not share an appreciable sequence homology. The satRNAs of Bamboo mosaic virus (satBaMV, the only satRNAs of the genus Potexvirus, have become one of the models of how satRNAs can modulate HV replication and virus-induced symptoms. In this review, we summarize the molecular mechanisms underlying the interaction of interfering satBaMV and BaMV. Like other satRNAs, satBaMV mimics the secondary structures of 5′- and 3′-untranslated regions (UTRs of BaMV as a molecular pretender. However, a conserved apical hairpin stem loop (AHSL in the 5′-UTR of satBaMV was found as the key determinant for downregulating BaMV replication. In particular, two unique nucleotides (C60 and C83 in the AHSL of satBaMVs determine the satBaMV interference ability by competing for the replication machinery. Thus, transgenic plants expressing interfering satBaMV could confer resistance to BaMV, and interfering satBaMV could be used as biological-control agent. Unlike two major anti-viral mechanisms, RNA silencing and salicylic acid-mediated immunity, our findings in plants by in vivo competition assay and RNA deep sequencing suggested replication competition is involved in this transgenic satBaMV-mediated BaMV interference. We propose how a single nucleotide of satBaMV can make a great change in BaMV pathogenicity and the underlying mechanism.

  3. Micro RNAs and Short-interfering RNAs in Plants

    Institute of Scientific and Technical Information of China (English)

    Ramanjulu Sunkar; Jian-Kang Zhu

    2007-01-01

    Gene silencing can occur either at the transcriptional level or post-transcriptional level or both. Many instances of sequence-specific silencing requires small RNAs that can be divided into two major classes: microRNAs (miRNAs) and short-interfering RNAs (siRNAs). miRNAs function in post-transcriptional gene silencing by guiding mRNA degradation or translational repression. Endogenous siRNAs are more diverse in plants than in animals and can direct post-transcriptional gene silencing through mRNA degradation or transcriptional gene silencing by triggering DNA methylation and histone modifications. This review discusses recent advances in the field of small RNA-guided gene silencing in plants including rice.

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

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    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.

  5. Therapeutic potential of small interfering RNAs/micro interfering RNA in hepatocellular carcinoma.

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    Farra, Rossella; Grassi, Mario; Grassi, Gabriele; Dapas, Barbara

    2015-08-14

    Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective, especially for the advanced forms of the disease. In the last year, short double stranded RNA molecules termed small interfering RNAs (siRNAs) and micro interfering RNAs (miRNA), emerged as interesting molecules with potential therapeutic value for HCC. The practical use of these molecules is however limited by the identification of optimal molecular targets and especially by the lack of effective and targeted HCC delivery systems. Here we focus our discussion on the most recent advances in the identification of siRNAs/miRNAs molecular targets and on the development of suitable siRNA/miRNAs delivery systems.

  6. Delivery of Small Interfering RNAs to Cells via Exosomes.

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    Wahlgren, Jessica; Statello, Luisa; Skogberg, Gabriel; Telemo, Esbjörn; Valadi, Hadi

    2016-01-01

    Exosomes are small membrane bound vesicles between 30 and 100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. Exosomes play a role in intercellular communication by transferring proteins, lipids, and RNAs to recipient cells.Exosomes from human cells could be used as vectors to provide cells with therapeutic RNAs. Here we describe how exogenous small interfering RNAs may successfully be introduced into various kinds of human exosomes using electroporation and subsequently delivered to recipient cells. Methods used to confirm the presence of siRNA inside exosomes and cells are presented, such as flow cytometry, confocal microscopy, and Northern blot.

  7. 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

    ). But evidence of specific siRNA inhibition in living fish is still needed. Using the small interfering RNAs (siRNAs), messenger RNA (mRNA) can be targeted resulting in degradation of targeted transcript or translational repression. Reporter genes such as luciferase and green fluorescence protein (GFP) can......RNA interference is a mechanism for silencing specific genes. It has been applied in cell culture to inhibit expression of genes involved in disease including viral genes as recently shown for the fish pathogenic rhabdovirus viral haemorrhagic septicaemia virus or VHSV (Bohle et al., 2011...... 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...

  8. 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

    ). But evidence of specific siRNA inhibition in living fish is still needed. Using the small interfering RNAs (siRNAs), messenger RNA (mRNA) can be targeted resulting in degradation of targeted transcript or translational repression. Reporter genes such as luciferase and green fluorescence protein (GFP) can......RNA interference is a mechanism for silencing specific genes. It has been applied in cell culture to inhibit expression of genes involved in disease including viral genes as recently shown for the fish pathogenic rhabdovirus viral haemorrhagic septicaemia virus or VHSV (Bohle et al., 2011...... 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...

  9. Development of Therapeutic-Grade Small Interfering RNAs by Chemical Engineering.

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    Bramsen, Jesper B; Kjems, Jørgen

    2012-01-01

    Recent successes in clinical trials have provided important proof of concept that small interfering RNAs (siRNAs) indeed constitute a new promising class of therapeutics. Although great efforts are still needed to ensure efficient means of delivery in vivo, the siRNA molecule itself has been successfully engineered by chemical modification to meet initial challenges regarding specificity, stability, and immunogenicity. To date, a great wealth of siRNA architectures and types of chemical modification are available for promoting safe siRNA-mediated gene silencing in vivo and, consequently, the choice of design and modification types can be challenging to individual experimenters. Here we review the literature and devise how to improve siRNA performance by structural design and specific chemical modification to ensure potent and specific gene silencing without unwarranted side-effects and hereby complement the ongoing efforts to improve cell targeting and delivery by other carrier molecules.

  10. Effect of small interfering RNAs on matrix metalloproteinase 1 expression

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    Gen-Hung Chen

    2014-12-01

    Full Text Available Three small double strand siRNAs (506-MMP1, 859-MMP1 and 891-MMP1, each contains 25–26 nucleotides, with high specific to human MMP1 were designed according to mRNA sequence of human MMP1 (NCBI, NM_002421. To monitor the MMP1 gene expression, the total RNAs of human skin fibroblast (Detroit 551, BCRC 60118 were extracted. One human matrix metalloproteinase 1 (MMP1 partial sequence cDNA, included all the three siRNA target sequences, amplified specifically via RT-PCR and PCR reactions, and three synthesized siRNA target DNAs were cloned individually into pAcGFP1-N3 with green fluorescent protein (GFP. These reporter plasmids were then transfected individually into malignant melanoma (MeWo, BCRC 60540 and the GFP was detected after 48 h. Fluorescence results indicated that the 859 siRNA revealed highest inhibitory ability (almost 90%, and was, accordingly, transfected into MeWo cells. According to the real-time quantitative PCR and western blot, the exhibition ability to silence MMP1 gene expression was 85–89%.

  11. Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs.

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    Mutisya, Daniel; Selvam, Chelliah; Lunstad, Benjamin D; Pallan, Pradeep S; Haas, Amanda; Leake, Devin; Egli, Martin; Rozners, Eriks

    2014-06-01

    RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3'-CH2-CO-NH-5' amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P-OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5'-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications.

  12. In vitro inhibition of field isolates of feline calicivirus with short interfering RNAs (siRNAs).

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    McDonagh, Phillip; Sheehy, Paul A; Fawcett, Anne; Norris, Jacqueline M

    2015-05-15

    Feline calicivirus (FCV) is a common infection of domestic cats. Most infections are mild and self-limiting; however more severe disease manifestations, such as FCV-associated virulent systemic disease, may be associated with significant morbidity and mortality. There is currently a lack of effective antiviral treatments for these disease manifestations. In this study, a panel of eight siRNAs were designed to target four conserved regions of the FCV genome. siRNAs were screened for in vitro antiviral efficacy against the reference strain FCV F9 by determination of extracellular virus titres and morphological assessment of protection from cytopathic effect. Three of the siRNA (FCV3.7, FCV4.1, and FCV4.2) demonstrated a marked antiviral effect with a greater than 99% reduction in extracellular viral titre. Titration of these effective siRNAs demonstrated a clear concentration-response relationship, with IC50 values of approximately 1 nM, and combination treatment with multiple siRNAs demonstrated additive or synergistic effects. To assess the potential usefulness of the compounds in a clinical setting, siRNAs were screened against a panel of six recent Australian FCV isolates from cats with FCV-related disease. The siRNAs shown to be effective against the reference strain FCV F9 were active against the majority of the isolates tested, although some variability was noted. Taken together these data suggest potential therapeutic application of antiviral RNAi for treating FCV-associated disease in cats. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression

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    CHEN Zhe; XU Ze-feng; YE Jing-jia; YAO Hang-ping; ZHENG Shu; DING Jia-yi

    2005-01-01

    Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region ofHBV genome were designed and chemically synthesized.(2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR(Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs(siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs.Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

  14. In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs

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    Martínez de Alba, Angel Emilio; Moreno, Ana Beatriz; Gabriel, Marc; Mallory, Allison C.; Christ, Aurélie; Bounon, Rémi; Balzergue, Sandrine; Aubourg, Sebastien; Gautheret, Daniel; Crespi, Martin D.; Vaucheret, Hervé; Maizel, Alexis

    2015-01-01

    Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5′ cap structure and their subsequent 5′ to 3′ degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs. PMID:25694514

  15. Transfer and Expression of Small Interfering RNAs in Mammalian Cells Using Lentiviral Vectors.

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    Lebedev, T D; Spirin, P V; Prassolov, V S

    2013-04-01

    RNA interference is a convenient tool for modulating gene expression. The widespread application of RNA interference is made difficult because of the imperfections of the methods used for efficient target cell delivery of whatever genes are under study. One of the most convenient and efficient gene transfer and expression systems is based on the use of lentiviral vectors, which direct the synthesis of small hairpin RNAs (shRNAs), the precursors of siRNAs. The application of these systems enables one to achieve sustainable and long-term shRNA expression in cells. This review considers the adaptation of the processing of artificial shRNA to the mechanisms used by cellular microRNAs and simultaneous expression of several shRNAs as potential approaches for producing lentiviral vectors that direct shRNA synthesis. Approaches to using RNA interference for the treatment of cancer, as well as hereditary and viral diseases, are under active development today. The improvement made to the methods for constructing lentiviral vectors and the investigation into the mechanisms of processing of small interfering RNA allow one to now consider lentiviral vectors that direct shRNA synthesis as one of the most promising tools for delivering small interfering RNAs.

  16. De novo generation of defective interfering-like RNAs in broad bean mottle bromovirus.

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    Pogany, J; Romero, J; Huang, Q; Sgro, J Y; Shang, H; Bujarski, J J

    1995-10-01

    Broad been mottle virus (BBMV) is the only member of the bromoviruses that is known to accumulate defective-interfering (DI) RNAs (Romero et al., Virology 194, 576-584, 1993). De novo generation of DI-like RNAs was demonstrated during serial passages of BBMV in broad bean using either DI RNA-free virion RNA preparations or transcribed genomic RNA inocula. As for previously described DI RNAs, all but one of the characterized de novo generated DI-like RNAs were derived by a single in-frame deletion from the RNA2 component. The sole exception was derived by two shorter in-frame deletions from RNA2. The maintenance of an open reading frame by all DI-like RNAs suggests the importance of coding capacity and/or the shortened 2a protein in the accumulation of these RNAs during infection. The deletion junction sites were between nucleotides 1152 and 2366, suggesting that the retained regions are essential for the efficient accumulation of BBMV DI-like RNAs in planta. Short regions of sequence similarity and/or complementarity were revealed at the 5' and 3' junction borders. We speculate that these regions can facilitate DI (DI-like) RNA formation. In addition to DI-like RNAs, the full-length nucleotide sequences of RNA2 components of the Type and Morocco strains of BBMV are presented.

  17. Evaluation of short-interfering RNAs treatment in experimental rabies due to wild-type virus

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    Camila Michele Appolinario

    2015-10-01

    Full Text Available ABSTRACTWe have evaluated the efficacy of short-interfering RNAs targeting the nucleoprotein gene and also the brain immune response in treated and non-treated infected mice. Mice were inoculated with wild-type virus, classified as dog (hv2 or vampire bat (hv3 variants and both groups were treated or leaved as controls. No difference was observed in the lethality rate between treated and non-treated groups, although clinical evaluation of hv2 infected mice showed differences in the severity of clinical disease (p = 0.0006. Evaluation of brain immune response 5 days post-inoculation in treated hv2 group showed no difference among the analyzed genes, whereas after 10 days post-inoculation there was increased expression of 2',5'-oligoadenylate synthetase 1, tumor necrosis factor alpha, interleukin 12, interferon gamma, and C-X-C motif chemokine 10 associated with higher expression of Ngene in the same period (p < 0.0001. In hv2 non-treated group only higher interferon beta expression was found at day 5. The observed differences in results of the immune response genes between treated and non-treated groups is not promising as they had neither impact on mortality nor even a reduction in the expression of N gene in siRNA treated animals. This finding suggests that the use of pre-designed siRNA alone may not be useful in rabies treatment.

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

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    Noah Fahlgren

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

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

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

    2013-01-01

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

  20. Non-Viral Delivery and Therapeutic Application of Small Interfering RNAs.

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    Nikitenko, N A; Prassolov, V S

    2013-07-01

    RNA interference (RNAi) is a powerful method used for gene expression regulation. The increasing knowledge about the molecular mechanism of this phenomenon creates new avenues for the application of the RNAi technology in the treatment of various human diseases. However, delivery of RNA interference mediators, small interfering RNAs (siRNAs), to target cells is a major hurdle. Effective and safe pharmacological use of siRNAs requires carriers that can deliver siRNA to its target site and the development of methods for protection of these fragile molecules from in vivo degradation. This review summarizes various strategies for siRNA delivery, including chemical modification and non-viral approaches, such as the polymer-based, peptide-based, lipid-based techniques, and inorganic nanosystems. The advantages, disadvantages, and prospects for the therapeutic application of these methods are also examined in this paper.

  1. In vitro inhibition of feline coronavirus replication by small interfering RNAs.

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    McDonagh, Phillip; Sheehy, Paul A; Norris, Jacqueline M

    2011-06-01

    Infection with virulent biotypes of feline coronavirus (FCoV) can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. In this study we demonstrate the ability of small interfering RNA (siRNA) mediated RNA interference (RNAi) to inhibit the replication of virulent FCoV strain FIPV WSU 79-1146 in an immortalised feline cell line. A panel of eight synthetic siRNAs targeting four different regions of the FCoV genome were tested for antiviral effects. Efficacy was determined by qRT-PCR of intracellular viral genomic and messenger RNA, TCID50 infectivity assay of extracellular virus, and direct IFA for viral protein expression. All siRNAs demonstrated an inhibitory effect on viral replication in vitro. The two most effective siRNAs, targeting the untranslated 5' leader sequence (L2) and the nucleocapsid gene (N1), resulted in a >95% reduction in extracellular viral titre. Further characterisation of these two siRNAs demonstrated their efficacy when used at low concentrations and in cells challenged with high viral loads. Taken together these findings provide important information for the potential therapeutic application of RNAi in treating FIP.

  2. Delivery of small interfering RNAs in human cervical cancer cells by polyethylenimine-functionalized carbon nanotubes

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    Huang, Yuan-Pin; Lin, I.-Jou; Chen, Chih-Chen; Hsu, Yi-Chiang; Chang, Chi-Chang; Lee, Mon-Juan

    2013-06-01

    Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method. PEI functionalization increased the positive charge on the surface of SWNTs and MWNTs, allowing carbon nanotubes to interact electrostatically with the negatively charged small interfering RNAs (siRNAs) and to serve as nonviral gene delivery reagents. PEI-NH-MWNTs and PEI-NH-SWNTs had a better solubility in water than pristine carbon nanotubes, and further removal of large aggregates by centrifugation produced a stable suspension of reduced particle size and improved homogeneity and dispersity. The amount of grafted PEI estimated by thermogravimetric analysis was 5.08% ( w/ w) and 5.28% ( w/ w) for PEI-NH-SWNTs and PEI-NH-MWNTs, respectively. For the assessment of cytotoxicity, various concentrations of PEI-NH-SWNTs and PEI-NH-MWNTs were incubated with human cervical cancer cells, HeLa-S3, for 48 h. PEI-NH-SWNTs and PEI-NH-MWNTs induced cell deaths in a dose-dependent manner but were less cytotoxic compared to pure PEI. As determined by electrophoretic mobility shift assay, siRNAs directed against glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) were completely associated with PEI-NH-SWNTs or PEI-NH-MWNTs at a PEI-NH-SWNT/siGAPDH or PEI-NH-MWNT/siGAPDH mass ratio of 80:1 or 160:1, respectively. Furthermore, PEI-NH-SWNTs and PEI-NH-MWNTs successfully delivered siGAPDH into HeLa-S3 cells at PEI-NH-SWNT/siGAPDH and PEI-NH-MWNT/siGAPDH mass ratios of 1:1 to 20:1, resulting in suppression of the mRNA level of GAPDH to an extent similar to that of DharmaFECT, a common transfection

  3. Cell type-specific delivery of short interfering RNAs by dye-functionalised theranostic nanoparticles

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    Press, Adrian T.; Traeger, Anja; Pietsch, Christian; Mosig, Alexander; Wagner, Michael; Clemens, Mark G.; Jbeily, Nayla; Koch, Nicole; Gottschaldt, Michael; Bézière, Nicolas; Ermolayev, Volodymyr; Ntziachristos, Vasilis; Popp, Jürgen; Kessels, Michael M.; Qualmann, Britta; Schubert, Ulrich S.; Bauer, Michael

    2014-12-01

    Efficient delivery of short interfering RNAs reflects a prerequisite for the development of RNA interference therapeutics. Here, we describe highly specific nanoparticles, based on near infrared fluorescent polymethine dye-derived targeting moieties coupled to biodegradable polymers. The fluorescent dye, even when coupled to a nanoparticle, mimics a ligand for hepatic parenchymal uptake transporters resulting in hepatobiliary clearance of approximately 95% of the dye within 45 min. Body distribution, hepatocyte uptake and excretion into bile of the dye itself, or dye-coupled nanoparticles can be tracked by intravital microscopy or even non-invasively by multispectral optoacoustic tomography. Efficacy of delivery is demonstrated in vivo using 3-hydroxy-3-methyl-glutaryl-CoA reductase siRNA as an active payload resulting in a reduction of plasma cholesterol levels if siRNA was formulated into dye-functionalised nanoparticles. This suggests that organ-selective uptake of a near infrared dye can be efficiently transferred to theranostic nanoparticles allowing novel possibilities for personalised silencing of disease-associated genes.

  4. Viral small interfering RNAs target host genes to mediate disease symptoms in plants.

    Directory of Open Access Journals (Sweden)

    Neil A Smith

    2011-05-01

    Full Text Available The Cucumber mosaic virus (CMV Y-satellite RNA (Y-Sat has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco. How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide (nt complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants, CHLI expression is dramatically down-regulated. Small RNA sequencing and 5' RACE analyses confirmed that this 22-nt sequence was targeted for mRNA cleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNA interference (RNAi vector targeting CHLI induced Y-Sat-like symptoms. In addition, the symptoms of Y-Sat infection can be completely prevented by transforming tobacco with a silencing-resistant variant of the CHLI gene. These results suggest that siRNA-directed silencing of CHLI is solely responsible for the Y-Sat-induced symptoms. Furthermore, we demonstrate that two Nicotiana species, which do not develop yellowing symptoms upon Y-Sat infection, contain a single nucleotide polymorphism within the siRNA-targeted CHLI sequence. This suggests that the previously observed species specificity of Y-Sat-induced symptoms is due to natural sequence variation in the CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-Sat siRNA. Taken together, these findings provide the first demonstration of small RNA-mediated viral disease symptom production and offer an explanation of the species specificity of the viral disease.

  5. Endogenous, tissue-specific short interfering RNAs silence the chalcone synthase gene family in glycine max seed coats.

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    Tuteja, Jigyasa H; Zabala, Gracia; Varala, Kranthi; Hudson, Matthew; Vodkin, Lila O

    2009-10-01

    Two dominant alleles of the I locus in Glycine max silence nine chalcone synthase (CHS) genes to inhibit function of the flavonoid pathway in the seed coat. We describe here the intricacies of this naturally occurring silencing mechanism based on results from small RNA gel blots and high-throughput sequencing of small RNA populations. The two dominant alleles of the I locus encompass a 27-kb region containing two perfectly repeated and inverted clusters of three chalcone synthase genes (CHS1, CHS3, and CHS4). This structure silences the expression of all CHS genes, including CHS7 and CHS8, located on other chromosomes. The CHS short interfering RNAs (siRNAs) sequenced support a mechanism by which RNAs transcribed from the CHS inverted repeat form aberrant double-stranded RNAs that become substrates for dicer-like ribonuclease. The resulting primary siRNAs become guides that target the mRNAs of the nonlinked, highly expressed CHS7 and CHS8 genes, followed by subsequent amplification of CHS7 and CHS8 secondary siRNAs by RNA-dependent RNA polymerase. Most remarkably, this silencing mechanism occurs only in one tissue, the seed coat, as shown by the lack of CHS siRNAs in cotyledons and vegetative tissues. Thus, production of the trigger double-stranded RNA that initiates the process occurs in a specific tissue and represents an example of naturally occurring inhibition of a metabolic pathway by siRNAs in one tissue while allowing expression of the pathway and synthesis of valuable secondary metabolites in all other organs/tissues of the plant.

  6. Rational Design of Micro-RNA-like Bifunctional siRNAs Targeting HIV and the HIV Coreceptor CCR5

    OpenAIRE

    2010-01-01

    Small-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are distinguished by their modes of action. SiRNAs serve as guides for sequence-specific cleavage of complementary mRNAs and the targets can be in coding or noncoding regions of the target transcripts. MiRNAs inhibit translation via partially complementary base-pairing to 3′ untranslated regions (UTRs) and are generally ineffective when targeting coding regions of a transcript. In this study, we deliberately designed siRNAs that simultan...

  7. 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

    RNA interference by small interfering RNAs (siRNAs) is considered to be a highly specific method for knockdown of gene expression in eukaryotic cells via degradation of target mRNA. Mutated siRNA molecules with 1–4 mismatching nucleotides compared to the target mRNA are regularly used as specific...

  8. Rational design of micro-RNA-like bifunctional siRNAs targeting HIV and the HIV coreceptor CCR5.

    Science.gov (United States)

    Ehsani, Ali; Saetrom, Pål; Zhang, Jane; Alluin, Jessica; Li, Haitang; Snøve, Ola; Aagaard, Lars; Rossi, John J

    2010-04-01

    Small-interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are distinguished by their modes of action. SiRNAs serve as guides for sequence-specific cleavage of complementary mRNAs and the targets can be in coding or noncoding regions of the target transcripts. MiRNAs inhibit translation via partially complementary base-pairing to 3' untranslated regions (UTRs) and are generally ineffective when targeting coding regions of a transcript. In this study, we deliberately designed siRNAs that simultaneously direct cleavage and translational suppression of HIV RNAs, or cleavage of the mRNA encoding the HIV coreceptor CCR5 and suppression of translation of HIV. These bifunctional siRNAs trigger inhibition of HIV infection and replication in cell culture. The design principles have wide applications throughout the genome, as about 90% of genes harbor sites that make the design of bifunctional siRNAs possible.

  9. Parallel ClickSeq and Nanopore sequencing elucidates the rapid evolution of defective-interfering RNAs in Flock House virus.

    Directory of Open Access Journals (Sweden)

    Elizabeth Jaworski

    2017-05-01

    Full Text Available Defective-Interfering RNAs (DI-RNAs have long been known to play an important role in virus replication and transmission. DI-RNAs emerge during virus passaging in both cell-culture and their hosts as a result of non-homologous RNA recombination. However, the principles of DI-RNA emergence and their subsequent evolution have remained elusive. Using a combination of long- and short-read Next-Generation Sequencing, we have characterized the formation of DI-RNAs during serial passaging of Flock House virus (FHV in cell-culture over a period of 30 days in order to elucidate the pathways and potential mechanisms of DI-RNA emergence and evolution. For short-read RNAseq, we employed 'ClickSeq' due to its ability to sensitively and confidently detect RNA recombination events with nucleotide resolution. In parallel, we used the Oxford Nanopore Technologies's (ONT MinION to resolve full-length defective and wild-type viral genomes. Together, these accurately resolve both rare and common RNA recombination events, determine the correlation between recombination events, and quantifies the relative abundance of different DI-RNAs throughout passaging. We observe the formation of a diverse pool of defective RNAs at each stage of viral passaging. However, many of these 'intermediate' species, while present in early stages of passaging, do not accumulate. After approximately 9 days of passaging we observe the rapid accumulation of DI-RNAs with a correlated reduction in specific infectivity and with the Nanopore data find that DI-RNAs are characterized by multiple RNA recombination events. This suggests that intermediate DI-RNA species are not competitive and that multiple recombination events interact epistatically to confer 'mature' DI-RNAs with their selective advantage allowing for their rapid accumulation. Alternatively, it is possible that mature DI-RNA species are generated in a single event involving multiple RNA rearrangements. These insights have

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

    Directory of Open Access Journals (Sweden)

    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

  11. Synthesis of small interfering RNAs containing acetal-type nucleoside analogs at their 3'-ends and analysis of their silencing activity and their ability to bind to the Argonaute2 PAZ domain.

    Science.gov (United States)

    Inada, Natsumi; Nakamoto, Kosuke; Yokogawa, Takashi; Ueno, Yoshihito

    2015-10-20

    In this study, we aimed to create small interfering RNAs (siRNAs) with increased silencing activities and nuclease resistance properties. Therefore, we designed and synthesized five types of siRNA containing acetal-type nucleoside analogs at their 3'-dangling ends. We found that the siRNA containing 1-O-(2,2,2-trifluoroethyl)-β-D-ribofuranose at the 3'-dangling end was the most potent among the synthesized siRNAs and showed more resistance to nucleolytic degradation by a 3' exonuclease than a natural RNA did. Thus, modification of siRNAs by addition of 1-O-(2,2,2-trifluoroethyl)-β-D-ribofuranose may hold promise as a means of improving the silencing activity and nuclease resistance of siRNAs.

  12. Molecular analysis of central feeding regulation by neuropeptide Y (NPY) neurons with NPY receptor small interfering RNAs (siRNAs).

    Science.gov (United States)

    Higuchi, Hiroshi

    2012-11-01

    Hypothalamic neuropeptides play important roles in central feeding behavior. Among them, neuropeptide Y (NPY) has the strongest orexigenic action. It is synthesized in NPY-expressing neurons in the arcuate nucleus (ARC), which projects to other nuclei, mainly to the paraventricular nucleus (PVN). PVN, which possesses NPY-Y1, -Y2 and -Y4, -Y5 receptors, is considered as feeding center for central feeding behavior. Herein I review recent results on feeding behavior obtained by gene knockdown technologies. The small interfering RNA (siRNA) plasmid-based vectors, which drive transcription of siRNA by U6 RNA polymerase III promoter to produce knockdown of the NPY and its receptor (Y1, Y2, Y4 and Y5) genes, were stereotaxically injected into mouse ARC and PVN. Feeding behaviors were measured for 6days after siRNA vector injection. NPY and its receptor mRNA levels were decreased, which were measured by RT-PCR and in situ hybridization, and simultaneous decrease in their proteins was also detected in separate nuclei by immunohistochemistry. In the NPY system, decrease in NPY, Y1 and Y5 expressions in specialized nuclei diminished central feeding behavior, whereas decrease in Y2 or Y4 expression in both ARC or PVN did not affect feeding behavior. Thus, specialized change in expressions of NPY and its receptors (especially Y1 and Y5) are important for regulation of endogenous feeding behavior in central regulation. Further analysis of NPY receptors may provide better understanding of feeding behavior and of potential therapeutic targets.

  13. 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

    Full Text Available BACKGROUND: 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. PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Fengfeng; Ruan, Hongjiang [Department of Orthopaedics, The Sixth Affiliated People' s Hospital, Shanghai Jiaotong University School of Medicine, 600 Yishan Road, Shanghai 200233 (China); Fan, Cunyi, E-mail: fancunyi888@hotmail.com [Department of Orthopaedics, The Sixth Affiliated People' s Hospital, Shanghai Jiaotong University School of Medicine, 600 Yishan Road, Shanghai 200233 (China); Zeng, Bingfang; Wang, Chunyang; Wang, Xiang [Department of Orthopaedics, The Sixth Affiliated People' s Hospital, Shanghai Jiaotong University School of Medicine, 600 Yishan Road, Shanghai 200233 (China)

    2010-01-01

    Transforming growth factor-{beta}1 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.

  15. Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs

    Institute of Scientific and Technical Information of China (English)

    Yi SHI; De Hua YANG; Jie XIONG; Jie JIA; Bing HUANG; You Xin JIN

    2005-01-01

    RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0~60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.

  16. Novel and Recently Evolved MicroRNA Clusters Regulate Expansive F-BOX Gene Networks through Phased Small Interfering RNAs in Wild Diploid Strawberry.

    Science.gov (United States)

    Xia, Rui; Ye, Songqing; Liu, Zongrang; Meyers, Blake C; Liu, Zhongchi

    2015-09-01

    The wild strawberry (Fragaria vesca) has recently emerged as an excellent model for cultivated strawberry (Fragaria × ananassa) as well as other Rosaceae fruit crops due to its short seed-to-fruit cycle, diploidy, and sequenced genome. Deep sequencing and parallel analysis of RNA ends were used to identify F. vesca microRNAs (miRNAs) and their target genes, respectively. Thirty-eight novel and 31 known miRNAs were identified. Many known miRNAs targeted not only conserved mRNA targets but also developed new target genes in F. vesca. Significantly, two new clusters of miRNAs were found to collectively target 94 F-BOX (FBX) genes. One of the miRNAs in the new cluster is 22 nucleotides and triggers phased small interfering RNA production from six FBX genes, which amplifies the silencing to additional FBX genes. Comparative genomics revealed that the main novel miRNA cluster evolved from duplications of FBX genes. Finally, conserved trans-acting siRNA pathways were characterized and confirmed with distinct features. Our work identified novel miRNA-FBX networks in F. vesca and shed light on the evolution of miRNAs/phased small interfering RNA networks that regulate large gene families in higher plants. © 2015 American Society of Plant Biologists. All Rights Reserved.

  17. Small interfering RNAs targeting peste des petits ruminants virus M mRNA increase virus-mediated fusogenicity and inhibit viral replication in vitro.

    Science.gov (United States)

    Liu, Fuxiao; Wu, Xiaodong; Zou, Yanli; Li, Lin; Liu, Shan; Chi, Tianying; Wang, Zhiliang

    2015-11-01

    Peste des petits ruminants (PPR), caused by peste des petits ruminants virus (PPRV), is an acute or subacute, highly contagious and economically important disease of small ruminants. The PPRV is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) protein possesses an intrinsic ability to bind to lipid membranes, and plays a crucial role in viral assembly and further budding. In this study, three different small interfering RNAs (siRNA) were designed on the basis of translated region for PPRV Nigeria 75/1M mRNA, and were subsequently synthesized for their transfection into Vero-SLAM cells, followed by infection with PPRVs. The results showed that two out of three siRNAs robustly induced cell-to-cell fusion as early as 36h post-infection with PPRVs, effectively suppressed expression of the M protein by interference for the M mRNA, and eventually inhibited viral replication in vitro. These findings led us to speculate that siRNA-mediated knockdown of the M protein would alter its interaction with viral glycoproteins, thus exacerbating intercellular fusion but hampering virus release. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs

    Science.gov (United States)

    Coffman, Stephanie R.; Lu, Jinfeng; Guo, Xunyang; Zhong, Jing; Broitman-Maduro, Gina; Li, Wan-Xiang; Lu, Rui; Maduro, Morris

    2017-01-01

    ABSTRACT Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans. Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans. Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors. PMID:28325765

  19. Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells

    Institute of Scientific and Technical Information of China (English)

    Xiao-Min Xin; Gui-Qiu U; Ying-Yu Jin; Min Zhuang; Di Li

    2008-01-01

    AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression in HepG2.2.15 cells by combination of small interfering RNAs (siRNAs).METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells.At 48 h,72 h and 96 h after transfection,culture media were collected and cells were harvested for HBV replication assay.HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay (ELISA).Intracellular viral DNA and covalently closed circular DNA (cccDNA)were quantified by real-time polymerase chain reaction (PCR).HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR (RT-PCR).RESULTS: siRNAs showed marked anti-HBV effects.siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner.Furthermore,combination of siRNAs,compared with individual use of each siRNA,exerted a stronger inhibition on antigen expression and viral replication.More importantly,combination of siRNAs significantly suppressed HBV cccDNA amplification.CONCLUSION: Combination of siRNAs mediates a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells,especially on cccDNA amplification.

  20. Synthetic RNAs for gene regulation: design principles and computational tools

    Directory of Open Access Journals (Sweden)

    Alessandro eLaganà

    2014-12-01

    Full Text Available The use of synthetic non-coding RNAs for post-transcriptional regulation of gene expression has not only become a standard laboratory tool for gene functional studies, but it has also opened up new perspectives in the design of new and potentially promising therapeutic strategies. Bioinformatics has provided researchers with a variety of tools for the design, the analysis and the evaluation of RNAi agents such as small-interfering RNA (siRNA, short-hairpin RNA (shRNA, artificial microRNA (a-miR and microRNA sponges. More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats, was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way. In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches.

  1. Inhibition of vaccinia virus replication by two small interfering RNAs targeting B1R and G7L genes and their synergistic combination with cidofovir.

    Science.gov (United States)

    Vigne, Solenne; Duraffour, Sophie; Andrei, Graciela; Snoeck, Robert; Garin, Daniel; Crance, Jean-Marc

    2009-06-01

    In view of the threat of the potential use of variola virus in a terrorist attack, considerable efforts have been performed to develop new antiviral strategies against orthopoxviruses. Here we report on the use of RNA interference, either alone or in combination with cidofovir, as an approach to inhibit orthopoxvirus replication. Two selected small interfering RNAs (siRNAs), named siB1R-2 and siG7L-1, and a previously reported siRNA, i.e., siD5R-2 (which targets the viral D5R mRNA), were evaluated for antiviral activity against vaccinia virus (VACV) by plaque reduction and virus yield assays. siB1R-2 and siG7L-1, administered before or after viral infection, reduced VACV replication by more than 90%. Also, these two siRNAs decreased monkeypox virus replication by 95% at a concentration of 1 nM. siB1R-2 and siG7L-1 were demonstrated to specifically silence their corresponding transcripts, i.e., B1R and G7L mRNAs, without induction of a beta interferon response. Strong synergistic effects were observed when siB1R-2, siG7L-1, or siD5R-2 was combined with cidofovir. In addition, the antiviral activities of these three siRNAs were evaluated against VACV resistant to cidofovir and other acyclic nucleoside phosphonates. siG7L-1 and siD5R-2 remained active against four of five VACV mutants, while siB1R-2 showed activity against only one of the mutants. Our results showed that siRNAs are potent inhibitory agents in vitro, not only against wild-type VACV but also against several cidofovir-resistant VACV. Furthermore, we showed that a combined therapy using siRNA and cidofovir may be useful in the treatment of poxvirus infections.

  2. An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

    Science.gov (United States)

    Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

    2015-12-01

    The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

  3. In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes.

    Directory of Open Access Journals (Sweden)

    Mahmoud ElHefnawi

    Full Text Available RNA interference (RNAi is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs targeting highly conserved regions of the hepatitis C virus (HCV genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258 were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h; they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic

  4. An Artificially Designed Interfering lncRNA Expressed by Oncolytic Adenovirus Competitively Consumes OncomiRs to Exert Antitumor Efficacy in Hepatocellular Carcinoma.

    Science.gov (United States)

    Li, Xiaoya; Su, Yinghan; Sun, Bin; Ji, Weidan; Peng, Zhangxiao; Xu, Yang; Wu, Mengchao; Su, Changqing

    2016-07-01

    Endogenous miRNAs, especially oncogenic miRNAs (OncomiR), have been molecular targets for cancer therapy. We generated an artificially designed interfering long noncoding RNA (lncRNAi), which contains the sequences that can complementarily bind to multiple OncomiRs and is expressed by cancer-selectively replicating adenovirus. The adenovirus-expressed lncRNAi with high levels in hepatocellular carcinoma (HCC) cells competes with OncomiR target genes to bind to and consume OncomiRs, thereby achieving the targeted anti-HCC efficacy. With the targeting replication of adenovirus in HCC cells, lncRNAi was highly expressed and resulted in decreased abilities of proliferation, migration, and invasion, induced cell-cycle changes and apoptosis, and markedly changed the cellular mRNA and miRNA expression profiles in HCC cells. The optimal antitumor effect was also demonstrated on HCC cell line xenograft models and HCC patient-derived xenograft (PDX) tumor models in nude mice. This strategy has established a technology platform with a reliable therapeutic effect for HCC therapy. Mol Cancer Ther; 15(7); 1436-51. ©2016 AACR.

  5. Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs.

    Science.gov (United States)

    Kim, Su-Mi; Park, Jong-Hyeon; Lee, Kwang-Nyeong; Kim, Se-Kyung; You, Su-Hwa; Kim, Taeseong; Tark, Dongseob; Lee, Hyang-Sim; Seo, Min-Goo; Kim, Byounghan

    2015-08-01

    Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-αγ and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-αγ and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD. The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against FMDV, although the

  6. Design, Cloning, and In Vitro Screening of Artificial miRNAs to Silence Alpha-1 Antitrypsin.

    Science.gov (United States)

    Borel, Florie; Mueller, Christian

    2017-01-01

    This protocol describes the design, cloning, and in vitro screening of artificial microRNAs (miRNAs) to silence alpha-1 antitrypsin (AAT). This method would be of interest to silence AAT in a variety of in vitro or in vivo models, and prevalidated sequences against human AAT are provided. This simple 5-day protocol may more generally be used to design artificial miRNAs against any transcript.

  7. Co-injection of a targeted, reversibly masked endosomolytic polymer dramatically improves the efficacy of cholesterol-conjugated small interfering RNAs in vivo.

    Science.gov (United States)

    Wong, So C; Klein, Jason J; Hamilton, Holly L; Chu, Qili; Frey, Christina L; Trubetskoy, Vladimir S; Hegge, Julia; Wakefield, Darren; Rozema, David B; Lewis, David L

    2012-12-01

    Effective in vivo delivery of small interfering (siRNA) has been a major obstacle in the development of RNA interference therapeutics. One of the first attempts to overcome this obstacle utilized intravenous injection of cholesterol-conjugated siRNA (chol-siRNA). Although studies in mice revealed target gene knockdown in the liver, delivery was relatively inefficient, requiring 3 daily injections of 50 mg/kg of chol-siRNA to obtain measurable reduction in gene expression. Here we present a new delivery approach that increases the efficacy of the chol-siRNA over 500-fold and allows over 90% reduction in target gene expression in mice and, for the first time, high levels of gene knockdown in non-human primates. This improved efficacy is achieved by the co-injection of a hepatocyte-targeted and reversibly masked endosomolytic polymer. We show that knockdown is absolutely dependent on the presence of hepatocyte-targeting ligand on the polymer, the cognate hepatocyte receptor, and the cholesterol moiety of the siRNA. Importantly, we provide evidence that this increase in efficacy is not dependent on interactions between the chol-siRNA with the polymer prior to injection or in the bloodstream. The simplicity of the formulation and efficacy of this mode of siRNA delivery should prove beneficial in the use of siRNA as a therapeutic.

  8. Design of a Linear Variable Differential Transformer With High Rejection to External Interfering Magnetic Field

    CERN Document Server

    Martino, M; Losito, R; Masi, A; Danisi, A

    2010-01-01

    The sensitivity of linear variable differential transformer (LVDT) position sensors to external slowly varying magnetic fields represents a critical issue when these sensors are installed close to high-current cables or electrical motors with significant fringe fields. The resulting position error can reach several hundreds of micrometers against a specified uncertainty normally below a few micrometers. In this paper, the design of a LVDT position sensor with high rejection to external constant or slowly varying magnetic fields is addressed by exploiting the finite element method (FEM) simulator FLUX. A shield, isolated from the sensor's magnetic circuit, has been considered to reduce the effect of magnetic fields on the secondary voltages of the LVDT. In addition, a dc current is used in order to polarize the magnetic circuit to reduce the sensitivity of the sensor to external interferences.

  9. 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.

  10. Improvement of the design and generation of highly specific plant knockdown lines using primary synthetic microRNAs (pri-smiRNAs

    Directory of Open Access Journals (Sweden)

    Alves Leonardo

    2010-03-01

    Full Text Available Abstract Background microRNAs (miRNAs are endogenous small non-coding RNAs that post-transcriptionally regulate gene expression. In plants, they typically show high complementarity to a single sequence motif within their target mRNAs and act by catalyzing specific mRNA cleavage and degradation. miRNAs are processed from much longer primary transcripts via precursor miRNAs containing fold-back structures. Leaving these secondary structures intact, miRNAs can be re-designed experimentally to target mRNAs of choice. Results We designed primary synthetic miRNAs (pri-smiRNAs on the basis of the primary transcript of the Arabidopsis MIR159A gene by replacing the original miR159a and the corresponding miR159a* with novel sequences, keeping the overall secondary structure as predicted by the program RNAfold. We used the program RNAhybrid to optimize smiRNA design and to screen the complete Arabidopsis transcriptome for potential off-targets. To improve the molecular cloning of the pri-smiRNA we inserted restriction sites in the original MIR159A primary transcript to easily accommodate the smiRNA/smiRNA* DNA fragment. As a proof-of-concept, we targeted the single gene encoding chalcone synthase (CHS in Arabidopsis. We demonstrate smiRNA(CHS expression and CHS mRNA cleavage in different transgenic lines. Phenotypic changes in these lines were observed for seed color and flavonol derivatives, and quantified with respect to anthocyanin content. We also tested the effect of mismatches and excess G:U base pairs on knockdown efficiency. Conclusions RNAhybrid-assisted design of smiRNAs and generation of pri-smiRNAs using a novel vector containing restriction sites greatly improves specificity and speed of the generation of stable knockdown lines for functional analyses in plants.

  11. CRISPy-web: An online resource to design sgRNAs for CRISPR applications

    Directory of Open Access Journals (Sweden)

    Kai Blin

    2016-06-01

    Full Text Available CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology, allowing the targeted engineering of a wide range of genomes. The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections. It now sees widespread application in genome engineering workflows, especially using the Streptococcus pyogenes endonuclease Cas9. To utilize Cas9, so-called single guide RNAs (sgRNAs need to be designed for each target gene. While there are many tools available to design sgRNAs for the popular model organisms, only few tools that allow designing sgRNAs for non-model organisms exist. Here, we present CRISPy-web (http://crispy.secondarymetabolites.org/, an easy to use web tool based on CRISPy to design sgRNAs for any user-provided microbial genome. CRISPy-web allows researchers to interactively select a region of their genome of interest to scan for possible sgRNAs. After checks for potential off-target matches, the resulting sgRNA sequences are displayed graphically and can be exported to text files. All steps and information are accessible from a web browser without the requirement to install and use command line scripts.

  12. Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs.

    Science.gov (United States)

    Kim, Dal Young; Atasheva, Svetlana; McAuley, Alexander J; Plante, Jessica A; Frolova, Elena I; Beasley, David W C; Frolov, Ilya

    2014-07-22

    Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.

  13. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation

    Science.gov (United States)

    Doench, John G.; Hartenian, Ella; Graham, Daniel B.; Tothova, Zuzana; Hegde, Mudra; Smith, Ian; Sullender, Meagan; Ebert, Benjamin L.; Xavier, Ramnik J.; Root, David E.

    2014-01-01

    Components of the prokaryotic clustered regularly interspersed palindromic repeat (CRISPR) loci have recently been repurposed for use in mammalian cells1–6. The Cas9 protein can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system7,8. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the proto-spacer adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest. PMID:25184501

  14. Rational Design of Small Molecules Targeting Oncogenic Noncoding RNAs from Sequence.

    Science.gov (United States)

    Disney, Matthew D; Angelbello, Alicia J

    2016-12-20

    The discovery of RNA catalysis in the 1980s and the dissemination of the human genome sequence at the start of this century inspired investigations of the regulatory roles of noncoding RNAs in biology. In fact, the Encyclopedia of DNA Elements (ENCODE) project has shown that only 1-2% of the human genome encodes protein, yet 75% is transcribed into RNA. Functional studies both preceding and following the ENCODE project have shown that these noncoding RNAs have important roles in regulating gene expression, developmental timing, and other critical functions. RNA's diverse roles are often a consequence of the various folds that it adopts. The single-stranded nature of the biopolymer enables it to adopt intramolecular folds with noncanonical pairings to lower its free energy. These folds can be scaffolds to bind proteins or to form frameworks to interact with other RNAs. Not surprisingly, dysregulation of certain noncoding RNAs has been shown to be causative of disease. Given this as the background, it is easy to see why it would be useful to develop methods that target RNA and manipulate its biology in rational and predictable ways. The antisense approach has afforded strategies to target RNAs via Watson-Crick base pairing and has typically focused on targeting partially unstructured regions of RNA. Small molecule strategies to target RNA would be desirable not only because compounds could be lead optimized via medicinal chemistry but also because structured regions within an RNA of interest could be targeted to directly interfere with RNA folds that contribute to disease. Additionally, small molecules have historically been the most successful drug candidates. Until recently, the ability to design small molecules that target non-ribosomal RNAs has been elusive, creating the perception that they are "undruggable". In this Account, approaches to demystify targeting RNA with small molecules are described. Rather than bulk screening for compounds that bind to singular

  15. Designing synthetic RNAs to determine the relevance of structural motifs in picornavirus IRES elements

    Science.gov (United States)

    Fernandez-Chamorro, Javier; Lozano, Gloria; Garcia-Martin, Juan Antonio; Ramajo, Jorge; Dotu, Ivan; Clote, Peter; Martinez-Salas, Encarnacion

    2016-04-01

    The function of Internal Ribosome Entry Site (IRES) elements is intimately linked to their RNA structure. Viral IRES elements are organized in modular domains consisting of one or more stem-loops that harbor conserved RNA motifs critical for internal initiation of translation. A conserved motif is the pyrimidine-tract located upstream of the functional initiation codon in type I and II picornavirus IRES. By computationally designing synthetic RNAs to fold into a structure that sequesters the polypyrimidine tract in a hairpin, we establish a correlation between predicted inaccessibility of the pyrimidine tract and IRES activity, as determined in both in vitro and in vivo systems. Our data supports the hypothesis that structural sequestration of the pyrimidine-tract within a stable hairpin inactivates IRES activity, since the stronger the stability of the hairpin the higher the inhibition of protein synthesis. Destabilization of the stem-loop immediately upstream of the pyrimidine-tract also decreases IRES activity. Our work introduces a hybrid computational/experimental method to determine the importance of structural motifs for biological function. Specifically, we show the feasibility of using the software RNAiFold to design synthetic RNAs with particular sequence and structural motifs that permit subsequent experimental determination of the importance of such motifs for biological function.

  16. [siRNAs with high specificity to the target: a systematic design by CRM algorithm].

    Science.gov (United States)

    Alsheddi, T; Vasin, L; Meduri, R; Randhawa, M; Glazko, G; Baranova, A

    2008-01-01

    'Off-target' silencing effect hinders the development of siRNA-based therapeutic and research applications. Common solution to this problem is an employment of the BLAST that may miss significant alignments or an exhaustive Smith-Waterman algorithm that is very time-consuming. We have developed a Comprehensive Redundancy Minimizer (CRM) approach for mapping all unique sequences ("targets") 9-to-15 nt in size within large sets of sequences (e.g. transcriptomes). CRM outputs a list of potential siRNA candidates for every transcript of the particular species. These candidates could be further analyzed by traditional "set-of-rules" types of siRNA designing tools. For human, 91% of transcripts are covered by candidate siRNAs with kernel targets of N = 15. We tested our approach on the collection of previously described experimentally assessed siRNAs and found that the correlation between efficacy and presence in CRM-approved set is significant (r = 0.215, p-value = 0.0001). An interactive database that contains a precompiled set of all human siRNA candidates with minimized redundancy is available at http://129.174.194.243. Application of the CRM-based filtering minimizes potential "off-target" silencing effects and could improve routine siRNA applications.

  17. Designing Ago2-specific siRNA/shRNA to Avoid Competition with Endogenous miRNAs.

    Science.gov (United States)

    Ma, Hongming; Zhang, Junli; Wu, Haoquan

    2014-07-15

    Relatively large amounts of transfected siRNA can compete for Ago proteins and thus compromise endogenous miRNA function, potentially leading to toxicities. Here, we show that shRNA can also perturb endogenous miRNA function similarly. More importantly, we also show that the problem can be solved by designing shRNAs in the context of pre-miR-451 structure with completely complementary stem, which significantly improves the Ago2 specificity. This shRNA was shown to be Ago2-specific, and maintain target-silencing ability while avoiding competition with endogenous miRNAs by not competing for Agos 1, 3, and 4. We conclude that modified pre-miR-451 structure provides a general platform to design shRNAs that significantly reduce perturbation of miRNA function.

  18. New short interfering RNA-based therapies for glomerulonephritis.

    Science.gov (United States)

    Shimizu, Hideki; Fujita, Toshiro

    2011-05-24

    Current treatments for glomerulonephritis are not satisfactory, and the development of new therapies would be indispensable. Short interfering RNAs (siRNAs) are promising candidates for molecular therapy because of their strong and specific gene-silencing effects. Despite rapid progress in research into the therapeutic uses of siRNAs, however, many hurdles must be overcome before siRNA-based therapies can be brought to the clinic. Most in vivo studies of siRNA-based therapy have been limited to local administration or delivery to specific target organs, including the liver. Therapies based on siRNAs for patients with glomerulonephritis show promise, although tissue-specific protocols using siRNAs have not yet been established for this indication. This Review aims to provide an overview of the current challenges in siRNA-based therapy, primarily with respect to glomerular targeting. In addition, novel delivery approaches for glomerulus-targeted, siRNA-based therapies are described.

  19. 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...

  20. EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens.

    Science.gov (United States)

    Peng, Duo; Tarleton, Rick

    2015-10-01

    Recent development of CRISPR-Cas9 genome editing has enabled highly efficient and versatile manipulation of a variety of organisms and adaptation of the CRISPR-Cas9 system to eukaryotic pathogens has opened new avenues for studying these otherwise hard to manipulate organisms. Here we describe a webtool, Eukaryotic Pathogen gRNA Design Tool (EuPaGDT; available at http://grna.ctegd.uga.edu), which identifies guide RNA (gRNA) in input gene(s) to guide users in arriving at well-informed and appropriate gRNA design for many eukaryotic pathogens. Flexibility in gRNA design, accommodating unique eukaryotic pathogen (gene and genome) attributes and high-throughput gRNA design are the main features that distinguish EuPaGDT from other gRNA design tools. In addition to employing an array of known principles to score and rank gRNAs, EuPaGDT implements an effective on-target search algorithm to identify gRNA targeting multi-gene families, which are highly represented in these pathogens and play important roles in host-pathogen interactions. EuPaGDT also identifies and scores microhomology sequences flanking each gRNA targeted cut-site; these sites are often essential for the microhomology-mediated end joining process used for double-stranded break repair in these organisms. EuPaGDT also assists users in designing single-stranded oligonucleotides for homology directed repair. In batch processing mode, EuPaGDT is able to process genome-scale sequences, enabling preparation of gRNA libraries for large-scale screening projects.

  1. Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs

    Science.gov (United States)

    Ferry, Quentin R. V.; Lyutova, Radostina; Fulga, Tudor A.

    2017-01-01

    CRISPR-based transcription regulators (CRISPR-TRs) have transformed the current synthetic biology landscape by allowing specific activation or repression of any target gene. Here we report a modular and versatile framework enabling rapid implementation of inducible CRISPR-TRs in mammalian cells. This strategy relies on the design of a spacer-blocking hairpin (SBH) structure at the 5′ end of the single guide RNA (sgRNA), which abrogates the function of CRISPR-transcriptional activators. By replacing the SBH loop with ligand-controlled RNA-cleaving units, we demonstrate conditional activation of quiescent sgRNAs programmed to respond to genetically encoded or externally delivered triggers. We use this system to couple multiple synthetic and endogenous target genes with specific inducers, and assemble gene regulatory modules demonstrating parallel and orthogonal transcriptional programs. We anticipate that this ‘plug and play' approach will be a valuable addition to the synthetic biology toolkit, facilitating the understanding of natural gene circuits and the design of cell-based therapeutic strategies. PMID:28256578

  2. Breaking-Cas-interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes.

    Science.gov (United States)

    Oliveros, Juan C; Franch, Mònica; Tabas-Madrid, Daniel; San-León, David; Montoliu, Lluis; Cubas, Pilar; Pazos, Florencio

    2016-07-08

    The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5' or 3' and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request.

  3. Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA

    NARCIS (Netherlands)

    Eefting, D.; Grimbergen, J.M.; Vries, M.R. de; Weel, V. van; Kaijzel, E.L.; Que, I.; Moon, R.T.; Löwik, C.W.; Bockel, J.H. van; Quax, P.H.A.

    2007-01-01

    For the successful application of RNA interference in vivo, it is desired to achieve (local) delivery of small interfering RNAs (siRNAs) and long-term gene silencing. Nonviral electrodelivery is suitable to obtain local and prolonged expression of transgenes. By intramuscular electrodelivery of a pl

  4. 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%.

  5. The biogenesis and function of small RNAs in C. elegans

    NARCIS (Netherlands)

    Tops, B.B.J.

    2007-01-01

    RNAi is the process by which double-stranded RNA (dsRNA) induces sequence-specific mRNA degradation. DsRNA is diced into small interfering RNAs (siRNAs) of ~21-23 nt by a complex containing the RNaseIII enzyme DICER. The mature siRNAs are subsequently bound by Argonaute proteins and incorporated int

  6. The biogenesis and function of small RNAs in C. elegans

    NARCIS (Netherlands)

    Tops, B.B.J.

    2007-01-01

    RNAi is the process by which double-stranded RNA (dsRNA) induces sequence-specific mRNA degradation. DsRNA is diced into small interfering RNAs (siRNAs) of ~21-23 nt by a complex containing the RNaseIII enzyme DICER. The mature siRNAs are subsequently bound by Argonaute proteins and incorporated int

  7. Structural Dynamics of Human Argonaute2 and Its Interaction with siRNAs Designed to Target Mutant tdp43

    Directory of Open Access Journals (Sweden)

    Vishwambhar Bhandare

    2016-01-01

    Full Text Available The human Argonaute2 protein (Ago2 is a key player in RNA interference pathway and small RNA recognition by Ago2 is the crucial step in siRNA mediated gene silencing mechanism. The present study highlights the structural and functional dynamics of human Ago2 and the interaction mechanism of Ago2 with a set of seven siRNAs for the first time. The human Ago2 protein adopts two conformations such as “open” and “close” during the simulation of 25 ns. One of the domains named as PAZ, which is responsible for anchoring the 3′-end of siRNA guide strand, is observed as a highly flexible region. The interaction between Ago2 and siRNA, analyzed using a set of siRNAs (targeting at positions 128, 251, 341, 383, 537, 1113, and 1115 of mRNA designed to target tdp43 mutants causing Amyotrophic Lateral Sclerosis (ALS disease, revealed the stable and strong recognition of siRNA by the Ago2 protein during dynamics. Among the studied siRNAs, the siRNA341 is identified as a potent siRNA to recognize Ago2 and hence could be used further as a possible siRNA candidate to target the mutant tdp43 protein for the treatment of ALS patients.

  8. Structural Dynamics of Human Argonaute2 and Its Interaction with siRNAs Designed to Target Mutant tdp43.

    Science.gov (United States)

    Bhandare, Vishwambhar; Ramaswamy, Amutha

    2016-01-01

    The human Argonaute2 protein (Ago2) is a key player in RNA interference pathway and small RNA recognition by Ago2 is the crucial step in siRNA mediated gene silencing mechanism. The present study highlights the structural and functional dynamics of human Ago2 and the interaction mechanism of Ago2 with a set of seven siRNAs for the first time. The human Ago2 protein adopts two conformations such as "open" and "close" during the simulation of 25 ns. One of the domains named as PAZ, which is responsible for anchoring the 3'-end of siRNA guide strand, is observed as a highly flexible region. The interaction between Ago2 and siRNA, analyzed using a set of siRNAs (targeting at positions 128, 251, 341, 383, 537, 1113, and 1115 of mRNA) designed to target tdp43 mutants causing Amyotrophic Lateral Sclerosis (ALS) disease, revealed the stable and strong recognition of siRNA by the Ago2 protein during dynamics. Among the studied siRNAs, the siRNA341 is identified as a potent siRNA to recognize Ago2 and hence could be used further as a possible siRNA candidate to target the mutant tdp43 protein for the treatment of ALS patients.

  9. PCR Primer Design for 16S rRNAs for Experimental Horizontal Gene Transfer Test in Escherichia coli

    Science.gov (United States)

    Miyazaki, Kentaro; Sato, Mitsuharu; Tsukuda, Miyuki

    2017-01-01

    We recently demonstrated that the Escherichia coli ribosome is robust enough to accommodate foreign 16S rRNAs from diverse gamma- and betaproteobacteria bacteria (Kitahara et al., 2012). Therein, we used the common universal primers Bac8f and UN1541r to obtain a nearly full-length gene. However, we noticed that these primers overlap variable sites at 19[A/C] and 1527[U/C] in Bac8f and UN1541r, respectively, and thus, the amplicon could contain mutations. This is problematic, particularly for the former site, because the 19th nucleotide pairs with the 916th nucleotide, which is a part of the “central pseudoknot” and is critical for function. Therefore, we mutationally investigated the role of the base pair using several 16S rRNAs from gamma- and betaproteobacteria. We found that both the native base pairs (gammaproteobacterial 19A–916U and betaproteobacterial 19C–916G) and the non-native 19A–916G pair retained function, whereas the non-native 19C–916U was defective 16S rRNAs. We next designed a new primer set, Bac1f and UN1542r, so that they do not overlap the potential mismatch sites. 16S rRNA amplicons obtained from the environmental metagenome using the new primer set were dominated by proteobacterial species (~85%). Subsequent functional screening identified various 16S rRNAs from proteobacteria, all of which contained native 19A–916U or 19C–916G base pairs. The primers developed in this study are thus advantageous for functional characterization of foreign 16S rRNA in E. coli with no artifacts.

  10. Dissecting the role of microRNAs in prostate cancer metastasis: implications for the design of novel therapeutic approaches.

    Science.gov (United States)

    Doldi, Valentina; Pennati, Marzia; Forte, Barbara; Gandellini, Paolo; Zaffaroni, Nadia

    2016-07-01

    Metastatic prostate cancer is a lethal disease that remains incurable despite the recent approval of new drugs, thus making the development of alternative treatment approaches urgently needed. A more precise understanding of the molecular mechanisms underlying prostate cancer dissemination could lead to the identification of novel therapeutic targets for the design of efficient anti-metastatic strategies. MicroRNA (miRNAs) are endogenous, small non-coding RNA molecules acting as key regulators of gene expression at post-transcriptional level. It has been clearly established that altered miRNA expression is a common hallmark of cancer. In addition, emerging evidence suggests their direct involvement in the metastatic cascade. In this review, we present a comprehensive overview of the data generated in experimental tumor models indicating that specific miRNAs may impinge on the different stages of prostate cancer metastasis, including (i) the regulation of epithelial-to-mesenchymal transition and cell migration/invasion, (ii) the interplay between cancer cells and the surrounding stroma, (iii) the control of angiogenesis, (iv) the regulation of anoikis, and (v) the colonization of distant organs. Moreover, we show preliminary evidence of the clinical relevance of some of these miRNAs, in terms of association with tumor aggressiveness/dissemination and clinical outcome, as emerged from translation studies carried out in prostate cancer patient cohorts. We also discuss the potential and the current limitations of manipulating metastasis-related miRNAs, by mimicking or inhibiting them, as a strategy for the development of novel therapeutic approaches for the advanced disease.

  11. Sustained Small Interfering RNA-Mediated Human Immunodeficiency Virus Type 1 Inhibition in Primary Macrophages

    OpenAIRE

    2003-01-01

    Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor...

  12. Characterisation of siRNAs derived from new isolates of bamboo mosaic virus and their associated satellites in infected ma bamboo (Dendrocalamus latiflorus).

    Science.gov (United States)

    Lin, Wenwu; Yan, Wenkai; Yang, Wenting; Yu, Chaowei; Chen, Huihuang; Zhang, Wen; Wu, Zujian; Yang, Liang; Xie, Lianhui

    2017-02-01

    We characterised the virus-derived small interfering RNAs (vsiRNA) of bamboo mosaic virus (Ba-vsiRNAs) and its associated satellite RNA (satRNA)-derived siRNAs (satsiRNAs) in a bamboo plant (Dendrocalamus latiflorus) by deep sequencing. Ba-vsiRNAs and satsiRNAs of 21-22 nt in length, with both (+) and (-) polarity, predominated. The 5'-terminal base of Ba-vsiRNA was biased towards A, whereas a bias towards C/U was observed in sense satsiRNAs, and towards A in antisense satsiRNAs. A large set of bamboo genes were identified as potential targets of Ba-vsiRNAs and satsiRNAs, revealing RNA silencing-based virus-host interactions in plants. Moreover, we isolated and characterised new isolates of bamboo mosaic virus (BaMV; 6,350 nt) and BaMV-associated satRNA (satBaMV; 834 nt), designated BaMV-MAZSL1 and satBaMV-MAZSL1, respectively.

  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. New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Ruiz, S.; Schyth, Brian Dall; Encinas, P.

    2009-01-01

    Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect ...... stably expressing rhabdoviral specific siRNA sequences could be a strategy to further investigate the use of RNA interference for targeting costly fish pathogenic viruses.......Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect...... cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene...

  15. 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.

  16. Micro RNAs: an arguable appraisal in medicine

    Directory of Open Access Journals (Sweden)

    Voglova K.

    2016-04-01

    Full Text Available Micro RNAs (miRNAs represent a newly discovered class of regulatory molecules in the human body. miRNA is a short double stranded RNA sequence interfering with mRNA, causing in most cases, inhibition of translation. Synthesis of miRNAs shows an increasing developmental pattern and postnatally miRNAs are synthesized in all cells possessing transcriptional machinery. miRNAs usually target several mRNAs and therefore conclusive evidences proving their functions are not always ease to be acquired. In spite of this difficulty, functions of miRNAs were firmly established in the development, the cardiovascular and neural diseases, and cancer. Many miRNAs have been reported to be associated with physiological state of cells and/or tissues. This finding becomes fundamental, especially when consider that these miRNAs can be released from cell into intracellular space or circulation. Correlation between miRNA production in tissues and its contribution to multisource miRNA pool in the circulation is in a focus of biomarker-oriented researchers. Recently, circulating miRNAs have been suggested to be applicable as biomarkers in several types of cancer, cardiovascular injury, and diabetes. Role of miRNAs in the organism intercellular signaling is still under the broad investigation. Several miRNA mimics, intended for treatment of disease, are being currently tested in the clinical trials.

  17. Circular RNAs

    DEFF Research Database (Denmark)

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

    2017-01-01

    exhibiting tissue/developmental-stage-specific expression. CircRNAs are generated either from exons or introns by back splicing or lariat introns. CircRNAs play important roles as miRNA sponges, gene transcription and expression regulators, RNA-binding protein (RBP) sponges and protein/peptide translators....... Emerging evidence revealed the function of circRNAs in cancer and may potentially serve as a required novel biomarker and therapeutic target for cancer treatment. In this review, we discuss about the origins, characteristics and functions of circRNA and how they work as miRNA sponges, gene transcription...... 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....

  18. Competition between small RNAs: a quantitative view.

    Science.gov (United States)

    Loinger, Adiel; Shemla, Yael; Simon, Itamar; Margalit, Hanah; Biham, Ofer

    2012-04-18

    Two major classes of small regulatory RNAs--small interfering RNAs (siRNAs) and microRNA (miRNAs)--are involved in a common RNA interference processing pathway. Small RNAs within each of these families were found to compete for limiting amounts of shared components, required for their biogenesis and processing. Association with Argonaute (Ago), the catalytic component of the RNA silencing complex, was suggested as the central mechanistic point in RNA interference machinery competition. Aiming to better understand the competition between small RNAs in the cell, we present a mathematical model and characterize a range of specific cell and experimental parameters affecting the competition. We apply the model to competition between miRNAs and study the change in the expression level of their target genes under a variety of conditions. We show quantitatively that the amount of Ago and miRNAs in the cell are dominant factors contributing greatly to the competition. Interestingly, we observe what to our knowledge is a novel type of competition that takes place when Ago is abundant, by which miRNAs with shared targets compete over them. Furthermore, we use the model to examine different interaction mechanisms that might operate in establishing the miRNA-Ago complexes, mainly those related to their stability and recycling. Our model provides a mathematical framework for future studies of competition effects in regulation mechanisms involving small RNAs.

  19. MicroRNAs and spermatogenesis.

    Science.gov (United States)

    Kotaja, Noora

    2014-06-01

    In mammals, male gametes are produced inside the testis by spermatogenesis, which has three phases: mitotic proliferation of spermatogonia, meiosis of spermatocytes, and haploid differentiation of spermatids. The genome of male germ cells is actively transcribed to produce phase-specific gene expression patterns. Male germ cells have a complex transcriptome. In addition to protein-coding messenger RNAs, many noncoding RNAs, including microRNAs (miRNAs), are produced. The miRNAs are important regulators of gene expression. They function mainly post-transcriptionally to control the stability or translation of their target messenger RNAs. The miRNAs are expressed in a cell-specific manner during spermatogenesis to participate in the control of each step of male germ cell differentiation. Genetically modified mouse models have demonstrated the importance of miRNA pathways for normal spermatogenesis, and functional studies have been designed to dissect the roles of specific miRNAs in distinct cell types. Clinical studies have exploited the well-defined expression profiles of miRNAs, and human spermatozoal or seminal plasma miRNAs have been explored as potential biomarkers for male factor infertility. This review article discusses the current findings that support the central role of miRNAs in the regulation of spermatogenesis and male fertility.

  20. Riding in silence: a little snowboarding, a lot of small RNAs.

    Science.gov (United States)

    Ameres, Stefan L; Fukunaga, Ryuya

    2010-03-15

    The recent symposium, RNA silencing: Mechanism, Biology and Applications, organized by Phillip D. Zamore (University of Massachusetts Medical School) and Beverly Davidson (University of Iowa), and held in Keystone, Colorado, brought together scientists working on diverse aspects of RNA silencing, a field that comprises a multitude of gene regulatory pathways guided by microRNAs, small interfering RNAs and PIWI-interacting RNAs.

  1. Gene Knockdown in Human Rhinovirus 1B Using 2'-OMe-modified siRNAs Results in the Reactivation of the Interferon Response.

    Science.gov (United States)

    Xie, Guang Cheng; Zhang, Qing; Pang, Li Li; Li, Dan Di; Jin, Miao; Li, Hui Ying; Xu, Zi Qian; Kong, Xiang Yu; Wang, Hong; Lu, Shan; Duan, Zhao Jun

    2016-02-01

    The aim of this study was to investigate the knockdown efficiency of 2'-O-methylated (2'-OMe)-modified small interfering RNAs (siRNAs) on human rhinovirus 1B (HRV1B) replication and the interferon response. Thus, 24 2'-OMe-modified siRNAs were designed to target HRV1B. The RNA levels of HRV1B, Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and interferons were determined in HRV1B-infected HeLa and BEAS-2B epithelial cells transfected with 2'-OMe-modified siRNAs. The results revealed that all 2'-OMe-modified siRNAs interfered with the replication of HRV1B in a cell-specific and transfection efficiency-dependent manner. Viral activation of Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and the interferon response was detected. In conclusion, the 2'-OMe-modified siRNAs used in this study could interfere with HRV1B replication, possibly leading to the reactivation of the interferon response.

  2. Mutant p53 inhibits miRNA biogenesis by interfering with the microprocessor complex.

    Science.gov (United States)

    Garibaldi, F; Falcone, E; Trisciuoglio, D; Colombo, T; Lisek, K; Walerych, D; Del Sal, G; Paci, P; Bossi, G; Piaggio, G; Gurtner, A

    2016-07-21

    Downregulation of microRNAs (miRNAs) is commonly observed in cancers and promotes tumorigenesis suggesting that miRNAs may function as tumor suppressors. However, the mechanism through which miRNAs are regulated in cancer, and the connection between oncogenes and miRNA biogenesis remain poorly understood. The TP53 tumor-suppressor gene is mutated in half of human cancers resulting in an oncogene with gain-of-function activities. Here we demonstrate that mutant p53 (mutp53) oncoproteins modulate the biogenesis of a subset of miRNAs in cancer cells inhibiting their post-transcriptional maturation. Interestingly, among these miRNAs several are also downregulated in human tumors. By confocal, co-immunoprecipitation and RNA-chromatin immunoprecipitation experiments, we show that endogenous mutp53 binds and sequesters RNA helicases p72/82 from the microprocessor complex, interfering with Drosha-pri-miRNAs association. In agreement with this, the overexpression of p72 leads to an increase of mature miRNAs levels. Moreover, functional experiments demonstrate the oncosuppressive role of mutp53-dependent miRNAs (miR-517a, -519a, -218, -105). Our study highlights a previously undescribed mechanism by which mutp53 interferes with Drosha-p72/82 association leading, at least in part, to miRNA deregulation observed in cancer.

  3. Abundant primary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line.

    Science.gov (United States)

    Lau, Nelson C; Robine, Nicolas; Martin, Raquel; Chung, Wei-Jen; Niki, Yuzo; Berezikov, Eugene; Lai, Eric C

    2009-10-01

    Piwi proteins, a subclass of Argonaute-family proteins, carry approximately 24-30-nt Piwi-interacting RNAs (piRNAs) that mediate gonadal defense against transposable elements (TEs). We analyzed the Drosophila ovary somatic sheet (OSS) cell line and found that it expresses miRNAs, endogenous small interfering RNAs (endo-siRNAs), and piRNAs in abundance. In contrast to intact gonads, which contain mixtures of germline and somatic cell types that express different Piwi-class proteins, OSS cells are a homogenous somatic cell population that expresses only PIWI and primary piRNAs. Detailed examination of its TE-derived piRNAs and endo-siRNAs revealed aspects of TE defense that do not rely upon ping-pong amplification. In particular, we provide evidence that a subset of piRNA master clusters, including flamenco, are specifically expressed in OSS and ovarian follicle cells. These data indicate that the restriction of certain TEs in somatic gonadal cells is largely mediated by a primary piRNA pathway.

  4. Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs

    Directory of Open Access Journals (Sweden)

    Samreen Baila

    2010-11-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. Results The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed. Conclusions Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.

  5. Engineering of small interfering RNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles for highly efficient and safe gene silencing: A quality by design-based approach

    DEFF Research Database (Denmark)

    Thanki, Kaushik; Zeng, Xianghui; Justesen, Sarah

    2017-01-01

    Safety and efficacy of therapeutics based on RNA interference, e.g., small interfering RNA (siRNA), are dependent on the optimal engineering of the delivery technology, which is used for intracellular delivery of siRNA to the cytosol of target cells. We investigated the hypothesis that commonly u...

  6. Conditional Dicer substrate formation via shape and sequence transduction with small conditional RNAs.

    Science.gov (United States)

    Hochrein, Lisa M; Schwarzkopf, Maayan; Shahgholi, Mona; Yin, Peng; Pierce, Niles A

    2013-11-20

    RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) enables knockdown of a gene of choice, executing the logical operation: silence gene Y. The fact that the siRNA is constitutively active is a significant limitation, making it difficult to confine knockdown to a specific locus and time. To achieve spatiotemporal control over silencing, we seek to engineer small conditional RNAs (scRNAs) that mediate 'conditional RNAi' corresponding to the logical operation: if gene X is transcribed, silence independent gene Y. By appropriately selecting gene X, knockdown of gene Y could then be restricted in a tissue- and time-specific manner. To implement the logic of conditional RNAi, our approach is to engineer scRNAs that, upon binding to mRNA 'detection target' X, perform shape and sequence transduction to form a Dicer substrate targeting independent mRNA 'silencing target' Y, with subsequent Dicer processing yielding an siRNA targeting mRNA Y for destruction. Toward this end, here we design and experimentally validate diverse scRNA mechanisms for conditional Dicer substrate formation. Test tube studies demonstrate strong OFF/ON conditional response, with at least an order of magnitude increase in Dicer substrate production in the presence of the cognate mRNA detection target. By appropriately dimensioning and/or chemically modifying the scRNAs, only the product of signal transduction, and not the reactants or intermediates, is efficiently processed by Dicer, yielding siRNAs. These mechanism studies explore diverse design principles for engineering scRNA signal transduction cascades including reactant stability vs metastability, catalytic vs noncatalytic transduction, pre- vs post-transcriptional transduction, reactant and product molecularity, and modes of molecular self-assembly and disassembly.

  7. Expressing functional siRNAs in mammalian cells using convergent transcription

    Directory of Open Access Journals (Sweden)

    Dawes Ian W

    2003-11-01

    Full Text Available Abstract Background The use of small interfering RNAs (siRNAs as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi-mediated gene silencing. Results With the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells. Conclusion The experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences

  8. Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)

    OpenAIRE

    Devor, Eric J.; Huang, Lingyan; Abdukarimov, Abdusattor; Abdurakhmonov, Ibrokhim Y.

    2009-01-01

    The “RNA revolution” that started at the end of the 20th century with the discovery of post-transcriptional gene silencing and its mechanism via RNA interference (RNAi) placed tiny 21-24 nucleotide long noncoding RNAs (ncRNAs) in the forefront of biology as one of the most important regulatory elements in a host of physiologic processes. The discovery of new classes of ncRNAs including endogenous small interfering RNAs, microRNAs, and PIWI-interacting RNAs is a hallmark in the understanding o...

  9. Broad-Spectrum Antiviral Activity of Small Interfering RNA Targeting the Conserved RNA Termini of Lassa Virus▿

    OpenAIRE

    Müller, Stefanie; Günther, Stephan

    2007-01-01

    Small interfering RNAs targeting the conserved RNA termini upstream of NP and L gene were found to reduce reporter gene expression from Lassa virus replicon and Lassa virus mRNA expression construct and to inhibit replication of different Lassa virus strains, lymphocytic choriomeningitis virus, and Mopeia virus in cell culture.

  10. Gene silencing by siRNAs and antisense oligonucleotides in the laboratory and the clinic

    Science.gov (United States)

    Watts, Jonathan K.; Corey, David R.

    2014-01-01

    Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past twenty years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology. PMID:22069063

  11. Non-coding RNAs in neural networks, REST-assured

    Directory of Open Access Journals (Sweden)

    Michael eROSSBACH

    2011-02-01

    Full Text Available In the nervous system, several key steps in cellular complexity and development are regulated by non-coding RNAs (ncRNAs and the repressor element-1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF. REST recruits gene regulatory complexes to regulatory sequences, among them the repressor element-1/neuron restrictive silencer element (RE1/NRSE, and mediates developmental stage-specific gene expression or repression, chromatin (re-orga-nization or silencing for protein-coding genes as well as for several ncRNAs like microRNAs (miRNAs, short interfering RNAs (siRNAs or long ncRNAs. NcRNAs are far from being just transcriptional noise and are involved in chromatin accessibility, transcription and post-transcriptional processing, trafficking or RNA editing. REST and its cofactor CoREST are both highly regulated through various ncRNAs. The importance of the correct regulation within the ncRNA network, the ncRNAome, is demonstrated when it comes to a deregulation of REST and/or ncRNAs associated with molecular pathophysiology underlying diverse disorders including neurodegenerative diseases or brain tumors.

  12. Apple miRNAs and tasiRNAs with novel regulatory networks

    Science.gov (United States)

    2012-01-01

    Background MicroRNAs (miRNAs) and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown. Results We performed deep small RNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that 10 of the 19 miR828-targeted MYBs undergo small interfering RNA (siRNA) biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis. Conclusions Our work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family. PMID:22704043

  13. Inforna 2.0: A Platform for the Sequence-Based Design of Small Molecules Targeting Structured RNAs.

    Science.gov (United States)

    Disney, Matthew D; Winkelsas, Audrey M; Velagapudi, Sai Pradeep; Southern, Mark; Fallahi, Mohammad; Childs-Disney, Jessica L

    2016-06-17

    The development of small molecules that target RNA is challenging yet, if successful, could advance the development of chemical probes to study RNA function or precision therapeutics to treat RNA-mediated disease. Previously, we described Inforna, an approach that can mine motifs (secondary structures) within target RNAs, which is deduced from the RNA sequence, and compare them to a database of known RNA motif-small molecule binding partners. Output generated by Inforna includes the motif found in both the database and the desired RNA target, lead small molecules for that target, and other related meta-data. Lead small molecules can then be tested for binding and affecting cellular (dys)function. Herein, we describe Inforna 2.0, which incorporates all known RNA motif-small molecule binding partners reported in the scientific literature, a chemical similarity searching feature, and an improved user interface and is freely available via an online web server. By incorporation of interactions identified by other laboratories, the database has been doubled, containing 1936 RNA motif-small molecule interactions, including 244 unique small molecules and 1331 motifs. Interestingly, chemotype analysis of the compounds that bind RNA in the database reveals features in small molecule chemotypes that are privileged for binding. Further, this updated database expanded the number of cellular RNAs to which lead compounds can be identified.

  14. Virus B e interferón

    Directory of Open Access Journals (Sweden)

    Gerardo Avendaño-Alvarado

    2008-11-01

    Full Text Available La aparición de nuevas drogas y formas de diagnóstico, han transformado la hepatitis crónica B de una enfermedad fatal a una manejable y aún curable. Se distinguen dos tipos de enfermedad crónica por virus B, la que se desarrolla con antígeno e positivo y la que cursa con antígeno e negativo. La enfermedad crónica puede presentarse con ALT normal, ALT en continua elevación, fluctuaciones de ALT sin llegar a ser normales o elevaciones intermitentes. El éxito de la terapia antiviral para el virus B incluye, suprimir la replicación viral al nivel más bajo posible, lograr mejoría bioquímica e histológica y prevenir el desarrollo de complicaciones. Existen dos estrategias de tratamiento para el virus B, una de duración limitada (interferones y otra de largo plazo (análogos nucleós(tidos. Existen factores que influencian favorablemente la respuesta al tratamiento con interferón: niveles bajos de HBV DNA, niveles altos de ALT, niveles bajos de HBeAg y genotipos A y B. En el manejo de la enfermedad crónica tanto por virus B e ( + y e ( - se han utilizado interferón αlfa y actualmente el interferón pegilado α-2a. El interferón pegylado también ha mostrado ser superior al interferón simple en cuanto a normalización de ALT, pérdida del HBe y pérdida sostenida del HBV DNA. El interferón pegylado también ha mostrado ser superior a la terapia combinada o a la lamivudina sola en cuanto a rangos de respuesta y seroconversión en e (+ y e (-. En los pacientes e (- sigue existiendo controversia por ameritar tratamiento a largo plazo el uso de interferón o iniciar con análogos nucleósidos.

  15. Cancer Cachexia and MicroRNAs.

    Science.gov (United States)

    Camargo, Rodolfo Gonzalez; Quintas Teixeira Ribeiro, Henrique; Geraldo, Murilo Vieira; Matos-Neto, Emídio; Neves, Rodrigo Xavier; Carnevali, Luiz Carlos; Donatto, Felipe Fedrizzi; Alcântara, Paulo S M; Ottoch, José P; Seelaender, Marília

    2015-01-01

    Cancer cachexia is a paraneoplastic syndrome compromising quality of life and survival, mainly characterized by involuntary weight loss, fatigue, and systemic inflammation. The syndrome is described as a result of tumor-host interactions characterized by an inflammatory response by the host to the presence of the tumor. Indeed, systemic inflammation is considered a pivotal feature in cachexia progression and maintenance. Cytokines are intimately related to chronic systemic inflammation and the mechanisms underlying the release of these factors are not totally elucidated, the etiology of cachexia being still not fully understood. Therefore, the understanding of cachexia-related mechanisms, as well as the establishment of markers for the syndrome, is very relevant. MicroRNAs (miRNAs) are a class of noncoding RNAs interfering with gene regulation. Different miRNA expression profiles are associated with different diseases and inflammatory processes. miRNAs modulate adipose and skeletal muscle tissue metabolism in cancer cachexia and also tumor and tissue derived inflammation. Therefore, we propose a possible role for miRNAs in the modulation of the host inflammatory response during cachexia. Moreover, the establishment of a robust body of evidence in regard to miRNAs and the mechanisms underlying cachexia is mandatory, and shall contribute to the improvement of its diagnosis and treatment.

  16. Locking-to-unlocking system is an efficient strategy to design DNA/silver nanoclusters (AgNCs) probe for human miRNAs

    DEFF Research Database (Denmark)

    Shah, Pratik; Choi, Suk Won; Kim, Ho-jin;

    2016-01-01

    MicroRNAs (miRNAs), small non-coding RNA molecules, are important biomarkers for research and medical purposes. Here, we describe the development of a fast and simple method using highly fluorescent oligonucleotide-silver nanocluster probes (DNA/AgNCs) to efficiently detect specific miRNAs. Due t...... detect a number of cancer related miRNAs in RNA extracts from human cancer cell lines....

  17. Analyses of a Glycine max Degradome Library Identify microRNA Targets and MicroRNAs that Trigger Secondary SiRNA Biogenesis

    Institute of Scientific and Technical Information of China (English)

    Zheng Hu; Qiyan Jiang; Zhiyong Ni; Rui Chen; Shuo Xu; Hui Zhang

    2013-01-01

    Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs.In this study,a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced.428 potential targets of small interfering RNAs and 25 novel miRNA families were identified.A total of 211 potential miRNA targets,including 174 conserved miRNA targets and 37 soybeanspecific miRNA targets,were identified.Among them,121 targets were first discovered in soybean.The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing.The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean,and nine Auxin Response Factors were identified as TAS3 siRNA targets.Twentythree miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean.These targets were guided by five miRNAs:gma-miR393,gma-miR1508,gma-miR1510,gma-miR1514,and novel-11.Multiple targets of these secondary siRNAs were detected.These 23 miRNA targets may be the putative novel TAS genes in soybean.Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.

  18. Analyses of a Glycine max degradome library identify microRNA targets and microRNAs that trigger secondary siRNA biogenesis.

    Science.gov (United States)

    Hu, Zheng; Jiang, Qiyan; Ni, Zhiyong; Chen, Rui; Xu, Shuo; Zhang, Hui

    2013-02-01

    Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs. In this study, a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced. 428 potential targets of small interfering RNAs and 25 novel miRNA families were identified. A total of 211 potential miRNA targets, including 174 conserved miRNA targets and 37 soybean-specific miRNA targets, were identified. Among them, 121 targets were first discovered in soybean. The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing. The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean, and nine Auxin Response Factors were identified as TAS3 siRNA targets. Twenty-three miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean. These targets were guided by five miRNAs: gma-miR393, gma-miR1508, gma-miR1510, gma-miR1514, and novel-11. Multiple targets of these secondary siRNAs were detected. These 23 miRNA targets may be the putative novel TAS genes in soybean. Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.

  19. MicroRNAs as mediators of insect host-pathogen interactions and immunity.

    Science.gov (United States)

    Hussain, Mazhar; Asgari, Sassan

    2014-11-01

    Insects are the most successful group of animals on earth, owing this partly to their very effective immune responses to microbial invasion. These responses mainly include cellular and humoral responses as well as RNA interference (RNAi). Small non-coding RNAs (snRNAs) produced through RNAi are important molecules in the regulation of gene expression in almost all living organisms; contributing to important processes such as development, differentiation, immunity as well as host-microorganism interactions. The main snRNAs produced by the RNAi response include short interfering RNAs, microRNAs and piwi-interacting RNAs. In addition to the host snRNAs, some microorganisms encode snRNAs that affect the dynamics of host-pathogen interactions. In this review, we will discuss the latest developments in regards to the role of microRNA in insect host-pathogen interactions and provide some insights into this rapidly developing area of research.

  20. Small interfering RNA targeted to IGF-IR delays tumor growth and induces proinflammatory cytokines in a mouse breast cancer model.

    Directory of Open Access Journals (Sweden)

    Tiphanie Durfort

    Full Text Available Insulin-like growth factor I (IGF-I and its type I receptor (IGF-IR play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2'-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD. Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2'-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines.

  1. Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington's Disease.

    Science.gov (United States)

    Miniarikova, Jana; Zanella, Ilaria; Huseinovic, Angelina; van der Zon, Tom; Hanemaaijer, Evelyn; Martier, Raygene; Koornneef, Annemart; Southwell, Amber L; Hayden, Michael R; van Deventer, Sander J; Petry, Harald; Konstantinova, Pavlina

    2016-03-22

    Huntington's disease (HD) is a neurodegenerative disorder caused by accumulation of CAG expansions in the huntingtin (HTT) gene. Hence, decreasing the expression of mutated HTT (mtHTT) is the most upstream approach for treatment of HD. We have developed HTT gene-silencing approaches based on expression cassette-optimized artificial miRNAs (miHTTs). In the first approach, total silencing of wild-type and mtHTT was achieved by targeting exon 1. In the second approach, allele-specific silencing was induced by targeting the heterozygous single-nucleotide polymorphism (SNP) rs362331 in exon 50 or rs362307 in exon 67 linked to mtHTT. The miHTT expression cassette was optimized by embedding anti-HTT target sequences in ten pri-miRNA scaffolds and their HTT knockdown efficacy, allele selectivity, passenger strand activity, and processing patterns were analyzed in vitro. Furthermore, three scaffolds expressing miH12 targeting exon 1 were incorporated in an adeno-associated viral serotype 5 (AAV5) vector and their HTT knock-down efficiency and pre-miHTT processing were compared in the humanized transgenic Hu128/21 HD mouse model. Our data demonstrate strong allele-selective silencing of mtHTT by miSNP50 targeting rs362331 and total HTT silencing by miH12 both in vitro and in vivo. Ultimately, we show that HTT knock-down efficiency and guide strand processing can be enhanced by using different cellular pri-miRNA scaffolds.

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

    Directory of Open Access Journals (Sweden)

    Joao Trindade Marques

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

  3. The highly conserved 5' untranslated region as an effective target towards the inhibition of Enterovirus 71 replication by unmodified and appropriate 2'-modified siRNAs

    Directory of Open Access Journals (Sweden)

    Deng Jun-Xia

    2012-08-01

    Full Text Available Abstract Background Enterovirus 71 (EV71 is a highly infectious agent that plays an etiological role in hand, foot, and mouth disease. It is associated with severe neurological complications and has caused significant mortalities in recent large-scale outbreaks. Currently, no effective vaccine or specific clinical therapy is available against EV71. Methods Unmodified 21 nucleotide small interfering RNAs (siRNAs and classic 2′-modified (2′-O-methylation or 2′-fluoro modification siRNAs were designed to target highly conserved 5′ untranslated region (UTR of the EV71 genome and employed as anti-EV71 agents. Real-time TaqMan RT-PCR, western blot analysis and plaque assays were carried out to evaluate specific viral inhibition by the siRNAs. Results Transfection of rhabdomyosarcoma (RD cells with siRNAs targeting the EV71 genomic 5′ UTR significantly delayed and alleviated the cytopathic effects of EV71 infection, increased cell viability in EV71-infected RD cells. The inhibitory effect on EV71 replication was sequence-specific and dosage-dependent, with significant corresponding decreases in viral RNA, VP1 protein and viral titer. Appropriate 2′-modified siRNAs exhibited similar RNA interference (RNAi activity with dramatically increased serum stability in comparison with unmodified counterparts. Conclusion Sequences were identified within the highly conserved 5′ UTR that can be targeted to effectively inhibit EV71 replication through RNAi strategies. Appropriate 2′-modified siRNAs provide a promising approach to optimizing siRNAs to overcome barriers on RNAi-based antiviral therapies for broader administration.

  4. Un coronographe interf\\'erentiel achromatique coaxial

    CERN Document Server

    Gay, Jean; Rivet, Jean-Pierre; Rabbia, Yves; Buisset, Christophe

    2005-01-01

    On-axis achromatic interfero-coronagraph. We present a new type of stellar interfero-coronagraph, the "CIAXE", which is a variant of the "AIC", the Achromatic Interfero-Coronagraph [3,4]. The CIAXE is characterized by a very simple, compact and fully coaxial optical combination. Indeed, contrarily to the classical AIC which has a Michelson interferometer structure, the CIAXE delivers its output beam on the same axis as the input beam. This will ease its insertion in the focal instrumentation of existing telescopes or next generation ones. Such a device could be a step forward in the field of instrumental search for exoplanets. ----- Dans le but deparvenir \\`a l'imagerie \\`a haute dynamique d'objets comme les exoplan\\`etes, nous pr\\'esentons ici un nouveau concept de coronographe stellaire interf\\'erentiel, le "CIAXE". Il est d\\'eriv\\'e du "CIA", le Coronographe Interf\\'erentiel Achromatique. Le CIAXE se distingue de son pr\\'ed\\'ecesseur par une combinaison optique originale, simplifi\\'ee, tr\\`es compacte et t...

  5. A High Throughput In Vivo Model for Testing Delivery and Antiviral Effects of siRNAs in Vertebrates

    DEFF Research Database (Denmark)

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

    2007-01-01

    Despite the promise of small interfering RNAs (siRNAs) in antiviral therapy, few in vivo studies of them as inhibitors of viral replication and disease have been published, a lack that is most probably due to problems with obtaining successful delivery. Here we introduce a novel in vivomodel comp...

  6. Inhibition of vascular endothelial growth factor gene expression by T7-siRNAs in cultured human retinal pigment epithelial cells

    Institute of Scientific and Technical Information of China (English)

    LI Guang-yu; FAN Bin; WU Ya-zhen; WANG Xin-rui; WANG Yao-hui; WU Jia-xiang

    2005-01-01

    Background Retinal pigment epithelial (RPE) cells play an important role in the occurrence of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) as a positive regulatory growth factor is produced by the RPE in an autocrine or paracrine manner, promoting CNV development. Duplexes of 21 nt RNAs, known as short interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. We searched for an efficient siRNA to interfere with VEGF expression in RPE cells and shed light on the treatment of CNV.Methods Human primary RPE (hRPE) cells were cultured and identified. Three pairs of siRNAs were designed according to the sequence of VEGF 1-5 extrons and synthesized by T7 RNA polymerase transcription in vitro. To evaluate the inhibitory activity of T7-siRNAs, hRPE cells were transfected via siPORT Amine. The interfering effect of T7-siRNAs in hRPE cells was examined by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence. Results Three pairs of T7-siRNAs synthesized by in vitro transcription with T7 RNA polymerase suppressed VEGF gene expression with efficiency from 65% to 90%. T7-siRNA (B), targeted region at 207 nt to 228 nt and double stranded for 21 nt with 2 nt UU 3' overhangs, was the most effective sequence tested for inhibition of VEGF expression in hRPE cells. Compared with nontransfected cells, the mean fluorescence in hRPE cells transfected with T7-sRNAs was significantly less (P<0.01). siRNA with a single-base mismatch and ssRNA(+) did not show suppressing effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect (5 to 10 pmol).Conclusion T7-siRNA can effectively and specifically suppress VEGF expression in hRPE cells and may be a new way to treat CNV.

  7. Characterization of siRNAs derived from cucumber green mottle mosaic virus in infected cucumber plants.

    Science.gov (United States)

    Li, Yongqiang; Deng, Congliang; Shang, Qiaoxia; Zhao, Xiaoli; Liu, Xingliang; Zhou, Qi

    2016-02-01

    Virus-derived small interfering RNAs (vsiRNAs) of cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, were characterised in cucumber plants by deep sequencing. CGMMV vsiRNAs of 21-22 nt in length predominated, suggesting that there might be a conserved mechanism of DCL2 and DCL4 involvement in the biogenesis of vsiRNAs, as well as a common RNA silencing pathway in CGMMV-infected cucumber plants. The 5'-terminal base of vsiRNAs was biased towards C/A/U, suggesting that CGMMV vsiRNAs might be loaded into diverse AGO-containing RISCs to disturb the gene expression of host plants. Possible targets for some of the vsiRNAs were also predicted.

  8. Endogenous TasiRNAs mediate non-cell autonomous effects on gene regulation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Rebecca Schwab

    Full Text Available BACKGROUND: Different classes of small RNAs (sRNAs refine the expression of numerous genes in higher eukaryotes by directing protein partners to complementary nucleic acids, where they mediate gene silencing. Plants encode a unique class of sRNAs, called trans-acting small interfering RNAs (tasiRNAs, which post-transcriptionally regulate protein-coding transcripts, as do microRNAs (miRNAs, and both sRNA classes control development through their targets. TasiRNA biogenesis requires multiple components of the siRNA pathway and also miRNAs. But while 21mer siRNAs originating from transgenes can mediate silencing across several cell layers, miRNA action seems spatially restricted to the producing or closely surrounding cells. PRINCIPAL FINDINGS: We have previously described the isolation of a genetrap reporter line for TAS3a, the major locus producing AUXIN RESPONS FACTOR (ARF-regulating tasiRNAs in the Arabidopsis shoot. Its activity is limited to the adaxial (upper side of leaf primordia, thus spatially isolated from ARF-activities, which are located in the abaxial (lower side. We show here by in situ hybridization and reporter fusions that the silencing activities of ARF-regulating tasiRNAs are indeed manifested non-cell autonomously to spatially control ARF activities. CONCLUSIONS/SIGNIFICANCE: Endogenous tasiRNAs are thus mediators of a mobile developmental signal and might provide effective gene silencing at a distance beyond the reach of most miRNAs.

  9. Deregulated microRNAs in multiple myeloma.

    Science.gov (United States)

    Benetatos, Leonidas; Vartholomatos, George

    2012-02-15

    MicroRNAs are short noncoding RNAS involved in gene expression regulation under physiological and pathological situations. They bind to mRNA of target genes and are potential regulators of gene expression at a post-transcription level through the RNA interference pathway. They are estimated to represent 1% to 2% of the known eukaryotic genome, and it has been demonstrated that they are involved in the pathogenesis of neurodegenerative diseases, cancer, metabolism disorders, and heart disease. MicroRNAs are known to act as tumor suppressors or oncogenes in cancer biology. The authors describe the current knowledge on microRNA involvement in regulatory pathways that characterize multiple myeloma pathogenesis gained from in vitro and in vivo studies. These small molecules interact with important factors such as p53, SOCS1, IGF-1, IGF-1R, vascular endothelial growth factor, NF-κB, and others. As such, microRNAs represent an attractive therapeutic target in the context of multiple myeloma interfering with the myeloma regulatory networks. Further studies are needed to better understand their role in myelomagenesis and their therapeutic potential.

  10. Targeting leukemic fusion proteins with small interfering RNAs: recent advances and therapeutic potentials

    Institute of Scientific and Technical Information of China (English)

    Maria THOMAS; Johann GREIL; Olaf HEIDENREICH

    2006-01-01

    RNA interference has become an indispensable research tool to study gene functions in a wide variety of organisms.Because of their high efficacy and specificity,RNA interference-based approaches may also translate into new therapeutic strategies to treat human diseases.In particular,oncogenes such as leukemic fusion proteins,which arise from chromosomal translocations,are promising targets for such gene silencing approaches,because they are exclusively expressed in precancerous and cancerous tissues,and because they are frequently indispensable for maintaining the malignant phenotype.This review summarizes recent developments in targeting leukemia-specific genes and discusses problems and approaches for possible clinical applications.

  11. Delivery of small interfering RNAs in human cervical cancer cells by polyethylenimine-functionalized carbon nanotubes

    OpenAIRE

    Huang, Yuan-Pin; Lin, I-Jou; Chen, Chih-Chen; Hsu, Yi-Chiang; Chang, Chi-Chang; Lee, Mon-Juan

    2013-01-01

    Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method....

  12. Effects of small interfering RNAs targeting fascin on human esophageal squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Garcia Jose

    2010-06-01

    Full Text Available Abstract Background Fascin induces membrane protrusions and cell motility. Fascin overexpression was associated with poor prognosis, and its downregulation reduces cell motility and invasiveness in esophageal squamous cell carcinoma (ESCC. Using a stable knockdown cell line, we revealed the effect of fascin on cell growth, cell adhesion and tumor formation. Methods We examined whether fascin is a potential target in ESCC using in vitro and in vivo studies utilizing a specific siRNA. We established a stable transfectant with downregulated fascin from KYSE170 cell line. Results The fascin downregulated cell lines showed a slower growth pattern by 40.3% (p In vivo, the tumor size was significantly smaller in the tumor with fascin knockdown cells than in mock cells by 95% at 30 days after inoculation. Conclusions These findings suggest that fascin overexpression plays a role in tumor growth and progression in ESCC and that cell death caused by its downregulation might be induced by cell adhesion loss. This indicates that targeting fascin pathway could be a novel therapeutic strategy for the human ESCC.

  13. Rapid and efficient isolation of high-quality small RNAs from recalcitrant plant species rich in polyphenols and polysaccharides.

    Directory of Open Access Journals (Sweden)

    Jun Peng

    Full Text Available Small RNAs, including microRNAs (miRNAs and small interfering RNAs (siRNAs, are important regulators of plant development and gene expression. The acquisition of high-quality small RNAs is the first step in the study of its expression and function analysis, yet the extraction method of small RNAs in recalcitrant plant tissues with various secondary metabolites is not well established, especially for tropical and subtropical plant species rich in polysaccharides and polyphenols. Here, we developed a simple and efficient method for high quality small RNAs extraction from recalcitrant plant species. Prior to RNA isolation, a precursory step with a CTAB-PVPP buffer system could efficiently remove compounds and secondary metabolites interfering with RNAs from homogenized lysates. Then, total RNAs were extracted by Trizol reagents followed by a differential precipitation of high-molecular-weight (HMW RNAs using polyethylene glycol (PEG 8000. Finally, small RNAs could be easily recovered from supernatant by ethanol precipitation without extra elimination steps. The isolated small RNAs from papaya showed high quality through a clear background on gel and a distinct northern blotting signal with miR159a probe, compared with other published protocols. Additionally, the small RNAs extracted from papaya were successfully used for validation of both predicted miRNAs and the putative conserved tasiARFs. Furthermore, the extraction method described here was also tested with several other subtropical and tropical plant tissues. The purity of the isolated small RNAs was sufficient for such applications as end-point stem-loop RT-PCR and northern blotting analysis, respectively. The simple and feasible extraction method reported here is expected to have excellent potential for isolation of small RNAs from recalcitrant plant tissues rich in polyphenols and polysaccharides.

  14. Identification and characterization of microRNAs and endogenous siRNAs in Schistosoma japonicum

    Directory of Open Access Journals (Sweden)

    Wang Heng

    2010-01-01

    Full Text Available Abstract Background Small endogenous non-coding RNAs (sncRNAs such as small interfering RNA (siRNA, microRNA and other small RNA transcripts are derived from distinct loci in the genome and play critical roles in RNA-mediated gene silencing mechanisms in plants and metazoa. They are approximately 22 nucleotides long; regulate mRNA stability through perfect or imperfect match to the targets. The biological activities of sncRNAs have been related to many biological events, from resistance to microbe infections to cellular differentiation. The development of the zoonotic parasite Schistosoma japonicum parasite includes multiple steps of morphological alterations and biological differentiations, which provide a unique model for studies on the functions of small RNAs. Characterization of the genome-wide transcription of the sncRNAs will be a major step in understanding of the parasite biology. The objective of this study is to investigate the transcriptional profile and potential function of the small non-coding RNAs in the development of S. japanicum. Results The endogenous siRNAs were found mainly derived from transposable elements (TE or transposons and the natural antisense transcripts (NAT. In contrast to other organisms, the TE-derived siRNAs in S. japonicum were more predominant than other sncRNAs including microRNAs (miRNAs. Further, there were distinct length and 3'end variations in the sncRNAs, which were associated with the developmental differentiation of the parasite. Among the identified miRNA transcripts, there were 38 unique to S. japonicum and 16 that belonged to 13 miRNA families are common to other metazoan lineages. These miRNAs were either ubiquitously expressed, or they exhibited specific expression patterns related to the developmental stages or sex. Genes that encoded miRNAs are mainly located in clusters within the genome of S. japonicum. However, genes within one cluster could be differentially transcribed, which suggested

  15. Locking-to-unlocking system is an efficient strategy to design DNA/silver nanoclusters (AgNCs) probe for human miRNAs

    DEFF Research Database (Denmark)

    Shah, Pratik; Choi, Suk Won; Kim, Ho-jin;

    2016-01-01

    to the great sequence diversity of miRNAs in humans and other organisms, a uniform strategy for miRNA detection is attractive. The concept presented is an oligonucleotide-based locking-to-unlocking system that can be endowed with miRNA complementarity while maintaining the same secondary structure. The locking...... detect a number of cancer related miRNAs in RNA extracts from human cancer cell lines....

  16. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.

    Directory of Open Access Journals (Sweden)

    Jae-Su Moon

    Full Text Available The hepatitis C virus (HCV internal ribosome entry site (IRES that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343 where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.

  17. 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.

  18. Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.

    Science.gov (United States)

    Song, Erwei; Lee, Sang-Kyung; Dykxhoorn, Derek M; Novina, Carl; Zhang, Dong; Crawford, Keith; Cerny, Jan; Sharp, Phillip A; Lieberman, Judy; Manjunath, N; Shankar, Premlata

    2003-07-01

    Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

  19. Engineering of small interfering RNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles for highly efficient and safe gene silencing: A quality by design-based approach

    DEFF Research Database (Denmark)

    Thanki, Kaushik; Zeng, Xianghui; Justesen, Sarah

    2017-01-01

    ), eventually resulting in the identification of a robust, highly efficacious and safe formulation. A 17-run design of experiment with an I-optimal approach was performed to systematically assess the effect of selected variables on critical quality attributes (CQAs), i.e. physicochemical properties...... formulation is highly challenging due to a strong interplay between several contributing factors. Hence, critical formulation variables, i.e. the lipidoid content and siRNA:lipidoid ratio, were initially identified, followed by a systematic quality-by-design approach to define the optimal operating space (OOS...... (hydrodynamic size, zeta potential, siRNA encapsulation/loading) and the biological performance (in vitro gene silencing and cell viability). Model fitting of the obtained data to construct predictive models revealed non-linear relationships for all CQAs, which can be readily overlooked in one...

  20. lncRNAs: novel players in intervertebral disc degeneration and osteoarthritis.

    Science.gov (United States)

    Chen, Wen-Kang; Yu, Xiao-Hua; Yang, Wei; Wang, Cheng; He, Wen-Si; Yan, Yi-Guo; Zhang, Jian; Wang, Wen-Jun

    2017-02-01

    The term long non-coding RNA (lncRNA) refers to a group of RNAs with length more than 200 nucleotides, limited protein-coding potential, and having widespread biological functions, including regulation of transcriptional patterns and protein activity, formation of endogenous small interfering RNAs (siRNAs) and natural microRNA (miRNA) sponges. Intervertebral disc degeneration (IDD) and osteoarthritis (OA) are the most common chronic, prevalent and age-related degenerative musculoskeletal disorders. Numbers of lncRNAs are differentially expressed in human degenerative nucleus pulposus tissue and OA cartilage. Moreover, some lncRNAs have been shown to be involved in multiple pathological processes during OA, including extracellular matrix (ECM) degradation, inflammatory responses, apoptosis and angiogenesis. In this review, we summarize current knowledge concerning lncRNAs, from their biogenesis, classification and biological functions to molecular mechanisms and therapeutic potential in IDD and OA.

  1. Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs from cell lysate using on-chip isotachophoresis.

    Science.gov (United States)

    Schoch, Reto B; Ronaghi, Mostafa; Santiago, Juan G

    2009-08-07

    We present a technique which enables the separation of small RNAs-such as microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs)-from >or=66 nucleotide RNAs and other biomolecules contained in a cell lysate. In particular, the method achieves separation of small RNAs from precursor miRNAs (pre-miRNAs) in less than 3 min. We use on-chip isotachophoresis (ITP) for the simultaneous extraction, isolation, preconcentration and quantitation of small RNAs (approximately 22 nucleotides) and employ the high-efficiency sieving matrix Pluronic F-127; a thermo-responsive triblock copolymer which allows convenient microchannel loading at low temperature. We present the isolation of small RNAs from the lysate of 293A human kidney cells, and quantitate the number of short RNA molecules per cell to be 2.9x10(7). We estimate this quantity is an aggregate of roughly 500 types of short RNA molecules per 293A cell. Currently, the minimal cell number for small RNA extraction and detection with our method is approximately 900 (from a 5 microL sample volume), and we believe that small RNA analysis from tens of cells is realizable. Techniques for rapid and sensitive extraction and isolation of small RNAs from cell lysate are much-needed to further uncover their full range and functionality, including RNA interference studies.

  2. Micro RNAs in animal development.

    NARCIS (Netherlands)

    Plasterk, R.H.A.

    2006-01-01

    Micro RNAs (miRNAs) are approximately 22 nucleotide single-stranded noncoding RNA molecules that bind to target messenger RNAs (mRNAs) and silence their expression. This Essay explores the importance of miRNAs in animal development and their possible roles in disease and evolution.

  3. Micro RNAs in animal development.

    NARCIS (Netherlands)

    Plasterk, R.H.A.

    2006-01-01

    Micro RNAs (miRNAs) are approximately 22 nucleotide single-stranded noncoding RNA molecules that bind to target messenger RNAs (mRNAs) and silence their expression. This Essay explores the importance of miRNAs in animal development and their possible roles in disease and evolution.

  4. Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants.

    Science.gov (United States)

    Coruh, Ceyda; Cho, Sung Hyun; Shahid, Saima; Liu, Qikun; Wierzbicki, Andrzej; Axtell, Michael J

    2015-08-01

    Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs. © 2015 American Society of Plant Biologists. All rights reserved.

  5. 32 CFR 234.6 - Interfering with agency functions.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 2 2010-07-01 2010-07-01 false Interfering with agency functions. 234.6 Section 234.6 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS CONDUCT ON THE PENTAGON RESERVATION § 234.6 Interfering with agency functions. The following are...

  6. 36 CFR 1002.32 - Interfering with agency functions.

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Interfering with agency functions. 1002.32 Section 1002.32 Parks, Forests, and Public Property PRESIDIO TRUST RESOURCE PROTECTION, PUBLIC USE AND RECREATION § 1002.32 Interfering with agency functions. (a) The following are...

  7. Small RNAs derived from natural antisense transcripts%天然反义转录物(NAT)来源的小分子RNA

    Institute of Scientific and Technical Information of China (English)

    谢兆辉

    2009-01-01

    天然反义转录物(natural antisense transcript,NAT)通常指自然情况下生物体内生成的内源RNA,它们可以与其他RNA部分或完全互补.NAT在生物中非常普遍,并且可以产生多种具有调节作用的小RNA,如天然反义转录干扰小RNA、天然反义微RNA、长的十扰短RNA、扫描RNA和与Piwi相互作用的RNA等.这些小RNA或许是NAT调节基因表达的重要物质分子之一.本文就NAT来源的小RNA及其功能作一慨述.%Natural antisense transcripts (NATs) are endogenous RNA molecules that exhibit partial or complete complementarity to other RNAs. They are common in prokaryotes and eukaryotes, and can produce some small regulatory RNAs. Such as nat-small interfering RNAs (nat-siRNAs), nat-microRNAs (nat-miRNAs), long short interfering RNAs (lsiRNAs), scan RNA (scnRNAs) and PIWl-interacting RNAs (piRNAs). These small RNAs may be involved in the gene regulation by NATs, In this paper, small RNAs derived from NATs and their mechanisms are discussed.

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

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    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.

  9. An inside job for siRNAs.

    Science.gov (United States)

    Golden, Daniel E; Gerbasi, Vincent R; Sontheimer, Erik J

    2008-08-08

    Among the three main categories of small silencing RNAs in insects and mammals-siRNAs, miRNAs, and piRNAs-siRNAs were thought to arise primarily from exogenous sources, whereas miRNAs and piRNAs arise from endogenous loci. Recent work in flies and mice reveals several classes of endogenous siRNAs (endo-siRNAs) that contribute to functions previously reserved for miRNAs and piRNAs, including gene regulation and transposon suppression.

  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. Big impacts by small RNAs in plant development.

    Science.gov (United States)

    Chuck, George; Candela, Héctor; Hake, Sarah

    2009-02-01

    The identification and study of small RNAs, including microRNAs and trans-acting small interfering RNAs, have added a layer of complexity to the many pathways that regulate plant development. These molecules, which function as negative regulators of gene expression, are now known to have greatly expanded roles in a variety of developmental processes affecting all major plant structures, including meristems, leaves, roots, and inflorescences. Mutants with specific developmental phenotypes have also advanced our knowledge of the biogenesis and mode of action of these diverse small RNAs. In addition, previous models on the cell autonomy of microRNAs may have to be revised as more data accumulate supporting their long distance transport. As many of these small RNAs appear to be conserved across different species, knowledge gained from one species is expected to have general application. However, a few surprising differences in small RNA function seem to exist between monocots and dicots regarding meristem initiation and sex determination. Integrating these unique functions into the overall scheme for plant growth will give a more complete picture of how they have evolved as unique developmental systems.

  12. Ingested plant miRNAs regulate gene expression in animals

    Institute of Scientific and Technical Information of China (English)

    Hervé Vaucheret; Yves Chupeau

    2012-01-01

    The incidence of genetic material or epigenetic information transferred from one organism to another is an important biological question.A recent study demonstrated that plant small RNAs acquired orally through food intake directly influence gene expression in animals after migration through the plasma and delivery to specific organs.Non-protein coding RNAs,and in particular small RNAs,were recently revealed as master chief regulators of gene expression in all organisms.Endogenous small RNAs come in different flavors,depending on their mode of biogenesis.Most microRNAs (miRNA)and short interferring RNAs (siRNA)derive from long double-stranded RNA (dsRNA) precursors that are processed into small RNA duplexes,20 to 25-nt long,by RNaselll enzymes called Dicer [1].One strand of small RNA duplexes is loaded onto an Argonaute protein that executes silencing by cleaving or repressing the translation of homologous mRNA [2].In certain species,RNA cleavage is followed by DNA methylation and/or histone modification,leading to heritable epigenetic modification [3].

  13. Probing the MicroRNA and Small Interfering RNA Pathways with Virus-Encoded Suppressors of RNA SilencingW⃞

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    Dunoyer, Patrice; Lecellier, Charles-Henri; Parizotto, Eneida Abreu; Himber, Christophe; Voinnet, Olivier

    2004-01-01

    In plants, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effectors of RNA silencing, a process involved in defense through RNA interference (RNAi) and in development. Plant viruses are natural targets of RNA silencing, and as a counterdefensive strategy, they have evolved highly diverse silencing suppressor proteins. Although viral suppressors are usually thought to act at distinct steps of the silencing machinery, there had been no consensus system so far that allowed a strict side-by-side analysis of those factors. We have set up such a system in Arabidopsis thaliana and used it to compare the effects of five unrelated viral silencing suppressors on the siRNA and miRNA pathways. Although all the suppressors inhibited RNAi, only three of them induced developmental defects, indicating that the two pathways are only partially overlapping. These developmental defects were remarkably similar, and their penetrance correlated with inhibition of miRNA-guided cleavage of endogenous transcripts and not with altered miRNA accumulation per se. Among the suppressors investigated, the tombusviral P19 protein coimmunoprecipitated with siRNA duplexes and miRNA duplexes corresponding to the primary cleavage products of miRNA precursors. Thus, it is likely that P19 prevents RNA silencing by sequestering both classes of small RNAs. Moreover, the finding here that P19 binds siRNAs and suppresses RNAi in Hela cells also suggests that this factor may be useful to dissect the RNA silencing pathways in animals. Finally, the differential effects of the silencing suppressors tested here upon other types of Arabidopsis silencing-related small RNAs revealed a surprising variety of biosynthetic and, presumably, functional pathways for those molecules. Therefore, silencing suppressors are valuable probes of the complexity of RNA silencing. PMID:15084715

  14. Virus-Encoded microRNAs: Future Therapeutic Targets?

    Institute of Scientific and Technical Information of China (English)

    Peng Qi; Jinxiang Han; Yanqin Lu; Chuanxi Wang; Fanfeng Bu

    2006-01-01

    The discovery of microRNAs (miRNAs) is a remarkable breakthrough in the field of molecular genetics, as miRNAs are key actors which regulate gene expression in diverse cellular processes from unicellular yeast to human. The recent discovery of virus-encoded miRNAs indicates that viruses also use this fundamental mode of gene regulation. Research into viral miRNAs function demonstrates that some miRNAs play an important role in regulating both the viral life cycle and the interaction between viruses and their hosts. The first in vivo "antagomir" study provides an exciting first step towards miRNA therapy, and the potential for ultimately designing molecular medicines based on the modulation of miRNAs seems good.

  15. Dermal/transdermal delivery of small interfering RNA and antisense oligonucleotides- advances and hurdles.

    Science.gov (United States)

    Ita, Kevin

    2017-03-01

    A diverse array of nucleic acids has been studied by several researchers for the management of several diseases. Among these compounds, small interfering RNA and antisense oligonucleotides have attracted considerable attention. Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA while siRNAs, on the other hand, are double-stranded RNA molecules which can hybridize with a specific mRNA sequence and block the translation of numerous genes. One of the main obstacles in the dermal or transdermal delivery of these compounds is their low skin permeability. In this review, various techniques used to enhance the delivery of these molecules into or across the skin are described and in some cases, the correlation between enhanced dermal/transdermal delivery and therapeutic efficacy is highlighted. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  16. MicroRNAs

    DEFF Research Database (Denmark)

    Devaux, Yvan; Stammet, Pascal; Friberg, Hans

    2015-01-01

    cardiac arrest would allow subsequent health care delivery to be tailored to individual patients. However, currently available predictive methods and biomarkers lack sufficient accuracy and therefore cannot be generally recommended in clinical practice. MicroRNAs have recently emerged as potential...... biomarkers of cardiovascular diseases. While the biomarker value of microRNAs for myocardial infarction or heart failure has been extensively studied, less attention has been devoted to their prognostic value after cardiac arrest. This review highlights the recent discoveries suggesting that microRNAs may...

  17. Cloning of Novel Repeat-associated Small RNAs Derived from Hairpin Precursors in Oryza sativa

    Institute of Scientific and Technical Information of China (English)

    Chengguo YAO; Botao ZHAO; Wei LI; Yang LI; Wenming QIN; Bing HUANG; Youxin JIN

    2007-01-01

    Plant small non-coding RNAs including microRNAs (miRNAs), small interfering RNAs (siRNAs) and trans-acting siRNAs, play important roles in modulating gene expression in cells. Here we isolated 21 novel endogenous small RNA molecules, ranging from 18 to 24 nucleotides, in Oryza sativa that can be mapped to 111 hairpin precursors. Further analysis indicated that most of these hairpin sequences originated from putative miniature inverted-repeat transposable elements, a major type of DNA transposon.Considering that miRNA is characteristic of hairpin-like precursor and plant endogenous siRNAs are often located at transposon regions, we hypothesized that our cloned small RNAs might represent the intermediate product in the evolutionary process between siRNAs and miRNAs. Northern blot analysis indicated that five of them were much more abundantly expressed in flower compared to other tissues, implying their potential function in inflorescence. In conclusion, our results enrich rice small RNA data and provide a meaningful perspective for small RNA annotation in plants.

  18. Expression Signatures of Long Noncoding RNAs in Adolescent Idiopathic Scoliosis

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    Xiao-Yang Liu

    2015-01-01

    Full Text Available Purpose. Adolescent idiopathic scoliosis (AIS, the most common pediatric spinal deformity, is considered a complex genetic disease. Causing genes and pathogenesis of AIS are still unclear. This study was designed to identify differentially expressed long noncoding RNAs (lncRNAs involving the pathogenesis of AIS. Methods. We first performed comprehensive screening of lncRNA and mRNA in AIS patients and healthy children using Agilent human lncRNA + mRNA Array V3.0 microarray. LncRNAs expression in different AIS patients was further evaluated using quantitative PCR. Results. A total of 139 lncRNAs and 546 mRNAs were differentially expressed between AIS patients and healthy control. GO and Pathway analysis showed that these mRNAs might be involved in bone mineralization, neuromuscular junction, skeletal system morphogenesis, nucleotide and nucleic acid metabolism, and regulation of signal pathway. Four lncRNAs (ENST00000440778.1, ENST00000602322.1, ENST00000414894.1, and TCONS_00028768 were differentially expressed between different patients when grouped according to age, height, classification, severity of scoliosis, and Risser grade. Conclusions. This study demonstrates the abnormal expression of lncRNAs and mRNAs in AIS, and the expression of some lncRNAs was related to clinical features. This study is helpful for further understanding of lncRNAs in pathogenesis, treatment, and prognosis of AIS.

  19. Expression Signatures of Long Noncoding RNAs in Adolescent Idiopathic Scoliosis

    Science.gov (United States)

    Liu, Xiao-Yang; Wang, Liang; Yu, Bin; Zhuang, Qian-yu; Wang, Yi-Peng

    2015-01-01

    Purpose. Adolescent idiopathic scoliosis (AIS), the most common pediatric spinal deformity, is considered a complex genetic disease. Causing genes and pathogenesis of AIS are still unclear. This study was designed to identify differentially expressed long noncoding RNAs (lncRNAs) involving the pathogenesis of AIS. Methods. We first performed comprehensive screening of lncRNA and mRNA in AIS patients and healthy children using Agilent human lncRNA + mRNA Array V3.0 microarray. LncRNAs expression in different AIS patients was further evaluated using quantitative PCR. Results. A total of 139 lncRNAs and 546 mRNAs were differentially expressed between AIS patients and healthy control. GO and Pathway analysis showed that these mRNAs might be involved in bone mineralization, neuromuscular junction, skeletal system morphogenesis, nucleotide and nucleic acid metabolism, and regulation of signal pathway. Four lncRNAs (ENST00000440778.1, ENST00000602322.1, ENST00000414894.1, and TCONS_00028768) were differentially expressed between different patients when grouped according to age, height, classification, severity of scoliosis, and Risser grade. Conclusions. This study demonstrates the abnormal expression of lncRNAs and mRNAs in AIS, and the expression of some lncRNAs was related to clinical features. This study is helpful for further understanding of lncRNAs in pathogenesis, treatment, and prognosis of AIS. PMID:26421281

  20. Essential Role for endogenous siRNAs during meiosis in mouse oocytes.

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    Paula Stein

    2015-02-01

    Full Text Available The RNase III enzyme DICER generates both microRNAs (miRNAs and endogenous short interfering RNAs (endo-siRNAs. Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO family of proteins. In mammals, there are four AGO proteins (AGO1-4, of which only AGO2 possesses endonucleolytic activity. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice that express a catalytically inactive knock-in allele of Ago2 (Ago2ADH exclusively in oocytes and thereby disrupted the function of siRNAs. Oogenesis and hormonal response are normal in Ago2ADH oocytes, but meiotic maturation is impaired, with severe defects in spindle formation and chromosome alignment that lead to meiotic catastrophe. The transcriptome of these oocytes is widely perturbed and shows a highly significant correlation with the transcriptome of Dicer null and Ago2 null oocytes. Expression of the mouse transcript (MT, the most abundant transposable element in mouse oocytes, is increased. This study reveals that endo-siRNAs are essential during meiosis I in mouse females, demonstrating a role for endo-siRNAs in mammals.

  1. α/β oscillations indicate inhibition of interfering visual memories.

    Science.gov (United States)

    Waldhauser, Gerd T; Johansson, Mikael; Hanslmayr, Simon

    2012-02-08

    Selective retrieval of a specific target memory often leads to the forgetting of related but irrelevant memories. Current cognitive theory states that such retrieval-induced forgetting arises due to inhibition of competing memory traces. To date, however, direct neural evidence for this claim has not been forthcoming. Studies on selective attention suggest that cortical inhibition is mediated by increased brain oscillatory activity in the alpha/beta frequency band. The present study, testing 18 human subjects, investigated whether these mechanisms can be generalized to selective memory retrieval in which competing memories interfere with the retrieval of a target memory. Our experiment was designed so that each cue used to search memory was associated with a target memory and a competitor memory stored in separate brain hemispheres. Retrieval-induced forgetting was observed in a condition in which the competitor memory interfered with target retrieval. Increased oscillatory alpha/beta power was observed over the hemisphere housing the sensory representation of the competitor memory trace and predicted the amount of retrieval-induced forgetting in the subsequent memory test. These results provide the first direct evidence for inhibition of competing memories during episodic memory retrieval and suggest that competitive retrieval is governed by inhibitory mechanisms similar to those employed in selective attention.

  2. Structural and functional analysis of viral siRNAs.

    Directory of Open Access Journals (Sweden)

    Gyorgy Szittya

    2010-04-01

    Full Text Available A large amount of short interfering RNA (vsiRNA is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a

  3. Optimizations of siRNA design for the activation of gene transcription by targeting the TATA-box motif.

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    Miaomiao Fan

    Full Text Available Small interfering RNAs (siRNAs are widely used to repress gene expression by targeting mRNAs. Some reports reveal that siRNAs can also activate or inhibit gene expression through targeting the gene promoters. Our group has found that microRNAs (miRNAs could activate gene transcription via interaction with the TATA-box motif in gene promoters. To investigate whether siRNA targeting the same region could upregulate the promoter activity, we test the activating efficiency of siRNAs targeting the TATA-box motif of 16 genes and perform a systematic analysis to identify the common features of the functional siRNAs for effective activation of gene promoters. Further, we try various modifications to improve the activating efficiency of siRNAs and find that it is quite useful to design the promoter-targeting activating siRNA by following several rules such as (a complementary to the TATA-box-centered region; (b UA usage at the first two bases of the antisense strand; (c twenty-three nucleotides (nts in length; (d 2'-O-Methyl (2'-OMe modification at the 3' terminus of the antisense strand; (e avoiding mismatches at the 3' end of the antisense strand. The optimized activating siRNAs potently enhance the expression of interleukin-2 (IL-2 gene in human and mouse primary CD4+ T cells with a long-time effect. Taken together, our study provides a guideline for rational design the promoter-targeting siRNA to sequence-specifically enhance gene expression.

  4. Unzipping and binding of small interfering RNA with single walled Carbon Nanotube: a platform for small interfering RNA delivery

    CERN Document Server

    Santosh, Mogurampelly; Bhattacharyya, Dhananjay; Sood, A K; Maiti, Prabal K; 10.1063/1.3682780

    2012-01-01

    In an effort to design efficient platform for siRNA delivery, we combine all atom classical and quantum simulations to study the binding of small interfering RNA (siRNA) by pristine single wall carbon nanotube (SWCNT). Our results show that siRNA strongly binds to SWCNT surface via unzipping its base-pairs and the propensity of unzipping increases with the increase in the diameter of the SWCNTs. The unzipping and subsequent wrapping events are initiated and driven by van der Waals interactions between the aromatic rings of siRNA nucleobases and the SWCNT surface. However, MD simulations of double strand DNA (dsDNA) of the same sequence show that the dsDNA undergoes much less unzipping and wrapping on the SWCNT in the simulation time scale of 70 ns. This interesting difference is due to smaller interaction energy of thymidine of dsDNA with the SWCNT compared to that of uridine of siRNA, as calculated by dispersion corrected density functional theory (DFT) methods. After the optimal binding of siRNA to SWCNT, t...

  5. Silencing by small RNAs is linked to endosomal trafficking.

    Science.gov (United States)

    Lee, Young Sik; Pressman, Sigal; Andress, Arlise P; Kim, Kevin; White, Jamie L; Cassidy, Justin J; Li, Xin; Lubell, Kim; Lim, Do Hwan; Cho, Ik Sang; Nakahara, Kenji; Preall, Jonathan B; Bellare, Priya; Sontheimer, Erik J; Carthew, Richard W

    2009-09-01

    Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport) depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.

  6. Identification and characterization of small RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus.

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    Ning Xu

    Full Text Available The term RNA silencing (RNA interference, RNAi describes a set of mechanisms that regulate gene expression in eukaryotes. Small interfering RNAs (siRNA and microRNAs (miRNAs are two major types of RNAi-associated small RNAs (smRNAs found in most eukaryotic organisms. Despite the presence of a plethora of non-coding RNAs longer than 50-nucleotide (nt in length in various species of Archaea, little is known about smRNAs in archaea that resemble the 20-24-nt long smRNAs found in eukaryotes, which have been implicated in the post-transcriptional control of gene expression. Here, we report the finding of a large number of smRNAs approximatelly 20-nt in length, including phased smRNAs and potential miRNAs, from the hyperthermophilic archaeon Sulfolobus solfataricus p2 (Ssp2 based on deep sequencing. The expression of some of the miRNA candidates in Ssp2 was confirmed. Consistent with the Ssp2 hyperthermophilic properties, we found that higher temperatures more efficiently induced the production of the miRNA candidates in an in vitro system using the putative foldback precursor transcripts incubated with Ssp2 extract. Although we initially predicted putative target genes of some miRNA candidates, further analysis mapped the cleavage sites downstream of the miRNA candidate complementary regions, similar to those involved in plant miRNA-mediated TAS transcript cleavage. We also identified smRNAs from clustered, regularly interspaced, short palindromic repeat (CRISPR loci, which play important roles in prokaryotic microbial defense systems. Archaea represent a unique life form next to Bacteria and Eukarya, and our results may provide a useful resource for further in-depth study on the regulation and evolution of smRNAs in this special organism.

  7. Unique Gene-Silencing and Structural Properties of 2;#8242;-Fluoro-Modified siRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Manoharan, Muthiah; Akinc, Akin; Pandey, Rajendra K.; Qin, June; Hadwiger, Philipp; John, Matthias; Mills, Kathy; Charisse, Klaus; Maier, Martin A.; Nechev, Lubomir; Greene, Emily M.; Pallan, Pradeep S.; Rozners, Eriks; Rajeev, Kallanthottathil G.; Egli, Martin (Binghamton); (Alnylam Pharm.); (Vanderbilt)

    2015-10-15

    With little or no negative impact on the activity of small interfering RNAs (siRNAs), regardless of the number of modifications or the positions within the strand, the 2'-deoxy-2'-fluoro (2'-F) modification is unique. Furthermore, the 2'-F-modified siRNA (see crystal structure) was thermodynamically more stable and more nuclease-resistant than the parent siRNA, and produced no immunostimulatory response.

  8. Identification of small RNAs in late developmental stage of rice anthers.

    Science.gov (United States)

    Fujioka, Tomoaki; Kaneko, Fumi; Kazama, Tomohiko; Suwabe, Keita; Suzuki, Go; Makino, Amane; Mae, Tadahiko; Endo, Makoto; Kawagishi-Kobayashi, Makiko; Watanabe, Masao

    2008-06-01

    Small RNAs including microRNA (miRNA) and small interfering RNA (siRNA) are known as repressors of gene expression. There are many plant proteins involved in small RNA-mediated gene silencing, such as Dicer ribonucleases and RNA-dependent RNA polymerases. However, most of these proteins have been reported to be absent in the late developmental stage of the plant male gamete, pollen. In order to clarify the existence of the small RNAs during maturation of pollen, we cloned and sequenced small RNAs from rice anthers including tricellular pollen. From fifty six candidates of small RNAs, we identified two known miRNAs (miR166 and miR167), eight potential miRNAs, and ten putative heterochromatic siRNAs (hc-siRNAs). RNA gel blot analyses clearly showed that miR166 and miR167 were accumulated in the uninuclear pollen stage of anther development and remained until the tricellular pollen stage. Our cloning and RNA gel blot analyses of small RNAs led us to propose a possible function of small RNA-mediated gene regulation for the development of male gametes in rice.

  9. Discovery of mosquito saliva microRNAs during CHIKV infection.

    Science.gov (United States)

    Maharaj, Payal D; Widen, Steven G; Huang, Jing; Wood, Thomas G; Thangamani, Saravanan

    2015-01-01

    Mosquito borne pathogens are transmitted to humans via saliva during blood feeding. Mosquito saliva is a complex concoction of many secretory factors that modulate the feeding foci to enhance pathogen infection and establishment. Multiple salivary proteins/factors have been identified/characterized that enhance pathogen infection. Here, we describe, for the first time, the identification of exogenous microRNAs from mosquito saliva. MicroRNAs are short, 18-24 nucleotide, non-coding RNAs that regulate gene expression, and are generally intracellular. However, circulating miRNAs have been described from serum and saliva of humans. Exogenous miRNAs have not been reported from hematophagous arthropod saliva. We sought to identify miRNAs in the mosquito saliva and their role in Chikungunya virus (CHIKV) infection. Next generation sequencing was utilized to identify 103 exogenous miRNAs in mosquito saliva of which 31 miRNAs were previously unidentified and were designated novel. Several miRNAs that we have identified are expressed only in the CHIKV infected mosquitoes. Five of the saliva miRNAs were tested for their potential to regulated CHIKV infection, and our results demonstrate their functional role in the transmission and establishment of infection during blood feeding on the host.

  10. Discovery of Mosquito Saliva MicroRNAs during CHIKV Infection

    Science.gov (United States)

    Maharaj, Payal D.; Widen, Steven G.; Huang, Jing; Wood, Thomas G.; Thangamani, Saravanan

    2015-01-01

    Mosquito borne pathogens are transmitted to humans via saliva during blood feeding. Mosquito saliva is a complex concoction of many secretory factors that modulate the feeding foci to enhance pathogen infection and establishment. Multiple salivary proteins/factors have been identified/characterized that enhance pathogen infection. Here, we describe, for the first time, the identification of exogenous microRNAs from mosquito saliva. MicroRNAs are short, 18–24 nucleotide, non-coding RNAs that regulate gene expression, and are generally intracellular. However, circulating miRNAs have been described from serum and saliva of humans. Exogenous miRNAs have not been reported from hematophagous arthropod saliva. We sought to identify miRNAs in the mosquito saliva and their role in Chikungunya virus (CHIKV) infection. Next generation sequencing was utilized to identify 103 exogenous miRNAs in mosquito saliva of which 31 miRNAs were previously unidentified and were designated novel. Several miRNAs that we have identified are expressed only in the CHIKV infected mosquitoes. Five of the saliva miRNAs were tested for their potential to regulated CHIKV infection, and our results demonstrate their functional role in the transmission and establishment of infection during blood feeding on the host. PMID:25612225

  11. Laser-Triggered Small Interfering RNA Releasing Gold Nanoshells against Heat Shock Protein for Sensitized Photothermal Therapy.

    Science.gov (United States)

    Wang, Zhaohui; Li, Siwen; Zhang, Min; Ma, Yi; Liu, Yuxi; Gao, Weidong; Zhang, Jiaqi; Gu, Yueqing

    2017-02-01

    The resistance of cancer cells to photothermal therapy is closely related to the overexpression of heat shock proteins (HSPs), which are abnormally upregulated when cells are under lethal stresses. Common strategies that use small molecule inhibitors against HSPs to enhance hyperthermia effect lack spatial and temporal control of drug release, leading to unavoidable systemic toxicity. Herein, a versatile photothermal platform is developed which is composed of a hollow gold nanoshell core densely packed with small interfering RNAs against heat shock protein 70 (Hsp70). Upon near infrared light irradiation, the small interfering RNAs can detach from gold surface specifically and escape from endosomes for Hsp70 silencing. Meanwhile, the temperature increases for hyperthermia therapy due to the high photothermal efficiency of the nanoshells. Efficient downregulation of Hsp70 after light activation is achieved in vitro and in vivo. Ultimately, the light-controlled dual functional nanosystem, with the effects of Hsp70 silencing and temperature elevation, results in sensitized photothermal therapy in nude mice model under mild temperature. This strategy smartly combines the localized photothermal therapy with controlled Hsp70 silencing, and has great potential for clinical translation with a simple and easily controlled structure.

  12. microRNAs Databases: Developmental Methodologies, Structural and Functional Annotations.

    Science.gov (United States)

    Singh, Nagendra Kumar

    2017-09-01

    microRNA (miRNA) is an endogenous and evolutionary conserved non-coding RNA, involved in post-transcriptional process as gene repressor and mRNA cleavage through RNA-induced silencing complex (RISC) formation. In RISC, miRNA binds in complementary base pair with targeted mRNA along with Argonaut proteins complex, causes gene repression or endonucleolytic cleavage of mRNAs and results in many diseases and syndromes. After the discovery of miRNA lin-4 and let-7, subsequently large numbers of miRNAs were discovered by low-throughput and high-throughput experimental techniques along with computational process in various biological and metabolic processes. The miRNAs are important non-coding RNA for understanding the complex biological phenomena of organism because it controls the gene regulation. This paper reviews miRNA databases with structural and functional annotations developed by various researchers. These databases contain structural and functional information of animal, plant and virus miRNAs including miRNAs-associated diseases, stress resistance in plant, miRNAs take part in various biological processes, effect of miRNAs interaction on drugs and environment, effect of variance on miRNAs, miRNAs gene expression analysis, sequence of miRNAs, structure of miRNAs. This review focuses on the developmental methodology of miRNA databases such as computational tools and methods used for extraction of miRNAs annotation from different resources or through experiment. This study also discusses the efficiency of user interface design of every database along with current entry and annotations of miRNA (pathways, gene ontology, disease ontology, etc.). Here, an integrated schematic diagram of construction process for databases is also drawn along with tabular and graphical comparison of various types of entries in different databases. Aim of this paper is to present the importance of miRNAs-related resources at a single place.

  13. Bioinformatics of prokaryotic RNAs.

    Science.gov (United States)

    Backofen, Rolf; Amman, Fabian; Costa, Fabrizio; Findeiß, Sven; Richter, Andreas S; Stadler, Peter F

    2014-01-01

    The genome of most prokaryotes gives rise to surprisingly complex transcriptomes, comprising not only protein-coding mRNAs, often organized as operons, but also harbors dozens or even hundreds of highly structured small regulatory RNAs and unexpectedly large levels of anti-sense transcripts. Comprehensive surveys of prokaryotic transcriptomes and the need to characterize also their non-coding components is heavily dependent on computational methods and workflows, many of which have been developed or at least adapted specifically for the use with bacterial and archaeal data. This review provides an overview on the state-of-the-art of RNA bioinformatics focusing on applications to prokaryotes.

  14. Micro-RNAs

    DEFF Research Database (Denmark)

    Taipaleenmäki, H.; Hokland, L. B.; Chen, Li

    2012-01-01

    Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post-transcriptional mechanisms. Recently, a novel class of regulatory factors termed microRNAs has been identified as playing an important role in the regulation of many aspects of osteoblast biology...... including proliferation, differentiation, metabolism and apoptosis. Also, preliminary data from animal disease models suggest that targeting miRNAs in bone can be a novel approach to increase bone mass. This review highlights the current knowledge of microRNA biology and their role in bone formation...

  15. Unexpected Diversity of Chloroplast Noncoding RNAs as Revealed by Deep Sequencing of the Arabidopsis Transcriptome.

    Science.gov (United States)

    Hotto, Amber M; Schmitz, Robert J; Fei, Zhangjun; Ecker, Joseph R; Stern, David B

    2011-12-01

    Noncoding RNAs (ncRNA) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly micro- and small-interfering RNAs (18-25 nt) involved in posttranscriptional gene silencing, whereas prokaryotic ncRNAs vary in size and are involved in various aspects of gene regulation. Given the prokaryotic origin of organelles, the presence of ncRNAs might be expected; however, the full spectrum of organellar ncRNAs has not been determined systematically. Here, strand-specific RNA-Seq analysis was used to identify 107 candidate ncRNAs from Arabidopsis thaliana chloroplasts, primarily encoded opposite protein-coding and tRNA genes. Forty-eight ncRNAs were shown to accumulate by RNA gel blot as discrete transcripts in wild-type (WT) plants and/or the pnp1-1 mutant, which lacks the chloroplast ribonuclease polynucleotide phosphorylase (cpPNPase). Ninety-eight percent of the ncRNAs detected by RNA gel blot had different transcript patterns between WT and pnp1-1, suggesting cpPNPase has a significant role in chloroplast ncRNA biogenesis and accumulation. Analysis of materials deficient for other major chloroplast ribonucleases, RNase R, RNase E, and RNase J, showed differential effects on ncRNA accumulation and/or form, suggesting specificity in RNase-ncRNA interactions. 5' end mapping demonstrates that some ncRNAs are transcribed from dedicated promoters, whereas others result from transcriptional read-through. Finally, correlations between accumulation of some ncRNAs and the symmetrically transcribed sense RNA are consistent with a role in RNA stability. Overall, our data suggest that this extensive population of ncRNAs has the potential to underpin a previously underappreciated regulatory mode in the chloroplast.

  16. Viral small RNAs reveal the genomic variations of three grapevine vein clearing virus quasispecies populations.

    Science.gov (United States)

    Howard, Susanne; Qiu, Wenping

    2017-02-02

    Viral small RNAs (vsRNAs) include viral small interfering RNAs (vsiRNAs) that are initiators and products of RNA silencing, and small RNAs that are derived from viral RNAs with function still unknown. Sequencing of vsRNAs allows assembling of viral genomes and revelation of viral population variations at genomic levels. Grapevine vein clearing virus (GVCV) is a new member of the family Caulimoviridae whose DNA genome is replicated by reverse transcription of pre-genomic RNA molecules. In this short report, three genomic sequences of GVCV were assembled from vsRNAs that were isolated and sequenced from three individual grapevines in commercial vineyards and compared to the GVCV-CHA reference genome. Profiles of single nucleotide polymorphism among three viral populations indicated a closer relatedness between two populations in different grape cultivars at the same location than those in the same grape cultivar at different locations, suggesting the spread of GVCV populations among vineyards of close proximity. Classic types of vsiRNAs (21-nt, 22-nt, and 24-nt) were found in the three GVCV vsiRNA populations, but these did not produce alignment hotspots on the GVCV-CHA reference genome. The number of 36-nt reads is the highest among vsRNAs, the role of these vsRNAs remains unclear. The analysis of vsRNAs provides a first holistic picture of genomic variations among GVCV viral quasispecies populations that help monitor epidemics and evolution of GVCV populations, an emerging virus that is becoming a threat to grape production in the Midwest region of the USA.

  17. Primary and secondary siRNAs in geminivirus-induced gene silencing.

    Directory of Open Access Journals (Sweden)

    Michael Aregger

    2012-09-01

    Full Text Available In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL proteins producing short interfering (siRNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV, four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR. However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3'-untranslated region (UTR induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5'-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.

  18. Transcriptome of small regulatory RNAs in the development of the zoonotic parasite Trichinella spiralis.

    Directory of Open Access Journals (Sweden)

    Xiaolei Liu

    Full Text Available BACKGROUND: Trichinella spiralis is a parasite with unique features. It is a multicellular organism but with an intracellular parasitization and development stage. T. spiralis is the helminthic pathogen that causes zoonotic trichinellosis and afflicts more than 10 million people worldwide, whereas the parasite's biology, especially the developmental regulation is largely unknown. In other organisms, small non-coding RNAs, such as microRNAs (miRNA and small interfering RNAs (siRNA execute post-transcriptional regulation by translational repression or mRNA degradation, and a large number of miRNAs have been identified in diverse species. In T. spiralis, the profile of small non-coding RNAs and their function remains poorly understood. METHODOLOGY AND PRINCIPAL FINDINGS: Here, the transcriptional profiles of miRNA and siRNA in three developmental stages of T. spiralis in the rat host were investigated, and compared by high-throughput cDNA sequencing technique ("RNA-seq". 5,443,641 unique sequence tags were obtained. Of these, 21 represented conserved miRNAs related to 13 previously identified metazoan miRNA families and 213 were novel miRNAs so far unique to T. spiralis. Some of these miRNAs exhibited stage-specific expression. Expression of miRNAs was confirmed in three stages of the life cycle by qRT-PCR and northern blot analysis. In addition, endogenous siRNAs (endo-siRNAs were found mainly derived from natural antisense transcripts (NAT and transposable elements (TE in the parasite. CONCLUSIONS AND SIGNIFICANCE: We provide evidence for the presence of miRNAs and endo-siRNAs in T. spiralis. The miRNAs accounted for the major proportion of the small regulatory RNA population of T. spiralis, while fewer endogenous siRNAs were found. The finding of stage-specific expression patterns of the miRNAs in different developmental stages of T. spiralis suggests that miRNAs may play important roles in parasite development. Our data provide a basis for

  19. Designing siRNA that distinguish between genes that differ by a single nucleotide.

    Science.gov (United States)

    Schwarz, Dianne S; Ding, Hongliu; Kennington, Lori; Moore, Jessica T; Schelter, Janell; Burchard, Julja; Linsley, Peter S; Aronin, Neil; Xu, Zuoshang; Zamore, Phillip D

    2006-09-01

    Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.

  20. Designing siRNA that distinguish between genes that differ by a single nucleotide.

    Directory of Open Access Journals (Sweden)

    Dianne S Schwarz

    2006-09-01

    Full Text Available Small interfering RNAs (siRNAs, the guides that direct RNA interference (RNAi, provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1 gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.

  1. Satellite RNAs and Satellite Viruses.

    Science.gov (United States)

    Palukaitis, Peter

    2016-03-01

    Satellite RNAs and satellite viruses are extraviral components that can affect either the pathogenicity, the accumulation, or both of their associated viruses while themselves being dependent on the associated viruses as helper viruses for their infection. Most of these satellite RNAs are noncoding RNAs, and in many cases, have been shown to alter the interaction of their helper viruses with their hosts. In only a few cases have the functions of these satellite RNAs in such interactions been studied in detail. In particular, work on the satellite RNAs of Cucumber mosaic virus and Turnip crinkle virus have provided novel insights into RNAs functioning as noncoding RNAs. These effects are described and potential roles for satellite RNAs in the processes involved in symptom intensification or attenuation are discussed. In most cases, models describing these roles involve some aspect of RNA silencing or its suppression, either directly or indirectly involving the particular satellite RNA.

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

    NARCIS (Netherlands)

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

    2016-01-01

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

  3. Satellite RNAs interfere with the function of viral RNA silencing suppressors

    Directory of Open Access Journals (Sweden)

    Wanxia eShen

    2015-04-01

    Full Text Available Viral satellite RNAs (satRNAs are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs, Cucumber mosaic virus (CMV 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA-induced silencing of a β-glucuronidase (GUS gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat via the Y-Sat-derived small interfering RNAs (siRNAs, which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function.

  4. Identification of novel non-coding RNAs using profiles of short sequence reads from next generation sequencing data

    Directory of Open Access Journals (Sweden)

    Makunin Igor V

    2010-02-01

    Full Text Available Abstract Background The increasing interest in small non-coding RNAs (ncRNAs such as microRNAs (miRNAs, small interfering RNAs (siRNAs and Piwi-interacting RNAs (piRNAs and recent advances in sequencing technology have yielded large numbers of short (18-32 nt RNA sequences from different organisms, some of which are derived from small nucleolar RNAs (snoRNAs and transfer RNAs (tRNAs. We observed that these short ncRNAs frequently cover the entire length of annotated snoRNAs or tRNAs, which suggests that other loci specifying similar ncRNAs can be identified by clusters of short RNA sequences. Results We combined publicly available datasets of tens of millions of short RNA sequence tags from Drosophila melanogaster, and mapped them to the Drosophila genome. Approximately 6 million perfectly mapping sequence tags were then assembled into 521,302 tag-contigs (TCs based on tag overlap. Most transposon-derived sequences, exons and annotated miRNAs, tRNAs and snoRNAs are detected by TCs, which show distinct patterns of length and tag-depth for different categories. The typical length and tag-depth of snoRNA-derived TCs was used to predict 7 previously unrecognized box H/ACA and 26 box C/D snoRNA candidates. We also identified one snRNA candidate and 86 loci with a high number of tags that are yet to be annotated, 7 of which have a particular 18mer motif and are located in introns of genes involved in development. A subset of new snoRNA candidates and putative ncRNA candidates was verified by Northern blot. Conclusions In this study, we have introduced a new approach to identify new members of known classes of ncRNAs based on the features of TCs corresponding to known ncRNAs. A large number of the identified TCs are yet to be examined experimentally suggesting that many more novel ncRNAs remain to be discovered.

  5. Spin O decay angular distribution for interfering mesons in electroproduction

    Energy Technology Data Exchange (ETDEWEB)

    Funsten, H.; Gilfoyle, G.

    1994-04-01

    Self analyzing meson electroproduction experiments are currently being planned for the CEBAF CLAS detector. These experiments deduce the spin polarization of outgoing unstable spin s (?)0 mesons from their decay angular distribution, W({theta},{psi}). The large angular acceptance of the CLAS detector permits kinematic tracking of a sufficient number of these events to accurately determine electroproduction amplitudes from the deduced polarization. Maximum polarization information is obtained from W({theta},{psi}) for decay into spin 0 daughters. The helicity of the decaying meson is transferred to the daughter`s relative orbital angular momentum m-projection; none is {open_quotes}absorbed{close_quotes} into daughter helicities. The decaying meson`s helicity maximally appears in W({theta},{psi}). W({theta},{psi}) for spin 0 daughters has been derived for (1) vector meson electroproduction and (2) general interfering mesons produced by incident pions. This paper derives W({theta},{psi}) for electroproduction of two interfering mesons that decay into spin 0 daughters. An application is made to the case of interfering scalar and vector mesons. The derivation is an extension of work by Schil using the general decay formalism of Martin. The expressions can be easily extended to the case of N interfering mesons since interference occurs pairwise in the observable W ({theta},{psi}), a quadratic function of the meson amplitudes. The derivation uses the virtual photon density matrix of Schil which is transformed by a meson electroproduction transition operator, T. The resulting density matrix for the interfering mesons is then converted into a corresponding statistical tensor and contracted into the efficiency tensor for spin 0 daughters.

  6. Long noncoding RNAs and atherosclerosis.

    Science.gov (United States)

    Zhou, Tian; Ding, Jia-wang; Wang, Xin-an; Zheng, Xia-xia

    2016-05-01

    Atherosclerosis is universally recognized as a chronic lipid-induced inflammation of the vessel wall in response to dyslipidemia and haemodynamic stress involving dysfunction and activation of resident vascular cells as well as infiltration of leukocytes. As members of nonprotein-coding RNAs, the long noncoding RNAs (lncRNAs) are implicated in various biological processes. Accumulating evidences suggest that lncRNAs regulate the function of vascular wall, activation of macrophages, lipid metabolism and immune response. Here, we review the effects of lncRNAs on the progress of atherosclerosis.

  7. Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations.

    Science.gov (United States)

    Sakaguchi, Aisa; Sarkies, Peter; Simon, Matt; Doebley, Anna-Lisa; Goldstein, Leonard D; Hedges, Ashley; Ikegami, Kohta; Alvares, Stacy M; Yang, Liwei; LaRocque, Jeannine R; Hall, Julie; Miska, Eric A; Ahmed, Shawn

    2014-10-14

    Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of late-generation rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing.

  8. Plant Mobile Small RNAs

    OpenAIRE

    Dunoyer, Patrice; Melnyk, Charles; Molnar, Attila; Slotkin, R Keith

    2013-01-01

    In plants, RNA silencing is a fundamental regulator of gene expression, heterochromatin formation, suppression of transposable elements, and defense against viruses. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. Although the spreading of RNA silencing across the plant has been recognized for nearly two decades, only recently have sRNAs been formally demonstrated as the mobile silencing signals. Here, we discuss the various types of mobile sRNA mol...

  9. Immunostimulatory motifs enhance antiviral siRNAs targeting highly pathogenic avian influenza H5N1.

    Directory of Open Access Journals (Sweden)

    Cameron R Stewart

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 virus is endemic in many regions around the world and remains a significant pandemic threat. To date H5N1 has claimed almost 300 human lives worldwide, with a mortality rate of 60% and has caused the death or culling of hundreds of millions of poultry since its initial outbreak in 1997. We have designed multi-functional RNA interference (RNAi-based therapeutics targeting H5N1 that degrade viral mRNA via the RNAi pathway while at the same time augmenting the host antiviral response by inducing host type I interferon (IFN production. Moreover, we have identified two factors critical for maximising the immunostimulatory properties of short interfering (siRNAs in chicken cells (i mode of synthesis and (ii nucleoside sequence to augment the response to virus. The 5-bp nucleoside sequence 5'-UGUGU-3' is a key determinant in inducing high levels of expression of IFN-α, -β, -λ and interleukin 1-β in chicken cells. Positioning of this 5'-UGUGU-3' motif at the 5'-end of the sense strand of siRNAs, but not the 3'-end, resulted in a rapid and enhanced induction of type I IFN. An anti-H5N1 avian influenza siRNA directed against the PB1 gene (PB1-2257 tagged with 5'-UGUGU-3' induced type I IFN earlier and to a greater extent compared to a non-tagged PB1-2257. Tested against H5N1 in vitro, the tagged PB1-2257 was more effective than non-tagged PB1-2257. These data demonstrate the ability of an immunostimulatory motif to improve the performance of an RNAi-based antiviral, a finding that may influence the design of future RNAi-based anti-influenza therapeutics.

  10. Small interfering RNA targeted to secretory clusterin blocks tumor growth, motility, and invasion in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Zhaohe Niu; Xinhui Li; Bin Hu; Rong Li; Ligang Wang; Lilin Wu; Xingang Wang

    2012-01-01

    Clusterin/apolipoprotein J (Clu) is a ubiquitously expressed secreted heterodimeric glycoprotein that is implicated in several physiological processes.It has been reported that the elevated level of secreted clusterin (sClu) protein is associated with poor survival in breast cancer patients and can induce metastasis in rodent models.In this study,we investigated the effects of sClu inhibition with small interfering RNAs (siRNAs) on cell motility,invasion,and growth in vitro and in vivo.MDA-MB-231 cells were transfected with pSuper-siRNA/sClu.Cell survival and proliferation were examined by 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and clonogenic survival assay.The results showed that sClu silencing significantly inhibited the proliferation of MDA-MB-231 cells.The invasion and migration ability were also dramatically decreased,which was detected by matrigel assays.TUNEL staining and caspase-3 activity assay demonstrated that sClu silencing also could increase the apoptosis rate of cells,resulting in the inhibition of cell growth.We also determined the effects of sClu silencing on tumor growth and metastatic progression in an orthotopic breast cancer model.The results showed that orthotopic primary tumors derived from MDA-MB-231/pSuper sClu siRNA cells grew significantly slower than tumors derived from parental MDA-MB-231 or MDA-MB-231/pSuper scramble siRNA cells,and metastasize less to the lungs.These data suggest that secretory clusterin plays a significant role in tumor growth and metastatic progression.Knocking-down sClu gene expression may provide a valuable method for breast cancer therapy.

  11. On the ambiguity of determination of interfering resonances parameters

    CERN Document Server

    Malyshev, V M

    2015-01-01

    The general form of solutions for parameters of interfering Breit-Wigner resonances is found. The number of solutions is determined by the properties of roots of corresponding characteristic equation and does not exceed $2^{N-1}$, where $N$ is the number of resonances. For resonances of more complicated form, provided that their amplitudes satisfy certain conditions, for any $N\\ge2$ multiple solutions also exist.

  12. Interferência das estatinas em exames laboratoriais

    Directory of Open Access Journals (Sweden)

    Danyelle Cristine MARINI

    2017-01-01

    Full Text Available Medicamentos assume importante papel na rotina laboratorial das Análises Clinicas, pela probabilidade de interferir nos ensaios e modificar o diagnóstico clínico-laboratorial. Cabe ao profissional analista clínico responsável detectar essas interferências a fim de evitar diagnósticos equivocados e intervenções clínicas errôneas. O objetivo do trabalho foi avaliar as possíveis interferências do fármaco sinvastatina nos exames laboratoriais bioquímicos. Foi realizado um estudo descritivo retrospectivo, no período de abril a julho de 2015. Foram avaliados os exames laboratoriais realizados pelos pacientes portadores de dislipidemia e usuários de medicamento específico, que utilizaram os serviços prestados pelo Laboratório Municipal de Análises Clínicas de Santo Antônio de Posse (SP. Aos participantes voluntários foi aplicado um questionário para avaliar o estilo de vida e o tempo de uso do medicamento. Foram analisadas as enzimas Fosfatase Alcalina, Creatinofosfoquinase (CPK, Aspartato Aminotransferase (AST e Alanina lminotransferase (ALT. O estudo mostrou que dos 43 pacientes avaliados, 69,8% apresentaram alterações em pelo menos um dos exames avaliados. Foi observada alteração da enzima ALT em 53% dos pacientes avaliados e nenhuma modificação no valor da CPK em 51% dos sujeitos. As interferências nos resultados de exames causadas por fármacos é um problema real, devido à grande utilização por toda a população, e comprova a necessidade de mais estudos relacionados às possíveis interferências nos exames clínicos.

  13. Different virus-derived siRNAs profiles between leaves and fruits in Cucumber green mottle mosaic virus-infected Lagenaria siceraria

    Directory of Open Access Journals (Sweden)

    Junmin Li

    2016-11-01

    Full Text Available RNA silencing is an evolutionarily conserved antiviral mechanism, through which virus-derived small interfering RNAs (vsiRNAs playing roles in host antiviral defence are produced in virus-infected plant. Deep sequencing technology has revolutionized the study on the interaction between virus and plant host through the analysis of vsiRNAs profile. However, comparison of vsiRNA profiles in different tissues from a same host plant has been rarely reported. In this study, the profiles of virus-derived small interfering RNAs (vsiRNAs from leaves and fruits of Lagenaria siceraria plants infected with Cucumber green mottle mosaic virus (CGMMV were comprehensively characterized and compared. Many more vsiRNAs were present in infected leaves than in fruits. vsiRNAs from both leaves and fruits were mostly 21- and 22-nt in size as previously described in other virus-infected plants. Interestingly, vsiRNAs were predominantly produced from the viral positive strand RNAs in infected leaves, whereas in infected fruits they were derived equally from the positive and negative strands. Many leaf-specific positive vsiRNAs with lengths of 21-nt (2,058 or 22-nt (3,996 were identified but only six (21-nt and one (22-nt positive vsiRNAs were found to be specific to fruits. vsiRNAs hotspots were only present in the 5’-terminal and 3’-terminal of viral positive strand in fruits, while multiple hotspots were identified in leaves. Differences in GC content and 5'-terminal nucleotide of vsiRNAs were also observed in the two organs. To our knowledge, this provides the first high-resolution comparison of vsiRNA profiles between different tissues of the same host plant.

  14. MicroRNAs (miRNAs) in neurodegenerative diseases.

    Science.gov (United States)

    Nelson, Peter T; Wang, Wang-Xia; Rajeev, Bernard W

    2008-01-01

    Aging-related neurodegenerative diseases (NDs) are the culmination of many different genetic and environmental influences. Prior studies have shown that RNAs are pathologically altered during the inexorable course of some NDs. Recent evidence suggests that microRNAs (miRNAs) may be a contributing factor in neurodegeneration. miRNAs are brain-enriched, small ( approximately 22 nucleotides) non-coding RNAs that participate in mRNA translational regulation. Although discovered in the framework of worm development, miRNAs are now appreciated to play a dynamic role in many mammalian brain-related biochemical pathways, including neuroplasticity and stress responses. Research about miRNAs in the context of neurodegeneration is accumulating rapidly, and the goal of this review is to provide perspective for these new data that may be helpful to specialists in either field. An overview is provided about the normal functions for miRNAs, including some of the newer concepts related to the human brain. Recently published studies pertaining to the roles of miRNAs in NDs--including Alzheimer's disease, Parkinson's disease and triplet repeat disorders-are described. Finally, a discussion is included with theoretical syntheses and possible future directions in exploring the nexus between miRNA and ND research.

  15. Expression and localization of microRNAs in perinatal rat pancreas

    DEFF Research Database (Denmark)

    Larsen, Louise; Rosenstierne, Maiken Worsøe; Gaarn, Louise;

    2011-01-01

    ABSTRACT Objective: To investigate the expression of pancreatic microRNAs (miRNAs) during the period of perinatal beta-cell expansion and maturation in rats, determine the localization of these miRNAs and perform a pathway analysis with predicted target mRNAs expressed in perinatal pancreas....... Research design and methods: RNA was extracted from whole pancreas at embryonic day 20 (E20), on the day of birth (P0) and two days after birth (P2) and hybridized to miRNA microarrays. Differentially expressed miRNAs were verified by northern blotting and their pancreatic localization determined...... by in situ hybridization. Pathway analysis was done using regulated sets of mRNAs predicted as targets of the miRNAs. Possible target genes were tested using reporter-gene analysis in INS-1E cells. Results: Nine miRNAs were differentially expressed perinatally, seven were confirmed to be regulated...

  16. Expression and Localization of microRNAs in Perinatal Rat Pancreas

    DEFF Research Database (Denmark)

    Larsen, Louise; Rosenstierne, Maiken Worsøe; Gaarn, Louise Winkel

    2011-01-01

    OBJECTIVE: To investigate the expression of pancreatic microRNAs (miRNAs) during the period of perinatal beta-cell expansion and maturation in rats, determine the localization of these miRNAs and perform a pathway analysis with predicted target mRNAs expressed in perinatal pancreas. RESEARCH DESIGN...... AND METHODS: RNA was extracted from whole pancreas at embryonic day 20 (E20), on the day of birth (P0) and two days after birth (P2) and hybridized to miRNA microarrays. Differentially expressed miRNAs were verified by northern blotting and their pancreatic localization determined by in situ hybridization....... Pathway analysis was done using regulated sets of mRNAs predicted as targets of the miRNAs. Possible target genes were tested using reporter-gene analysis in INS-1E cells. RESULTS: Nine miRNAs were differentially expressed perinatally, seven were confirmed to be regulated at the level of the mature miRNA...

  17. Methodologies for in vitro cloning of small RNAs and application for plant genome(s).

    Science.gov (United States)

    Devor, Eric J; Huang, Lingyan; Abdukarimov, Abdusattor; Abdurakhmonov, Ibrokhim Y

    2009-01-01

    The "RNA revolution" that started at the end of the 20th century with the discovery of post-transcriptional gene silencing and its mechanism via RNA interference (RNAi) placed tiny 21-24 nucleotide long noncoding RNAs (ncRNAs) in the forefront of biology as one of the most important regulatory elements in a host of physiologic processes. The discovery of new classes of ncRNAs including endogenous small interfering RNAs, microRNAs, and PIWI-interacting RNAs is a hallmark in the understanding of RNA-dependent gene regulation. New generation high-throughput sequencing technologies further accelerated the studies of this "tiny world" and provided their global characterization and validation in many biological systems with sequenced genomes. Nevertheless, for the many "yet-unsequenced" plant genomes, the discovery of small RNA world requires in vitro cloning from purified cellular RNAs. Thus, reproducible methods for in vitro small RNA cloning are of paramount importance and will remain so into the foreseeable future. In this paper, we present a description of existing small RNA cloning methods as well as next-generation sequencing methods that have accelerated this research along with a description of the application of one in vitro cloning method in an initial small RNA survey in the "still unsequenced" allotetraploid cotton genome.

  18. Long noncoding RNAs regulate adipogenesis.

    Science.gov (United States)

    Sun, Lei; Goff, Loyal A; Trapnell, Cole; Alexander, Ryan; Lo, Kinyui Alice; Hacisuleyman, Ezgi; Sauvageau, Martin; Tazon-Vega, Barbara; Kelley, David R; Hendrickson, David G; Yuan, Bingbing; Kellis, Manolis; Lodish, Harvey F; Rinn, John L

    2013-02-26

    The prevalence of obesity has led to a surge of interest in understanding the detailed mechanisms underlying adipocyte development. Many protein-coding genes, mRNAs, and microRNAs have been implicated in adipocyte development, but the global expression patterns and functional contributions of long noncoding RNA (lncRNA) during adipogenesis have not been explored. Here we profiled the transcriptome of primary brown and white adipocytes, preadipocytes, and cultured adipocytes and identified 175 lncRNAs that are specifically regulated during adipogenesis. Many lncRNAs are adipose-enriched, strongly induced during adipogenesis, and bound at their promoters by key transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (CEBPα). RNAi-mediated loss of function screens identified functional lncRNAs with varying impact on adipogenesis. Collectively, we have identified numerous lncRNAs that are functionally required for proper adipogenesis.

  19. Plant Mobile Small RNAs

    Science.gov (United States)

    Dunoyer, Patrice; Melnyk, Charles; Molnar, Attila; Slotkin, R. Keith

    2013-01-01

    In plants, RNA silencing is a fundamental regulator of gene expression, heterochromatin formation, suppression of transposable elements, and defense against viruses. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. Although the spreading of RNA silencing across the plant has been recognized for nearly two decades, only recently have sRNAs been formally demonstrated as the mobile silencing signals. Here, we discuss the various types of mobile sRNA molecules, their short- and long-range movement, and their function in recipient cells. PMID:23818501

  20. Diversity in parasitic nematode genomes: the microRNAs of Brugia pahangi and Haemonchus contortus are largely novel

    Directory of Open Access Journals (Sweden)

    Winter Alan D

    2012-01-01

    Full Text Available Abstract Background MicroRNAs (miRNAs play key roles in regulating post-transcriptional gene expression and are essential for development in the free-living nematode Caenorhabditis elegans and in higher organisms. Whether microRNAs are involved in regulating developmental programs of parasitic nematodes is currently unknown. Here we describe the the miRNA repertoire of two important parasitic nematodes as an essential first step in addressing this question. Results The small RNAs from larval and adult stages of two parasitic species, Brugia pahangi and Haemonchus contortus, were identified using deep-sequencing and bioinformatic approaches. Comparative analysis to known miRNA sequences reveals that the majority of these miRNAs are novel. Some novel miRNAs are abundantly expressed and display developmental regulation, suggesting important functional roles. Despite the lack of conservation in the miRNA repertoire, genomic positioning of certain miRNAs within or close to specific coding genes is remarkably conserved across diverse species, indicating selection for these associations. Endogenous small-interfering RNAs and Piwi-interacting (piRNAs, which regulate gene and transposon expression, were also identified. piRNAs are expressed in adult stage H. contortus, supporting a conserved role in germline maintenance in some parasitic nematodes. Conclusions This in-depth comparative analysis of nematode miRNAs reveals the high level of divergence across species and identifies novel sequences potentially involved in development. Expression of novel miRNAs may reflect adaptations to different environments and lifestyles. Our findings provide a detailed foundation for further study of the evolution and function of miRNAs within nematodes and for identifying potential targets for intervention.

  1. Comparison of non-coding RNAs in human and canine cancer

    Directory of Open Access Journals (Sweden)

    Siegfried eWagner

    2013-04-01

    Full Text Available The discovery of the post-transcriptional gene silencing by small non-protein-coding RNAs is considered as a major breakthrough in biology. In the last decade we just started to realize the biologic function and complexity of gene regulation by small non-coding RNAs. Post-transcriptional gene silencing (PTGS is a conserved phenomenon which was observed in various species such as fungi, worms, plants and mammals. Micro RNAs (miRNA and small interfering RNAs (siRNAs are two gene silencing mediators constituting an evolutionary conserved class of non-coding RNAs regulating many biological processes in eukaryotes. As this small RNAs appear to regulate gene expression at translational and transcriptional level it is not surprising that during the last decade many human diseases among them Alzheimer's disease, cardiovascular diseases and various cancer types were associated with deregulated miRNA expression. Consequently small RNAs are considered to hold big promises as therapeutic agents. However despite of the enormous therapeutic potential many questions remain unanswered. A major critical point, when evaluating novel therapeutic approaches, is the transfer of in vitro settings to an in vivo model. Classical animal models rely on the laboratory kept animals under artificial conditions and often missing an intact immune system. Model organisms with spontaneously occurring tumors as e.g. dogs provide the possibility to evaluate therapeutic agents under the surveillance of an in intact immune system and thereby providing an authentic tumor reacting scenario. Considering the genomic similarity between canines and humans and the advantages of the dog as cancer model system for human neoplasias the analyses of the complex role of small RNAs in canine tumor development could be of major value for both species. Herein we discuss comparatively the role of miRNAs in human and canine cancer development and highlight the potential and advantages of the model

  2. Noncoding RNAs in Cancer Immunology.

    Science.gov (United States)

    Li, Qian; Liu, Qiang

    2016-01-01

    Cancer immunology is the study of interaction between cancer cells and immune system by the application of immunology principle and theory. With the recent approval of several new drugs targeting immune checkpoints in cancer, cancer immunology has become a very attractive field of research and is thought to be the new hope to conquer cancer. This chapter introduces the aberrant expression and function of noncoding RNAs, mainly microRNAs and long noncoding RNAs, in tumor-infiltrating immune cells, and their significance in tumor immunity. It also illustrates how noncoding RNAs are shuttled between tumor cells and immune cells in tumor microenvironments via exosomes or other microvesicles to modulate tumor immunity.

  3. Specific expression of short-interfering RNA driven by human telomerase reverse transcriptase promoter in tumor cells

    Institute of Scientific and Technical Information of China (English)

    Xuejing Luan; Limin Guo; Zuozhen Yang; Min Liu; Xin Li; Hua Tang

    2008-01-01

    RNA interference (RNAi) has been shown to be an effective method for inhibiting the expression of a given gene in human cells by targeting with short duplex RNA (short-interfering RNA or siRNA). However, more and more studies suggest that non-specific effects can be induced by siRNAs, such as off-target inhibition, activation of interferon response, and saturation of cellular silencing machinery. It has been known that more than 90% of human tumors exhibit teiomerase activity. Consequently, teiomerase is believed to be a broadspectrum molecular marker of malignancies. In the present study we attempt to develop a tumor-specific RNAi system using the human telomerase reverse transcriptase promoter.This system may provide a basis for RNAi therapy.

  4. Promoter-associated RNAs and promoter-targeted RNAs.

    Science.gov (United States)

    Yan, Bing-Xue; Ma, Jin-Xia

    2012-09-01

    The world of RNAs is much more complex than previously thought, and has rapidly emerged as one of the most actively researched topics in the life sciences. Recently, two findings in this field were reported and given special attention: promoter-associated RNAs (paRNAs), a novel class of RNAs with numerous potential functions; and promoter-targeted RNA-induced transcriptional gene regulation, a new regulatory mechanism to control transcription. In this review, we summarize the studies in these two areas, and outline the current understanding with respect to the potential biological functions of paRNAs, and the molecular mechanisms of promoter-targeted RNA-induced transcriptional gene silencing and activation. Additionally, we seek to integrate these two areas, as paRNAs may have potential biological links with promoter-targeted RNA-induced transcriptional gene regulation. Finally, we will discuss the significance of identifying paRNAs and the possible use of promoter-targeted RNAs in gene regulation and therapy.

  5. microRNAs-powerful repression comes from small RNAs

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    microRNAs (miRNAs) encode a novel class of small, non-coding RNAs that regulate gene expression post-trancriptionally. miRNAs comprise one of the major non-coding RNA families, whose diverse bio- logical functions and unusual capacity for gene regulation have attracted enormous interests in the RNA world. Over the past 16 years, genetic, biochemical and computational approaches have greatly shaped the growth of the field, leading to the identification of thousands of miRNA genes in nearly all metazoans. The key molecular machinery for miRNA biogenesis and silencing has been identified, yet the precise biochemical and regulatory mechanisms still remain elusive. However, recent findings have shed new light on how miRNAs are generated and how they function to repress gene expression. miRNAs provide a paradigm for endogenous small RNAs that mediate gene silencing at a genome-wide level. The gene silencing mediated by these small RNAs constitutes a major component of gene regu- lation during various developmental and physiological processes. The accumulating knowledge about their biogenesis and gene silencing mechanism will add a new dimension to our understanding about the complex gene regulatory networks.

  6. microRNAs- powerful repression comes from small RNAs

    Institute of Scientific and Technical Information of China (English)

    MA Cong; LIU YuFei; HE Lin

    2009-01-01

    microRNAs (miRNAs) encode a novel class of small, non-coding RNAs that regulate gene expression post-trancriptionally, miRNAs comprise one of the major non-coding RNA families, whose diverse bio-logical functions and unusual capacity for gene regulation have attracted enormous interests in the RNA world. Over the past 16 years, genetic, biochemical and computational approaches have greatly shaped the growth of the field, leading to the identification of thousands of miRNA genes in nearly all metazoans. The key molecular machinery for miRNA biogenesis and silencing has been identified, yet the precise biochemical and regulatory mechanisms still remain elusive. However, recent findings have shed new light on how miRNAs are generated and how they function to repress gene expression.miRNAs provide a paradigm for endogenous small RNAs that mediate gene silencing at a genome-wide level. The gene silencing mediated by these small RNAs constitutes a major component of gene regu-lation during various developmental and physiological processes. The accumulating knowledge about their biogenesis and gene silencing mechanism will add a now dimension to our understanding about the complex gene regulatory networks.

  7. Inhibition of HIV-1 replication by small interfering RNAs directed against Glioma Pathogenesis Related Protein (GliPR expression

    Directory of Open Access Journals (Sweden)

    Ottmann Oliver G

    2010-03-01

    Full Text Available Abstract Background Previously, we showed that glioma pathogenesis related protein (GliPR is induced in CEM T cells upon HIV-1 infection in vitro. To examine whether GliPR plays a role as HIV dependency factor (HDF, we tested the effect of GliPR suppression by siRNA on HIV-1 replication. Results Induction of GliPR expression by HIV-1 was confirmed in P4-CCR5 cells. When GliPR was suppressed by siRNA, HIV-1 replication was significantly reduced as measured by HIV-1 transcript levels, HIV-1 p24 protein levels, and HIV-1 LTR-driven reporter gene expression, suggesting that GliPR is a cellular co-factor of HIV-1. Microarray analysis of uninfected HeLa cells following knockdown of GliPR revealed, among a multitude of gene expression alterations, a down-regulation of syndecan-1, syndecan-2, protein kinase C alpha (PRKCA, the catalytic subunit β of cAMP-dependent protein kinase (PRKACB, nuclear receptor co-activator 3 (NCOA3, and cell surface protein CD59 (protectin, all genes having relevance for HIV-1 pathology. Conclusions The up-regulation of GliPR by HIV-1 and the early significant inhibition of HIV-1 replication mediated by knockdown of GliPR reveal GliPR as an important HIV-1 dependency factor (HDF, which may be exploited for HIV-1 inhibition.

  8. Potential MiRNAs recognition site identification in 3' UTR regions by DSP methods.

    Science.gov (United States)

    Maggi, Norbert; Arrigo, Patrizio; Ruggiero, Carmelina

    2012-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate fundamental cellular processes in diverse organisms and that have an important function in gene expression regulation. miRNAs seem capable to concurrently modulate hundreds of target genes. Their abnormal expression is emerging as important element in many pathological conditions. The identification of microRNA binding sites on those proteins that can be disease biomarker is fundamental to design synthetic artificial oligomers. In this paper we suggest a method, based on signal processing, to filter out potential miRNA recognition sites in the 3' UTR region of mRNAs.

  9. Expression and Localization of microRNAs in Perinatal Rat Pancreas

    DEFF Research Database (Denmark)

    Larsen, Louise; Rosenstierne, Maiken Worsøe; Gaarn, Louise Winkel;

    2011-01-01

    OBJECTIVE: To investigate the expression of pancreatic microRNAs (miRNAs) during the period of perinatal beta-cell expansion and maturation in rats, determine the localization of these miRNAs and perform a pathway analysis with predicted target mRNAs expressed in perinatal pancreas. RESEARCH DESIGN...... AND METHODS: RNA was extracted from whole pancreas at embryonic day 20 (E20), on the day of birth (P0) and two days after birth (P2) and hybridized to miRNA microarrays. Differentially expressed miRNAs were verified by northern blotting and their pancreatic localization determined by in situ hybridization...

  10. Interferência por RNA: uma nova alternativa para terapia nas doenças reumáticas RNA interference: a new alternative for rheumatic diseases therapy

    Directory of Open Access Journals (Sweden)

    Natália Regine de França

    2010-12-01

    Full Text Available A interferência por RNA (RNAi é um mecanismo de silenciamento gênico pós-transcricional conservado durante a evolução. Esse mecanismo, recentemente descrito, é mediado por pequenos RNAs de fita dupla (dsRNAs capazes de reconhecer especificamente uma sequência de mRNA-alvo e mediar sua clivagem ou repressão traducional. O emprego da RNAi como uma ferramenta de terapia gênica tem sido muito estudado, especialmente em infecções virais, câncer, desordens genéticas herdadas, doenças cardiovasculares e mesmo em doenças reumáticas. Aliados aos dados do genoma humano, os conhecimentos do silenciamento gênico mediado por RNAi podem permitir a determinação funcional de praticamente qualquer gene expresso em uma célula e sua implicação para o funcionamento e homeostase celular. Vários estudos terapêuticos in vitro e in vivo em modelos de doenças autoimunes vêm sendo realizados com resultados encorajadores. As vias de quebra de tolerância e inflamação são alvos potenciais para terapia com RNAi em doenças inflamatórias e autoimunes. Nesta revisão vamos recordar os princípios básicos da RNAi e discutir os aspectos que levaram ao desenvolvimento de propostas terapêuticas baseadas em RNAi, começando pelos estudos in vitro de desenvolvimento de ferramentas e identificação de alvos, chegando até os estudos pré-clínicos de disponibilização da droga in vivo, e testes em células humanas e modelos animais de doenças autoimunes. Por fim, vamos revisar os últimos avanços da experiência clínica da terapia com RNAiRNA interference (RNAi is a post-transcriptional gene silencing mechanism preserved during evolution. This mechanism, recently described, is mediated by small double-stranded RNAs (dsRNAs that can specifically recognize a target mRNA sequence and mediate its cleavage or translational repression. The use of RNAi as a tool for gene therapy has been extensively studied, especially in viral infections, cancer

  11. Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes

    Directory of Open Access Journals (Sweden)

    Sharbati Soroush

    2012-01-01

    Full Text Available Abstract Background Small interfering and non-coding RNAs regulate gene expression across all kingdoms of life. MicroRNAs constitute an important group of metazoan small RNAs regulating development but also disease. Accordingly, in functional genomics microRNA expression analysis sheds more and more light on the dynamic regulation of gene expression in various cellular processes. Results We have developed custom RT-qPCR arrays allowing for accurate quantification of 31 small RNAs in triplicate using a 96 well format. In parallel, we provide accurate normalisation of microRNA expression data based on the quantification of 5 reference snRNAs. We have successfully employed such arrays to study microRNA regulation during human monocyte differentiation as well as Salmonella infection. Besides well-known protagonists such as miR-146 or miR-155, we identified the up-regulation of miR-21, miR-222, miR-23b, miR-24, miR-27a as well as miR-29 upon monocyte differentiation or infection, respectively. Conclusions The provided protocol for RT-qPCR arrays enables straight-forward microRNA expression analysis. It is fully automatable, compliant with the MIQE guidelines and can be completed in only 1 day. The application of these arrays revealed microRNAs that may mediate monocyte host defence mechanisms by regulating the TGF-β signalling upon Salmonella infection. The introduced arrays are furthermore suited for customised quantification of any class of small non-coding RNAs as exemplified by snRNAs and thus provide a versatile tool for ubiquitous applications.

  12. Long Non-coding RNAs Expression Profile in HepG2 Cells Reveals the Potential Role of Long Non-coding RNAs in the Cholesterol Metabolism

    Directory of Open Access Journals (Sweden)

    Gang Liu

    2015-01-01

    Full Text Available Background: Green tea has been shown to improve cholesterol metabolism in animal studies, but the molecular mechanisms underlying this function have not been fully understood. Long non-coding RNAs (lncRNAs have recently emerged as a major class of regulatory molecules involved in a broad range of biological processes and complex diseases. Our aim was to identify important lncRNAs that might play an important role in contributing to the benefits of epigallocatechin-3-gallate (EGCG on cholesterol metabolism. Methods: Microarrays was used to reveal the lncRNA and mRNA profiles in green tea polyphenol(--epigallocatechin gallate in cultured human liver (HepG2 hepatocytes treated with EGCG and bioinformatic analyses of the predicted target genes were performed to identify lncRNA-mRNA targeting relationships. RNA interference was used to investigate the role of lncRNAs in cholesterol metabolism. Results: The expression levels of 15 genes related to cholesterol metabolism and 285 lncRNAs were changed by EGCG treatment. Bioinformatic analysis found five matched lncRNA-mRNA pairs for five differentially expressed lncRNAs and four differentially expressed mRNA. In particular, the lncRNA AT102202 and its potential targets mRNA-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR were identified. Using a real-time polymerase chain reaction technique, we confirmed that EGCG down-regulated mRNA expression level of the HMGCR and up-regulated expression of AT102202. After AT102202 knockdown in HepG2, we observed that the level of HMGCR expression was significantly increased relative to the scrambled small interfering RNA control (P < 0.05. Conclusions: Our results indicated that EGCG improved cholesterol metabolism and meanwhile changed the lncRNAs expression profile in HepG2 cells. LncRNAs may play an important role in the cholesterol metabolism.

  13. MicroRNAs in neurodegeneration.

    Science.gov (United States)

    Bushati, Natascha; Cohen, Stephen M

    2008-06-01

    microRNAs (miRNAs) act as post-transcriptional regulators of gene expression in diverse cellular and developmental processes. Many miRNAs are expressed specifically in the central nervous system, where they have roles in differentiation, neuronal survival, and potentially also in plasticity and learning. The absence of miRNAs in a variety of specific postmitotic neurons can lead to progressive loss of these neurons and behavioral defects reminiscent of the phenotypes seen in the pathologies of neurodegenerative diseases. Here, we review recent studies which provide a link between miRNA function and neurodegeneration. We also discuss evidence which might suggest involvement of miRNAs in the emergence or progression of neurodegenerative diseases.

  14. Non-Protein Coding RNAs

    CERN Document Server

    Walter, Nils G; Batey, Robert T

    2009-01-01

    This book assembles chapters from experts in the Biophysics of RNA to provide a broadly accessible snapshot of the current status of this rapidly expanding field. The 2006 Nobel Prize in Physiology or Medicine was awarded to the discoverers of RNA interference, highlighting just one example of a large number of non-protein coding RNAs. Because non-protein coding RNAs outnumber protein coding genes in mammals and other higher eukaryotes, it is now thought that the complexity of organisms is correlated with the fraction of their genome that encodes non-protein coding RNAs. Essential biological processes as diverse as cell differentiation, suppression of infecting viruses and parasitic transposons, higher-level organization of eukaryotic chromosomes, and gene expression itself are found to largely be directed by non-protein coding RNAs. The biophysical study of these RNAs employs X-ray crystallography, NMR, ensemble and single molecule fluorescence spectroscopy, optical tweezers, cryo-electron microscopy, and ot...

  15. Profiling of microRNA-mRNA reveals roles of microRNAs in cervical cancer

    Institute of Scientific and Technical Information of China (English)

    MA Ding; ZHANG You-yi; GUO Yan-li; LI Zi-jian; GENG Li

    2012-01-01

    Background Cervical cancer is one of the most common malignant tumors in women.This study was designed to explore the expression profiles of microRNAs (miRNAs) and mRNAs and the gene regulation network in cervical tumorigenesis and to find candidate molecular markers and key tumorigenic genes in cervical cancer.Methods miRNAs and mRNAs expression microarrays were used to detect the expression of miRNAs and mRNAs in normal and cancer cervical tissues.TargetScan 5.0 database (UK) was used to predict the target genes of the miRNAs,analyze their intersection with differentially expressed mRNAs and negatively correlate the intersection with miRNAs.Bioinformatic approaches were used to analyze functions and pathways of the target genes and establish miRNA-gene network.Results Twenty-nine miRNAs and 2036 mRNAs were differentially expressed in normal and cervical tumor tissues.Among them,13 miRNAs and 754 mRNAs were up-regulated in cervical tumor tissues and 16 miRNAs and 1282 RNA were down-regulated.The 327 target genes negatively related to miRNAs in the intersection were involved in functions and signal pathways.Down-regulated miRNAs targeted genes and up-regulated miRNAs targeted genes were involved in 415 and 163 functions,respectively,and in 37 and 17 significant pathways,respectively (P <0.05,false discovery rate (FDR) <0.05).We constructed the miRNAs-gene network and found that hsa-miR-15a,hsa-miR-106b and hsa-miR-20b were key nodes in the network.Conclusions The differentially expressed miRNAs and mRNAs in cervical cancer and related miRNA-gene network have been identified.They play important roles in cervical tumorigenesis and are involved in many important biological functions and signal transduction pathways.These findings lay a foundation for research on the molecular mechanism of miRNAs in the pathogenesis of cervical cancer.

  16. The miRNAome of globe artichoke: conserved and novel micro RNAs and target analysis

    Directory of Open Access Journals (Sweden)

    De Paola Domenico

    2012-01-01

    Full Text Available Abstract Background Plant microRNAs (miRNAs are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA libraries can detect even poorly expressed miRNAs. No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots. Results Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to Arabidopsis proteins; the 43 targets (23 for novel miRNAs identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1. Conclusions The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant

  17. Screening for N-AHSL-Based-Signaling Interfering Enzymes.

    Science.gov (United States)

    Uroz, Stéphane; Oger, Phil M

    2017-01-01

    Quorum sensing (QS)-based signaling is a widespread pathway used by bacteria for the regulation of functions involved in their relation to the environment or their host. QS relies upon the production, accumulation and perception of small diffusable molecules by the bacterial population, hence linking high gene expression with high cell population densities. Among the different QS signal molecules, an important class of signal molecules is the N-acyl homoserine lactone (N-AHSL). In pathogens such as Erwinia or Pseudomonas, N-AHSL based QS is crucial to overcome the host defenses and ensure a successful infection. Interfering with QS-regulation allows the algae Delisea pulcra to avoid surface colonization by bacteria. Thus, interfering the QS-regulation of pathogenic bacteria is a promising antibiotic-free antibacterial therapeutic strategy. To date, two N-AHSL lactonases and one amidohydrolase families of N-ASHL degradation enzymes have been characterized and have proven to be efficient in vitro to control N-AHSL-based QS-regulated functions in pathogens. In this chapter, we provide methods to screen individual clones or bacterial strains as well as pool of clones for genomic and metagenomic libraries, that can be used to identify strains or clones carrying N-ASHL degradation enzymes.

  18. Defective interfering virus protects elderly mice from influenza

    Directory of Open Access Journals (Sweden)

    Easton Andrew J

    2011-05-01

    Full Text Available Abstract Background We have identified and characterised a defective-interfering (DI influenza A virus particles containing a highly deleted segment 1 RNA that has broad-spectrum antiviral activity. In young adult mice it exerts protection against several different subtypes of influenza A virus (defined here as homologous or genetically compatible protection and against a paramyxovirus and an influenza B virus (heterologous or genetically unrelated protection. Homologous protection is mediated by replication competition between the deleted and full-length genomes, and heterologous protection occurs through stimulation of innate immunity, especially interferon type I. Methods A single dose of the protective DI virus was administered intranasally to elderly mice at -7, -1 and +1 days relative to intranasal challenge with influenza A virus. Results A single dose of the DI virus given 1 or 7 days protected elderly mice, reducing a severe, sometimes fatal disease to a subclinical or mild infection. In contrast, all members of control groups treated with inactivated DI virus before challenge became extremely ill and most died. Despite the subclinical/mild nature of their infection, protected mice developed solid immunity to a second infectious challenge. Conclusions The defective interfering virus is effective in preventing severe influenza A in elderly mice and may offer a new approach to protection of the human population.

  19. Joint Network Coding for Interfering Wireless Multicast Networks

    CERN Document Server

    Qureshi, Jalaluddin; Cai, Jianfei

    2011-01-01

    Interference in wireless networks is one of the key-capacity limiting factor. The multicast capacity of an ad- hoc wireless network decreases with an increasing number of transmitting and/or receiving nodes within a fixed area. Digital Network Coding (DNC) has been shown to improve the multicast capacity of non-interfering wireless network. However recently proposed Physical-layer Network Coding (PNC) and Analog Network Coding (ANC) has shown that it is possible to decode an unknown packet from the collision of two packet, when one of the colliding packet is known a priori. Taking advantage of such collision decoding scheme, in this paper we propose a Joint Network Coding based Cooperative Retransmission (JNC- CR) scheme, where we show that ANC along with DNC can offer a much higher retransmission gain than that attainable through either ANC, DNC or Automatic Repeat reQuest (ARQ) based retransmission. This scheme can be applied for two wireless multicast groups interfering with each other. Because of the broa...

  20. MysiRNA-designer: a workflow for efficient siRNA design.

    Directory of Open Access Journals (Sweden)

    Mohamed Mysara

    Full Text Available The design of small interfering RNA (siRNA is a multi factorial problem that has gained the attention of many researchers in the area of therapeutic and functional genomics. MysiRNA score was previously introduced that improves the correlation of siRNA activity prediction considering state of the art algorithms. In this paper, a new program, MysiRNA-Designer, is described which integrates several factors in an automated work-flow considering mRNA transcripts variations, siRNA and mRNA target accessibility, and both near-perfect and partial off-target matches. It also features the MysiRNA score, a highly ranked correlated siRNA efficacy prediction score for ranking the designed siRNAs, in addition to top scoring models Biopredsi, DISR, Thermocomposition21 and i-Score, and integrates them in a unique siRNA score-filtration technique. This multi-score filtration layer filters siRNA that passes the 90% thresholds calculated from experimental dataset features. MysiRNA-Designer takes an accession, finds conserved regions among its transcript space, finds accessible regions within the mRNA, designs all possible siRNAs for these regions, filters them based on multi-scores thresholds, and then performs SNP and off-target filtration. These strict selection criteria were tested against human genes in which at least one active siRNA was designed from 95.7% of total genes. In addition, when tested against an experimental dataset, MysiRNA-Designer was found capable of rejecting 98% of the false positive siRNAs, showing superiority over three state of the art siRNA design programs. MysiRNA is a freely accessible (Microsoft Windows based desktop application that can be used to design siRNA with a high accuracy and specificity. We believe that MysiRNA-Designer has the potential to play an important role in this area.

  1. The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA- and microRNA-mediated pathways.

    Science.gov (United States)

    Csorba, Tibor; Bovi, Aurelie; Dalmay, Tamás; Burgyán, József

    2007-11-01

    One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3' overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised.

  2. Structural and Functional Characterization of Noncoding Repetitive RNAs Transcribed in Stressed Human CellsD⃞

    Science.gov (United States)

    Valgardsdottir, Rut; Chiodi, Ilaria; Giordano, Manuela; Cobianchi, Fabio; Riva, Silvano; Biamonti, Giuseppe

    2005-01-01

    Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA. In response to stress, these domains change their epigenetic status from heterochromatin to euchromatin and are transcribed in poly-adenylated RNAs that remain associated with nSBs. In this article, we describe the cloning, sequencing, and functional characterization of these transcripts. They are composed of SatIII repeats and originate from the transcription of multiple sites within the SatIII arrays. Interestingly, the level of SatIII RNAs can be down-regulated both by antisense oligonucleotides and small interfering RNAs (siRNA). Knockdown of SatIII RNA by siRNAs requires the activity of Argonaute 2, a component of the RNA-induced silencing complex. Down-regulation of satellite III RNAs significantly affects the recruitment of RNA processing factors to nSBs without altering the association of HSF-1 with these structures nor the presence of acetylated histones within nSBs. Thus, satellite III RNAs have a major role in the formation of nSBs. PMID:15788562

  3. Circular RNAs in heart failure.

    Science.gov (United States)

    Devaux, Yvan; Creemers, Esther E; Boon, Reinier A; Werfel, Stanislas; Thum, Thomas; Engelhardt, Stefan; Dimmeler, Stefanie; Squire, Iain

    2017-06-01

    Cardiovascular disease, and particularly heart failure, is still a serious health care issue for which novel treatments and biomarkers are needed. The RNA family comprises different subgroups, among which the small-sized microRNAs and the larger long non-coding RNAs have shown some potential to aid in moving personalized health care of heart failure patients a step forward. Here, members of the Cardiolinc network review the recent findings suggesting that the less well-known circular RNAs may constitute a novel reservoir of therapeutic targets and biomarkers of heart failure. The knowledge of the mode of biogenesis of circular RNAs will first be reported, followed by a description of different features that make these RNA molecules of interest for the heart failure community. The functions of circular RNAs in the heart will be described, with some emphasis given to their regulation in the failing heart. Circulating in the bloodstream, circular RNAs have appeared as potential biomarkers and recent findings associated with the use of circular RNAs as heart failure biomarkers will be discussed. Finally, some directions for future research will be provided. © 2017 The Authors. European Journal of Heart Failure © 2017 European Society of Cardiology.

  4. DGCR8-mediated production of canonical microRNAs is critical for regulatory T cell function and stability.

    Directory of Open Access Journals (Sweden)

    Lukas T Jeker

    Full Text Available Regulatory T cells (Treg are integral for immune homeostasis. Here we demonstrate that canonical microRNAs (miRNAs are required for Treg function because mice with DGCR8-deficient Treg cells spontaneously develop a scurfy-like disease. Using genetic lineage marking we show that absence of miRNAs leads to reduced FoxP3 expression in Treg cells in vivo. In vitro culture of purified DGCR8-deficient Treg leads to a loss of FoxP3 expression. We conclude that canonical miRNAs are essential to maintain stable FoxP3 expression and Treg function. Thus, signals interfering with miRNA homeostasis might contribute to autoimmune diseases.

  5. The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

    Science.gov (United States)

    Gaston, Kirk W; Limbach, Patrick A

    2014-01-01

    The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems. PMID:25616408

  6. Small interfering RNA targeting the nonstructural gene 1 transcript inhibits influenza A virus replication in experimental mice.

    Science.gov (United States)

    Rajput, Roopali; Khanna, Madhu; Kumar, Prashant; Kumar, Binod; Sharma, Sonal; Gupta, Neha; Saxena, Latika

    2012-12-01

    Nonstructural protein 1 (NS1) of influenza A viruses counteracts the host immune response against the influenza viruses by not only inhibiting the nuclear export and maturation of host cell messenger RNA (mRNA), but by also blocking the double-stranded RNA-activated protein kinase-mediated inhibition of viral RNA translation. Reduction of NS1 gene product in the host cell may be a potent antiviral strategy to provide protection against the influenza virus infection. We used small interfering RNAs (siRNAs) synthesized against the viral mRNA to down regulate the NS1 gene and observed its effect on inhibition of virus replication. When NS1 gene-specific siRNA were transfected in Madin Darby canine kidney (MDCK) cells followed by influenza A virus infection, approximately 60% inhibition in intracellular levels of NS1 RNA was observed. When siRNA was administered in BALB/c mice, 92% reduction in the levels of NS1 gene expression in mice lungs was observed. A significant reduction in the lung virus titers and cytokine levels was also detected in the presence of siRNAs as compared with the untreated control. The study was validated by the use of selectively disabled mutants of each set of siRNA. Our findings suggest that siRNA targeted against NS1 gene of influenza A virus can provide considerable protection to the virus-infected host cells and may be used as potential candidates for nucleic acid-based antiviral therapy for prevention of influenza A virus infection.

  7. MicroRNAs in inflammation and immune responses.

    Science.gov (United States)

    Contreras, J; Rao, D S

    2012-03-01

    MicroRNAs (miRNAs) are important regulators of gene expression in the immune system. In a few short years, their mechanism of action has been described in various cell lineages within the immune system, targets have been defined and their unique contributions to immune cell function have been examined. Certain miRNAs serve in important negative feedback loops in the immune system, whereas others serve to amplify the response of the immune system by repressing inhibitors of the response. Here, we review some of the better understood mechanisms as well as some emerging concepts of miRNA function. Future work will likely involve defining the function of specific miRNAs in specific immune cell lineages and to utilize them in the design of therapeutic strategies for diseases involving the immune system.

  8. Competitive inhibition of transcription factors by small interfering peptides.

    Science.gov (United States)

    Seo, Pil Joon; Hong, Shin-Young; Kim, Sang-Gyu; Park, Chung-Mo

    2011-10-01

    Combinatorial assortment by dynamic dimer formation diversifies gene transcriptional specificities of transcription factors. A similar but biochemically distinct mechanism is competitive inhibition in which small proteins act as negative regulators by competitively forming nonfunctional heterodimers with specific transcription factors. The most extensively studied is the negative regulation of auxin response factors by AUXIN/INDOLE-3-ACETIC ACID repressors. Similarly, Arabidopsis thaliana (Arabidopsis) little zipper and mini finger proteins act as competitive inhibitors of target transcription factors. Competitive inhibitors are also generated by alternative splicing and controlled proteolytic processing. Because they provide a way of attenuating transcription factors we propose to call them small interfering peptides (siPEPs). The siPEP-mediated strategy could be applied to deactivate specific transcription factors in crop plants.

  9. Long noncoding RNAs and neuroblastoma.

    Science.gov (United States)

    Pandey, Gaurav Kumar; Kanduri, Chandrasekhar

    2015-07-30

    Neuroblastoma is a disease that affects infants and despite intense multimodal therapy, high-risk patients have low survival rates (neuroblastoma have just begun to be elucidated. This review summarises where we are with regards to lncRNAs in neuroblastoma. The known mechanistic roles of lncRNAs during neuroblastoma pathogenesis are discussed, as well as the relationship between lncRNA expression and the differentiation capacity of neuroblastoma cells. We speculate about the use of some of these lncRNAs, such as those mapping to the 6p22 hotspot, as biomarkers for neuroblastoma prognosis and treatment. This novel way of thinking about both neuroblastoma and lncRNAs brings a new perspective to the prognosis and treatment of high-risk patients.

  10. [MicroRNAs in neurobiology].

    Science.gov (United States)

    Kawahara, Yukio

    2008-12-01

    MicroRNAs have emerged as a new regulatory factor of gene expression. They mediate translational repression or degradation of their target mRNAs by RNA interference (RNAi). The expression of each microRNA is tightly regulated in a development- and cell-specific manner by various mechanisms such as blockade of let-7 family expression by Lin-28 or RNA editing. They also act as regulatory switches for development, organogenesis, and cellular differentiation or for controlling distinct functions that are required for the maintenance of each tissue and cell subtypes. The abundant expression of microRNAs as well as the exclusive expression of certain microRNAs in the central nervous system highlights their biological importance at all stages of neural development and in postmitotic and differentiated neurons. Further, some microRNAs, such as miRNA-134, and miRNA-132 are localized and are synthesized in part at synaptic sites in dendrites to regulate synaptic formation and plasticity. In addition to the imparting of basic knowledge about the biogenesis and mechanism of action of microRNAs, this review focuses on the recent advances in microRNA studies in neurobiology, including the expression pattern of microRNAs in the mammalian brain, the role of microRNAs in neural differentiation and maturation, formation and plasticity of synaptic connections, and maintenance of neural function such as the synthesis of the neurotransmitters in selected neurons. Finally, the possible connection between microRNA dysfunction and neurological diseases, and future implications for diagnosis, and treatment of defects in human brain development and neurodegenerative diseases are discussed.

  11. Systematic Identification and Characterization of Long Non-Coding RNAs in the Silkworm, Bombyx mori.

    Science.gov (United States)

    Wu, Yuqian; Cheng, Tingcai; Liu, Chun; Liu, Duolian; Zhang, Quan; Long, Renwen; Zhao, Ping; Xia, Qingyou

    2016-01-01

    Long noncoding RNAs (lncRNAs) are emerging as important regulators in various biological processes. However, to date, no systematic characterization of lncRNAs has been reported in the silkworm Bombyx mori. In the present study, we generated eighteen RNA-seq datasets with relatively high depth. Using an in-house designed lncRNA identification pipeline, 11,810 lncRNAs were identified for 5,556 loci. Among these lncRNAs, 474 transcripts were intronic lncRNAs (ilncRNAs), 6,250 transcripts were intergenic lncRNAs (lincRNAs), and 5,086 were natural antisense lncRNAs (lncNATs). Compared with protein-coding mRNAs, silkworm lncRNAs are shorter in terms of full length but longer in terms of exon and intron length. In addition, lncRNAs exhibit a lower level of sequence conservation, more repeat sequences overlapped and higher tissue specificity than protein-coding mRNAs in the silkworm. We found that 69 lncRNA transcripts from 33 gene loci may function as miRNA precursors, and 104 lncRNA transcripts from 72 gene loci may act as competing endogenous RNAs (ceRNAs). In total, 49.47% of all gene loci (2,749/5,556) for which lncRNAs were identified showed sex-biased expression. Co-expression network analysis resulted in 19 modules, 12 of which revealed relatively high tissue specificity. The highlighted darkgoldenrod module was specifically associated with middle and posterior silk glands, and the hub lncRNAs within this module were co-expressed with proteins involved in translation, translocation, and secretory processes, suggesting that these hub lncRNAs may function as regulators of the biosynthesis, translocation, and secretion of silk proteins. This study presents the first comprehensive genome-wide analysis of silkworm lncRNAs and provides an invaluable resource for genetic, evolutionary, and genomic studies of B. mori.

  12. Platelet MicroRNAs: An Overview.

    Science.gov (United States)

    Dahiya, Neetu; Sarachana, Tewarit; Vu, Long; Becker, Kevin G; Wood, William H; Zhang, Yongqing; Atreya, Chintamani D

    2015-10-01

    MicroRNAs (miRNAs) are short ~22-nucleotide noncoding RNA that have been found to influence the expression of many genes and cellular processes by either repressing translation or degrading messenger RNA transcripts. Platelet miRNA expression has been shown to be perturbed during ex vivo storage of platelets and in platelet-associated disorders. Although bioinformatics-based miRNA target predictions have been established, direct experimental validation of the role of miRNAs in platelet biology has been rather slow. Target prediction studies are, nonetheless, valuable in directing the design of appropriate experiments to test specific miRNA:messenger RNA interactions relevant to the underlying mechanisms of platelet function in general and in disease as well as in ex vivo storage-associated "storage lesions," a collective term used to include physiologic, biochemical, and morphologic changes that occur in stored platelets. This brief review will focus on emerging human platelet miRNA studies to emphasize their potential role relevant to transfusion medicine field in terms of regulating platelet signaling pathways, markers of platelet associated disorders, and remote impactors of gene expression (intercellular biomodulators) as well as potential platelet quality markers of storage and pathogen reduction treatments.

  13. 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...

  14. Identifying cancer-causing noncoding RNAs

    NARCIS (Netherlands)

    le Sage, Carlos Karel

    2008-01-01

    To circumvent the dependency on prediction models, we developed a microRNA-screen-based assay to establish links between cellular phenotypes and microRNAs (miRNAs). To this end, a miRNA expression library (miR-Lib) was built consisting of 300 annotated miRNAs and around 100 candidate miRNAs. These m

  15. 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.

  16. MicroRNAs in Metabolism

    DEFF Research Database (Denmark)

    Vienberg, Sara; Geiger, Julian; Madsen, Søren

    2017-01-01

    MicroRNAs (miRNAs) have within the past decade emerged as key regulators of metabolic homeostasis. Major tissues in intermediary metabolism important during development of the metabolic syndrome, such as β-cells, liver, skeletal and heart muscle as well as adipose tissue have all been shown to be...... diabetes and atherosclerosis stresses their potential as therapeutic targets. This review emphasizes current ideas and controversies within miRNA research in metabolism. This article is protected by copyright. All rights reserved.......MicroRNAs (miRNAs) have within the past decade emerged as key regulators of metabolic homeostasis. Major tissues in intermediary metabolism important during development of the metabolic syndrome, such as β-cells, liver, skeletal and heart muscle as well as adipose tissue have all been shown...... to be affected by miRNAs. In the pancreatic β-cell a number of miRNAs are important in maintaining the balance between differentiation and proliferation (miR-200 and miR-29 families) and insulin exocytosis in the differentiated state is controlled by miR-7, miR-375 and miR-335. MiR-33a and -33b play crucial...

  17. Delivery of Therapeutic RNAs Into Target Cells IN VIVO

    Science.gov (United States)

    Ng, Mei Ying; Hagen, Thilo

    2014-02-01

    RNA-based therapy is one of the most promising approaches to treat human diseases. Specifically, the use of short interfering RNA (siRNA) siRNA and microRNA (miRNA) mimics for in vivo RNA interference has immense potential as it directly lowers the expression of the therapeutic target protein. However, there are a number of major roadblocks to the successful implementation of siRNA and other RNA based therapies in the clinic. These include the instability of RNAs in vivo and the difficulty to efficiently deliver the RNA into the target cells. Hence, various innovative approaches have been taken over the years to develop effective RNA delivery methods. These methods include liposome-, polymeric nanoparticle- and peptide-mediated cellular delivery. In a recent innovative study, bioengineered bacterial outer membrane vesicles were used as vehicles for effective delivery of siRNA into cells in vivo.

  18. Coexpression of Escherichia coli RNase Ⅲ in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

    Institute of Scientific and Technical Information of China (English)

    Jae Man Lee; Yoshito Kojin; Tsuneyuki Tatsuke; Hiroaki Mon; Yoshitaka Miyagawa; Takahiro Kusakabe

    2013-01-01

    Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells,although its gene silencing efficiency is lower than that of exogenously introduced double-stranded RNAs(dsRNAs).To solve this problem,we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA(siRNA).Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm,but were not effectively diced into siRNAs.Interestingly,RNAi with hairpin dsRNAs from genome-integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase Ⅲ,although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.

  19. HIVsirDB: a database of HIV inhibiting siRNAs.

    Directory of Open Access Journals (Sweden)

    Atul Tyagi

    Full Text Available BACKGROUND: Human immunodeficiency virus (HIV is responsible for millions of deaths every year. The current treatment involves the use of multiple antiretroviral agents that may harm patients due to their toxic nature. RNA interference (RNAi is a potent candidate for the future treatment of HIV, uses short interfering RNA (siRNA/shRNA for silencing HIV genes. In this study, attempts have been made to create a database HIVsirDB of siRNAs responsible for silencing HIV genes. DESCRIPTIONS: HIVsirDB is a manually curated database of HIV inhibiting siRNAs that provides comprehensive information about each siRNA or shRNA. Information was collected and compiled from literature and public resources. This database contains around 750 siRNAs that includes 75 partially complementary siRNAs differing by one or more bases with the target sites and over 100 escape mutant sequences. HIVsirDB structure contains sixteen fields including siRNA sequence, HIV strain, targeted genome region, efficacy and conservation of target sequences. In order to facilitate user, many tools have been integrated in this database that includes; i siRNAmap for mapping siRNAs on target sequence, ii HIVsirblast for BLAST search against database, iii siRNAalign for aligning siRNAs. CONCLUSION: HIVsirDB is a freely accessible database of siRNAs which can silence or degrade HIV genes. It covers 26 types of HIV strains and 28 cell types. This database will be very useful for developing models for predicting efficacy of HIV inhibiting siRNAs. In summary this is a useful resource for researchers working in the field of siRNA based HIV therapy. HIVsirDB database is accessible at http://crdd.osdd.net/raghava/hivsir/.

  20. Development and characterization of promoterless helper RNAs for the production of alphavirus replicon particle.

    Science.gov (United States)

    Kamrud, K I; Alterson, K; Custer, M; Dudek, J; Goodman, C; Owens, G; Smith, J F

    2010-07-01

    Alphavirus-based replicon systems are frequently used as preclinical vectors and as antigen discovery tools, and they have recently been assessed in clinical vaccine trials. Typically, alphavirus replicon RNAs are delivered within virus-like replicon particles (VRP) that are produced following transfection of replicon RNA and two helper RNAs into permissive cells in vitro. The non-structural proteins expressed from the replicon RNA amplify the replicon RNA in cis and the helper RNAs in trans, the latter providing the viral structural proteins necessary to package the replicon RNA into VRP. Current helper RNA designs incorporate the alphavirus 26S promoter to direct the transcription of high levels of structural gene mRNAs. We demonstrate here that the 26S promoter is not required on helper RNAs to produce VRP and propose that such promoterless helper RNAs, by design, reduce the probability of generating replication-competent virus that may otherwise result from RNA recombination.

  1. MicroRNAs in Human Pituitary Adenomas

    Directory of Open Access Journals (Sweden)

    Xu-Hui Li

    2014-01-01

    Full Text Available MicroRNAs (miRNAs are a class of recently identified noncoding RNAs that regulate gene expression at posttranscriptional level. Due to the large number of genes regulated by miRNAs, miRNAs play important roles in many cellular processes. Emerging evidence indicates that miRNAs are dysregulated in pituitary adenomas, a class of intracranial neoplasms which account for 10–15% of diagnosed brain tumors. Deregulated miRNAs and their targets contribute to pituitary adenomas progression and are associated with cell cycle control, apoptosis, invasion, and pharmacological treatment of pituitary adenomas. To provide an overview of miRNAs dysregulation and functions of these miRNAs in pituitary adenoma progression, we summarize the deregulated miRNAs and their targets to shed more light on their potential as therapeutic targets and novel biomarkers.

  2. Competitive Endogenous RNAs in Prostate Cancer

    Science.gov (United States)

    2015-01-01

    system in prostate cancer. The goal of this study was to determine whether lncRNAs Body The importance of lncRNAs in human diseases may have to do...exert their functions. For example, lncRNAs may exert their functions through interaction with regulatory proteins such as those chromatin remodeling ...ABSTRACT The human genome actively transcribes a large amount of non-coding genes, including microRNA and long non- coding RNAs (lncRNAs). Compared to

  3. Production of Small Noncoding RNAs from the flamenco Locus Is Regulated by the gypsy Retrotransposon of Drosophila melanogaster.

    Science.gov (United States)

    Guida, Vincenzo; Cernilogar, Filippo M; Filograna, Angela; De Gregorio, Roberto; Ishizu, Hirotsugu; Siomi, Mikiko C; Schotta, Gunnar; Bellenchi, Gian Carlo; Andrenacci, Davide

    2016-10-01

    Protective mechanisms based on RNA silencing directed against the propagation of transposable elements are highly conserved in eukaryotes. The control of transposable elements is mediated by small noncoding RNAs, which derive from transposon-rich heterochromatic regions that function as small RNA-generating loci. These clusters are transcribed and the precursor transcripts are processed to generate Piwi-interacting RNAs (piRNAs) and endogenous small interfering RNAs (endo-siRNAs), which silence transposable elements in gonads and somatic tissues. The flamenco locus is a Drosophila melanogaster small RNA cluster that controls gypsy and other transposable elements, and has played an important role in understanding how small noncoding RNAs repress transposable elements. In this study, we describe a cosuppression mechanism triggered by new euchromatic gypsy insertions in genetic backgrounds carrying flamenco alleles defective in gypsy suppression. We found that the silencing of gypsy is accompanied by the silencing of other transposons regulated by flamenco, and of specific flamenco sequences from which small RNAs against gypsy originate. This cosuppression mechanism seems to depend on a post-transcriptional regulation that involves both endo-siRNA and piRNA pathways and is associated with the occurrence of developmental defects. In conclusion, we propose that new gypsy euchromatic insertions trigger a post-transcriptional silencing of gypsy sense and antisense sequences, which modifies the flamenco activity. This cosuppression mechanism interferes with some developmental processes, presumably by influencing the expression of specific genes. Copyright © 2016 by the Genetics Society of America.

  4. 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.

  5. Circulating exosomal microRNAs as biomarkers of colon cancer.

    Directory of Open Access Journals (Sweden)

    Hiroko Ogata-Kawata

    Full Text Available PURPOSE: Exosomal microRNAs (miRNAs have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC. To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined. EXPERIMENTAL DESIGN: Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients. RESULTS: The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis. CONCLUSION: Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and

  6. Designing sgRNAs with CRISPy web

    DEFF Research Database (Denmark)

    Blin, Kai; Lee, Sang Yup; Weber, Tilmann

    2017-01-01

    Tilmann Weber’s group at the Novo Nordisk Foundation Center for Biosustainability developed a user-friendly, web server implementation of the sgRNA prediction software, CRISPy, for non-computer scientists.......Tilmann Weber’s group at the Novo Nordisk Foundation Center for Biosustainability developed a user-friendly, web server implementation of the sgRNA prediction software, CRISPy, for non-computer scientists....

  7. 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.

  8. The tRNA-Derived Small RNAs Regulate Gene Expression through Triggering Sequence-Specific Degradation of Target Transcripts in the Oomycete Pathogen Phytophthora sojae

    Science.gov (United States)

    Wang, Qinhu; Li, Tingting; Xu, Ke; Zhang, Wei; Wang, Xiaolong; Quan, Junli; Jin, Weibo; Zhang, Meixiang; Fan, Guangjin; Wang, Ming-Bo; Shan, Weixing

    2016-01-01

    Along with the well-studied microRNA (miRNA) and small interfering RNA (siRNA) is a new class of transfer RNA-derived small RNA (tsRNA), which has recently been detected in multiple organisms and is implicated in gene regulation. However, while miRNAs and siRNAs are known to repress gene expression through sequence-specific RNA cleavage or translational repression, how tsRNAs regulate gene expression remains unclear. Here we report the identification and functional characterization of tsRNAs in the oomycete pathogen Phytophthora sojae. We show that multiple tRNAs are processed into abundant tsRNAs, which accumulate in a similar developmental stage-specific manner and are negatively correlated with the expression of predicted target genes. Degradome sequencing and 5′ RLM RACE experiments indicate tsRNAs can trigger degradation of target transcripts. Transient expression assays using GUS sensor constructs confirmed the requirement of sequence complementarity in tsRNA-mediated RNA degradation in P. sojae. Our results show that the tsRNA are a class of functional endogenous sRNAs and suggest that tsRNA regulate gene expression through inducing sequence-specific degradation of target RNAs in oomycetes. PMID:28066490

  9. Functional nanostructures for effective delivery of small interfering RNA therapeutics.

    Science.gov (United States)

    Hong, Cheol Am; Nam, Yoon Sung

    2014-01-01

    Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA.

  10. Small Interfering RNA Transfection Across a Phospholipid Membrane

    Science.gov (United States)

    Ngo, Van; Choubey, Amit; Kalia, Rajiv; Nakano, Aiichiro; Vashishta, Priya

    2012-02-01

    Small interfering RNA (siRNA) molecules play a pivotal role in silencing gene expression via the RNA interference mechanism. We have performed steered MD simulations to study the transfection of a bare siRNA and siRNA/Oleic Acid (OA) complex across the dipalmitoylphosphatidycholine (DPPC) bilayer at T = 323 K. Bare siRNA induces the formation of frustrated lipid gel domains, whereas in the presence of siRNA/OA complex the membrane is found to be in the liquid-ordered phase. In both cases the stress profiles across the membrane indicate that the membrane is under tension near the head groups and highly compressed at the water-hydrophobic interface. During transfection, the membrane is deformed and the lateral stress is significantly lowered for the bare siRNA and siRNA/OA complex. The bare siRNA transfects through a lipid-nanopore of hydrophilic head-groups and hydrophobic carbon chains, whereas the siRNA/OA complex transfects through a lipid-nanopore of hydrophilic head groups.

  11. Small interfering RNA delivery through positively charged polymer nanoparticles

    Science.gov (United States)

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

    2016-03-01

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

  12. Exploring the Secrets of Long Noncoding RNAs

    Directory of Open Access Journals (Sweden)

    Mingyang Quan

    2015-03-01

    Full Text Available High-throughput sequencing has revealed that the majority of RNAs have no capacity to encode protein. Among these non-coding transcripts, recent work has focused on the roles of long noncoding RNAs (lncRNAs of >200 nucleotides. Although many of their attributes, such as patterns of expression, remain largely unknown, lncRNAs have key functions in transcriptional, post-transcriptional, and epigenetic gene regulation; Also, new work indicates their functions in scaffolding ribonuclear protein complexes. In plants, genome-wide identification of lncRNAs has been conducted in several species, including Zea mays, and recent research showed that lncRNAs regulate flowering time in the photoperiod pathway, and function in nodulation. In this review, we discuss the basic mechanisms by which lncRNAs regulate key cellular processes, using the large body of knowledge on animal and yeast lncRNAs to illustrate the significance of emerging work on lncRNAs in plants.

  13. Role of microRNAs in spermatogenesis

    Directory of Open Access Journals (Sweden)

    S. A. Rudneva

    2016-01-01

    Full Text Available Male germ cells have a complex transcriptome. In addition to proteincoding messenger RNAs, many noncoding RNAs, including microRNAs (miRNAs, are produced. The miRNAs are important regulators of gene expression. They function mainly post-transcriptionally to control the stability or translation of their target messenger RNAs. The miRNAs are expressed in a cell-specific manner during spermatogenesis to participate in the control of each stage of male germ cell differentiation. Clinical studies have exploited the well-defined expression profiles of miRNAs, and human spermatozoal or seminal plasma miRNAs have been explored as potential biomarkers for male factor infertility.

  14. miRNAs Related to Skeletal Diseases.

    Science.gov (United States)

    Seeliger, Claudine; Balmayor, Elizabeth R; van Griensven, Martijn

    2016-09-01

    miRNAs as non-coding, short, double-stranded RNA segments are important for cellular biological functions, such as proliferation, differentiation, and apoptosis. miRNAs mainly contribute to the inhibition of important protein translations through their cleavage or direct repression of target messenger RNAs expressions. In the last decade, miRNAs got in the focus of interest with new publications on miRNAs in the context of different diseases. For many types of cancer or myocardial damage, typical signatures of local or systemically circulating miRNAs have already been described. However, little is known about miRNA expressions and their molecular effect in skeletal diseases. An overview of published studies reporting miRNAs detection linked with skeletal diseases was conducted. All regulated miRNAs were summarized and their molecular interactions were illustrated. This review summarizes the involvement and interaction of miRNAs in different skeletal diseases. Thereby, 59 miRNAs were described to be deregulated in tissue, cells, or in the circulation of osteoarthritis (OA), 23 miRNAs deregulated in osteoporosis, and 107 miRNAs deregulated in osteosarcoma (OS). The molecular influences of miRNAs regarding OA, osteoporosis, and OS were illustrated. Specific miRNA signatures for skeletal diseases are described in the literature. Some overlapped, but also unique ones for each disease exist. These miRNAs may present useful targets for the development of new therapeutic approaches and are candidates for diagnostic evaluations.

  15. A Comprehensive NGS Data Analysis of Differentially Regulated miRNAs, piRNAs, lncRNAs and sn/snoRNAs in Triple Negative Breast Cancer

    Science.gov (United States)

    Koduru, Srinivas V; Tiwari, Amit K; Leberfinger, Ashley; Hazard, Sprague W; Kawasawa, Yuka Imamura; Mahajan, Milind; Ravnic, Dino J

    2017-01-01

    Cancer is the second leading cause of death in the United States and is a major public health concern worldwide. Basic, clinical and epidemiological research is leading to improved cancer detection, prevention, and outcomes. Recent technological advances have allowed unbiased and comprehensive screening of genome-wide gene expression. Small non-coding RNAs (sncRNAs) have been shown to play an important role in biological processes and could serve as a diagnostic, prognostic and therapeutic biomarker for specific diseases. Recent findings have begun to reveal and enhance our understanding of the complex architecture of sncRNA expression including miRNAs, piRNAs, lncRNAs, sn/snoRNAs and their relationships with biological systems. We used publicly available small RNA sequencing data that was derived from 24 triple negative breast cancers (TNBC) and 14 adjacent normal tissue samples to remap various types of sncRNAs. We found a total of 55 miRNAs were aberrantly expressed (p<0.005) in TNBC samples (8 miRNAs upregulated; 47 downregulated) compared to adjacent normal tissues whereas the original study reported only 25 novel miRs. In this study, we used pathway analysis of differentially expressed miRNAs which revealed TGF-beta signaling pathways to be profoundly affected in the TNBC samples. Furthermore, our comprehensive re-mapping strategy allowed us to discover a number of other differentially expressed sncRNAs including piRNAs, lncRNAs, sn/snoRNAs, rRNAs, miscRNAs and nonsense-mediated decay RNAs. We believe that our sncRNA analysis workflow is extremely comprehensive and suitable for discovery of novel sncRNAs changes, which may lead to the development of innovative diagnostic and therapeutic tools for TNBC.

  16. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses

    Directory of Open Access Journals (Sweden)

    Barik Sailen

    2001-12-01

    Full Text Available Abstract Background Post-transcriptional gene silencing (PTGS by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs for their ability to silence cytoplasmic RNA genomes. Results Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. Conclusions Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.

  17. MicroRNAs as potential therapeutics for treating spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Hualin Yan; Peiwei Hong; Mei Jiang; Hedong Li

    2012-01-01

    MicroRNAs are a class of recently discovered, small non-coding RNAs that have been shown to play essential roles in a vast majority of biological processes. Very little is known about the role of microRNAs during spinal cord injury. This review summarizes the changes in expression levels of microRNAs after spinal cord injury. These aberrant changes suggest that microRNAs play an important role in inflammation, oxidative stress, apoptosis, glial scar formation and axonal regeneration. Given their small size and specificity of action, microRNAs could be potential therapeutics for treating spinal cord injury in the future. There are rapidly developing techniques for manipulating microRNA levels in animals; we review different chemical modification and delivery strategies. These may provide platforms for designing efficient microRNA delivery protocols for use in the clinic.

  18. DASHR: database of small human noncoding RNAs.

    Science.gov (United States)

    Leung, Yuk Yee; Kuksa, Pavel P; Amlie-Wolf, Alexandre; Valladares, Otto; Ungar, Lyle H; Kannan, Sampath; Gregory, Brian D; Wang, Li-San

    2016-01-01

    Small non-coding RNAs (sncRNAs) are highly abundant RNAs, typically database provides searchable, unified annotation, and expression information for full sncRNA transcripts and mature RNA products derived from these larger RNAs. Here, we present the Database of small human noncoding RNAs (DASHR). DASHR contains the most comprehensive information to date on human sncRNA genes and mature sncRNA products. DASHR provides a simple user interface for researchers to view sequence and secondary structure, compare expression levels, and evidence of specific processing across all sncRNA genes and mature sncRNA products in various human tissues. DASHR annotation and expression data covers all major classes of sncRNAs including microRNAs (miRNAs), Piwi-interacting (piRNAs), small nuclear, nucleolar, cytoplasmic (sn-, sno-, scRNAs, respectively), transfer (tRNAs), and ribosomal RNAs (rRNAs). Currently, DASHR (v1.0) integrates 187 smRNA high-throughput sequencing (smRNA-seq) datasets with over 2.5 billion reads and annotation data from multiple public sources. DASHR contains annotations for ∼ 48,000 human sncRNA genes and mature sncRNA products, 82% of which are expressed in one or more of the curated tissues. DASHR is available at http://lisanwanglab.org/DASHR.

  19. Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system.

    Science.gov (United States)

    Taki, Masumi; Tokuda, Yasunori; Ohtsuki, Takashi; Sisido, Masahiko

    2006-12-01

    Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected. Mutated streptavidin mRNAs that contained different four-base codons were prepared and added to the insect cell-free system in the presence of various tRNAs possessing the corresponding four-base anticodons. The tRNAs were chemically aminoacylated with various types of nonnatural amino acids to examine their incorporation efficiencies. Using p-nitrophenylalanine as the nonnatural amino acid and streptavidin as the target protein, tRNA sequences and the types of four-base codons were optimized to maximize the yield of the nonnatural mutant and to minimize production of full-length proteins that do not contain the nonnatural amino acid. Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNA(Pyl) was the most efficient and orthogonal tRNA. Of the CGGN-type four-base codons, CGGA and CGGG were the most efficient ones for assigning the positions of nonnatural amino acids. p-Nitrophenylalanine and 2-naphthylalanine were efficiently incorporated as in the case of Escherichia coli and rabbit reticulocyte cell-free systems. Much less efficient incorporation was observed, however, for other nonnatural amino acids, indicating that the insect system is less tolerant to the structural diversity of amino acids than the E. coli cell-free system.

  20. Abundant primary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line

    NARCIS (Netherlands)

    Lau, N.C.; Robine, N.; Martin, R.; Chung, W.J.; Niki, Y.; Berezikov, E.; Lai, E.C

    2009-01-01

    Piwi proteins, a subclass of Argonaute-family proteins, carry approximately 24-30-nt Piwi-interacting RNAs (piRNAs) that mediate gonadal defense against transposable elements (TEs). We analyzed the Drosophila ovary somatic sheet (OSS) cell line and found that it expresses miRNAs, endogenous small

  1. [Expression silence of Physarum polycephalum serine/arginine protein kinase by small interfering RNA].

    Science.gov (United States)

    Tian, Sheng Li; Zheng, Shuo; Liu, Shi De; Zhang, Jian Hua; Xing, Miao

    2008-04-01

    Serine/arginine protein kinases are specific kinase family for phosphorylating SR protein regulating alternative splicing of SR protein and its distribution, localization in the nucleus. However, it is unclear how Physarum Polycephalum Serine/Arginine Protein Kinase(PSRPK) functions in the cells. In order to study its function, Oligonucleotides for transcribing siRNAs were designed and inserted into pSIREN-RetroQ vector to construct pSIREN-PSRPK-1, pSIREN-PSRPK-2, pSIREN-PSRPK-3, pSIREN-PSRPK-4, pSIREN-PSRPK-5 for expressing siRNAs targeting at PSRPK, as well as the negative control pSIREN-PSRPK-Neg. The PSRPK cDNA amplified by PCR was inserted into the pDsRed-N1 vector to construct a pPSRPK-DsRed plasmid. After the pPSRPK-DsRed was co-transfected into HEK293 cell with recombinant siRNA expression plasmids respectively, the PSRPK-DsRed fusion fluorescent protein was observed under fluorescent microscope after 72 hours co-transfection. The results indicated that pSIREN-PSRPK-2 and pSIREN-PSRPK-5 were able to inhibit the expression of PSRPK-DsRed fusion fluorescent protein efficiently. RT-PCR and Northern dot blot analysis further demonstrated that pSIREN-PSRPK-2 and pSIREN-PSRPK-5 can effectively inhibit PSRPK expression, which accorded with the results under the fluorescent microscope.

  2. The defective RNAs of Closteroviridae

    Directory of Open Access Journals (Sweden)

    Munir eMawassi

    2013-05-01

    Full Text Available The family Closteroviridae consists of two genera, Closterovirus and Ampelovirus with monopartite genomes transmitted respectively by aphids and mealybugs and the Crinivirus with bipartite genomes transmitted by whiteflies. The Closteroviridae consists of more than thirty virus species, which differ considerably in their phytopathological significance. Some, like Beet yellows virus (BYV and Citrus tristeza virus (CTV were associated for many decades with their respective hosts, sugar beets and citrus. Others, like the grapevine leafroll-associated ampeloviruses 1, and 3 were also associated with their grapevine hosts for long periods; however difficulties in virus isolation hampered their molecular characterization. The majority of the recently identified Closteroviridae were probably associated with their vegetative propagated host plants for long periods and only detected through the considerable advances in dsRNA isolation and sequencing of PCR amplified replicons. Molecular characterization of CTV and several other Closteroviridae revealed that, in addition to genomic and subgenomic RNAs, infected plants contain several different subviral defective RNAs (dRNAs. The roles and biological functions of dRNAs associated with Closteroviridae remain terra incognita.

  3. Population diversity of rice stripe virus-derived siRNAs in three different hosts and RNAi-based antiviral immunity in Laodelphgax striatellus.

    Directory of Open Access Journals (Sweden)

    Yi Xu

    Full Text Available BACKGROUND: Small RNA-mediated gene silencing plays evolutionarily conserved roles in gene regulation and defense against invasive nucleic acids. Virus-derived small interfering RNAs (vsiRNAs are one of the key elements involved in RNA silencing-based antiviral activities in plant and insect. vsiRNAs produced after viruses infecting hosts from a single kingdom (i.e., plant or animal are well described. In contrast, vsiRNAs derived from viruses capable of infecting both plants and their insect vectors have not been characterized. METHODOLOGY/PRINCIPAL FINDINGS: We examined Rice stripe virus (RSV-derived small interfering RNAs in three different hosts, Oryza sativa, Nicotiana benthamiana and a natural RSV transmitting vector Laodelphgax striatellus, through deep sequencing. Our results show that large amounts of vsiRNAs generated in these hosts after RSV infection. The vsiRNAs from N. benthamiana and L. striatellus mapped equally to the genomic- and antigenomic-strand of RSV RNAs. They showed, however, a significant bias in those from O. sativa. Furthermore, our results demonstrate that the number and size distributions of vsiRNAs in the three hosts were very different. In O. sativa and N. benthamiana, most vsiRNAs were mapped to the discrete regions in the RSV genome sequence, and most of the vsiRNAs from these two hosts were generated from RSV genomic RNAs 3 and 4. In contrast, the vsiRNAs identified in L. striatellus distributed uniformly along the whole genome of RSV. We have also shown that silencing Agronaute 2 in L. striatellus enhanced RSV accumulation in this host. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that the core RNA-induced gene silencing (RNAi machinery is present in L. striatellus. We also provide evidence that the RNAi-mediated immunity against RSV is present in L. striatellus. We propose that a common small RNA-mediated virus defense mechanism exists in both helipterum insects and plants, but the vsiRNAs are generated

  4. Different Virus-Derived siRNAs Profiles between Leaves and Fruits in Cucumber Green Mottle Mosaic Virus-Infected Lagenaria siceraria Plants.

    Science.gov (United States)

    Li, Junmin; Zheng, Hongying; Zhang, Chenhua; Han, Kelei; Wang, Shu; Peng, Jiejun; Lu, Yuwen; Zhao, Jinping; Xu, Pei; Wu, Xiaohua; Li, Guojing; Chen, Jianping; Yan, Fei

    2016-01-01

    RNA silencing is an evolutionarily conserved antiviral mechanism, through which virus-derived small interfering RNAs (vsiRNAs) playing roles in host antiviral defense are produced in virus-infected plant. Deep sequencing technology has revolutionized the study on the interaction between virus and plant host through the analysis of vsiRNAs profile. However, comparison of vsiRNA profiles in different tissues from a same host plant has been rarely reported. In this study, the profiles of vsiRNAs from leaves and fruits of Lagenaria siceraria plants infected with Cucumber green mottle mosaic virus (CGMMV) were comprehensively characterized and compared. Many more vsiRNAs were present in infected leaves than in fruits. vsiRNAs from both leaves and fruits were mostly 21- and 22-nt in size as previously described in other virus-infected plants. Interestingly, vsiRNAs were predominantly produced from the viral positive strand RNAs in infected leaves, whereas in infected fruits they were derived equally from the positive and negative strands. Many leaf-specific positive vsiRNAs with lengths of 21-nt (2058) or 22-nt (3996) were identified but only six (21-nt) and one (22-nt) positive vsiRNAs were found to be specific to fruits. vsiRNAs hotspots were only present in the 5'-terminal and 3'-terminal of viral positive strand in fruits, while multiple hotspots were identified in leaves. Differences in GC content and 5'-terminal nucleotide of vsiRNAs were also observed in the two organs. To our knowledge, this provides the first high-resolution comparison of vsiRNA profiles between different tissues of the same host plant.

  5. 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...... including cancer and cardiovascular diseases. Since miRNAs have a crucial role in fine-tuning the expression of their targets, they have been proposed as biomarkers of disease progression and prognostication.In this chapter we discuss different approaches for computational predictions of miRNA targets based...... on sequence complementarity and integration of expression data. In the last section of the chapter we discuss new opportunities in the study of miRNA regulatory networks in the context of temporal disease progression and comorbidities....

  6. MicroRNAs in nasopharyngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jeff P Bruce; Fei-Fei Liu

    2014-01-01

    MicroRNAs (miRNAs) provide insight into both the biology and clinical behavior of many human cancers, including nasopharyngeal carcinoma (NPC). The dysregulation of miRNAs in NPC results in a variety of tumor-promoting effects. Furthermore, several miRNAs are prognostic markers for NPC. In addition to celular miRNAs, NPC samples also often contain miRNAs encoded by Epstein-Barr virus, and these miRNAs may impact NPC biology by targeting both celular and viral genes. Given their numerous putative roles in NPC development and progression, a thorough understanding of the impact of miRNA dysregulation in NPC is expected to shed light on useful biomarkers and therapeutic targets for the clinical management of this disease. In this review, we describe the efforts to date to identify and characterize such miRNAs in the context of NPC.

  7. Uncovering growth-suppressive MicroRNAs in lung cancer

    DEFF Research Database (Denmark)

    Liu, Xi; Sempere, Lorenzo F; Galimberti, Fabrizio

    2009-01-01

    PURPOSE: MicroRNA (miRNA) expression profiles improve classification, diagnosis, and prognostic information of malignancies, including lung cancer. This study uncovered unique growth-suppressive miRNAs in lung cancer. EXPERIMENTAL DESIGN: miRNA arrays were done on normal lung tissues and adenocar...

  8. Sponging of Cellular Proteins by Viral RNAs

    OpenAIRE

    Charley, Phillida A.; Wilusz, Jeffrey

    2014-01-01

    Viral RNAs accumulate to high levels during infection and interact with a variety of cellular factors including miRNAs and RNA-binding proteins. Although many of these interactions exist to directly modulate replication, translation and decay of viral transcripts, evidence is emerging that abundant viral RNAs may in certain cases serve as a sponge to sequester host non coding RNAs and proteins. By effectively reducing the ability of cellular RNA binding proteins to regulate host cell gene exp...

  9. 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.

  10. Non-Coding RNAs in Retinal Development

    Directory of Open Access Journals (Sweden)

    Robert Hindges

    2012-01-01

    Full Text Available Retinal development is dependent on an accurately functioning network of transcriptional and translational regulators. Among the diverse classes of molecules involved, non-coding RNAs (ncRNAs play a significant role. Members of this family are present in the cell as transcripts, but are not translated into proteins. MicroRNAs (miRNAs are small ncRNAs that act as post-transcriptional regulators. During the last decade, they have been implicated in a variety of biological processes, including the development of the nervous system. On the other hand, long-ncRNAs (lncRNAs represent a different class of ncRNAs that act mainly through processes involving chromatin remodeling and epigenetic mechanisms. The visual system is a prominent model to investigate the molecular mechanisms underlying neurogenesis or circuit formation and function, including the differentiation of retinal progenitor cells to generate the seven principal cell classes in the retina, pathfinding decisions of retinal ganglion cell axons in order to establish the correct connectivity from the eye to the brain proper, and activity-dependent mechanisms for the functionality of visual circuits. Recent findings have associated ncRNAs in several of these processes and uncovered a new level of complexity for the existing regulatory mechanisms. This review summarizes and highlights the impact of ncRNAs during the development of the vertebrate visual system, with a specific focus on the role of miRNAs and a synopsis regarding recent findings on lncRNAs in the retina.

  11. Studying microRNAs in lymphoma

    NARCIS (Netherlands)

    Kluiver, Joost; Slezak-Prochazka, Izabella; van den Berg, Anke

    2013-01-01

    MicroRNAs (miRNAs) play important roles in development, differentiation, homeostasis, and also in diseases such as lymphoma. This chapter describes methods to study the role of miRNAs in lymphoma. First, we describe a multiplex RT reaction followed by qPCR that can be used to determine differential

  12. Gene function analysis by artificial microRNAs in Physcomitrella patens.

    KAUST Repository

    Khraiwesh, Basel

    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 sequences resulting in cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA sequence of endogenous MIR precursor genes, while maintaining the general pattern of matches and mismatches in the foldback. Thus, for functional gene analysis amiRNAs can be designed to target any gene of interest. During the last decade the moss Physcomitrella patens emerged as a model plant for functional gene analysis based on its unique ability to integrate DNA into the nuclear genome by homologous recombination which allows for the generation of targeted gene knockout mutants. In addition to this, we developed a protocol to express amiRNAs in P. patens that has particular advantages over the generation of knockout mutants and might be used to speed up reverse genetics approaches in this model species.

  13. Comparative Characterization of Cardiac Development Specific microRNAs: Fetal Regulators for Future.

    Science.gov (United States)

    Rustagi, Yashika; Jaiswal, Hitesh K; Rawal, Kamal; Kundu, Gopal C; Rani, Vibha

    2015-01-01

    MicroRNAs (miRNAs) are small, conserved RNAs known to regulate several biological processes by influencing gene expression in eukaryotes. The implication of miRNAs as another player of regulatory layers during heart development and diseases has recently been explored. However, there is no study which elucidates the profiling of miRNAs during development of heart till date. Very limited miRNAs have been reported to date in cardiac context. In addition, integration of large scale experimental data with computational and comparative approaches remains an unsolved challenge.The present study was designed to identify the microRNAs implicated in heart development using next generation sequencing, bioinformatics and experimental approaches. We sequenced six small RNA libraries prepared from different developmental stages of the heart using chicken as a model system to produce millions of short sequence reads. We detected 353 known and 703 novel miRNAs involved in heart development. Out of total 1056 microRNAs identified, 32.7% of total dataset of known microRNAs displayed differential expression whereas seven well studied microRNAs namely let-7, miR-140, miR-181, miR-30, miR-205, miR-103 and miR-22 were found to be conserved throughout the heart development. The 3'UTR sequences of genes were screened from Gallus gallus genome for potential microRNA targets. The target mRNAs were appeared to be enriched with genes related to cell cycle, apoptosis, signaling pathways, extracellular remodeling, metabolism, chromatin remodeling and transcriptional regulators. Our study presents the first comprehensive overview of microRNA profiling during heart development and prediction of possible cardiac specific targets and has a big potential in future to develop microRNA based therapeutics against cardiac pathologies where fetal gene re-expression is witnessed in adult heart.

  14. Comparative Characterization of Cardiac Development Specific microRNAs: Fetal Regulators for Future.

    Directory of Open Access Journals (Sweden)

    Yashika Rustagi

    Full Text Available MicroRNAs (miRNAs are small, conserved RNAs known to regulate several biological processes by influencing gene expression in eukaryotes. The implication of miRNAs as another player of regulatory layers during heart development and diseases has recently been explored. However, there is no study which elucidates the profiling of miRNAs during development of heart till date. Very limited miRNAs have been reported to date in cardiac context. In addition, integration of large scale experimental data with computational and comparative approaches remains an unsolved challenge.The present study was designed to identify the microRNAs implicated in heart development using next generation sequencing, bioinformatics and experimental approaches. We sequenced six small RNA libraries prepared from different developmental stages of the heart using chicken as a model system to produce millions of short sequence reads. We detected 353 known and 703 novel miRNAs involved in heart development. Out of total 1056 microRNAs identified, 32.7% of total dataset of known microRNAs displayed differential expression whereas seven well studied microRNAs namely let-7, miR-140, miR-181, miR-30, miR-205, miR-103 and miR-22 were found to be conserved throughout the heart development. The 3'UTR sequences of genes were screened from Gallus gallus genome for potential microRNA targets. The target mRNAs were appeared to be enriched with genes related to cell cycle, apoptosis, signaling pathways, extracellular remodeling, metabolism, chromatin remodeling and transcriptional regulators. Our study presents the first comprehensive overview of microRNA profiling during heart development and prediction of possible cardiac specific targets and has a big potential in future to develop microRNA based therapeutics against cardiac pathologies where fetal gene re-expression is witnessed in adult heart.

  15. The Tumor Cytosol miRNAs, Fluid miRNAs and Exosome miRNAs in Lung Cancer

    Directory of Open Access Journals (Sweden)

    Xin eQin

    2015-01-01

    Full Text Available The focus of this review is to provide an update on the progress of microRNAs (miRNAs as potential biomarkers for lung cancer. miRNAs are single-stranded, small noncoding RNAs that regulate gene expression and show tissue-specific signatures. Accumulating evidence indicates that miRNA expression patterns represent the in vivo status in physiology and disease. Moreover, miRNAs are stable in serum and other clinically convenient and available tissue sources, so they are being developed as biomarkers for cancer and other diseases. Cancer is currently the primary driver of the field, but miRNA biomarkers are being developed for many other diseases such as cardiovascular and central nervous system diseases. Here we examine the framework and scope of the miRNA landscape as it specifically relates to the translation of miRNA expression patterns/signatures into biomarkers for developing diagnostics for lung cancer. We focus on examining tumor cytosol miRNAs, fluid miRNAs, and exosome miRNAs in lung cancer, the connections among these miRNAs, and the potential of miRNA biomarkers for the development of diagnostics. In lung cancer, miRNAs have been studied in both cell populations and in the circulation. However, a major challenge is to develop biomarkers to monitor cancer development and to identify circulating miRNAs that are linked to cancer stage. Importantly, the fact that miRNAs can be successfully harvested from biological fluids allows for the development of biofluid biopsies, in which miRNAs as circulating biomarkers can be captured and analyzed ex vivo. Our hope is that these minimally invasive entities provide a window to the in vivo milieu of the patients without the need for costly, complex invasive procedures, rapidly moving miRNAs from research to the clinic.

  16. MicroRNAs in the Hypothalamus

    DEFF Research Database (Denmark)

    Meister, Björn; Herzer, Silke; Silahtaroglu, Asli

    2013-01-01

    MicroRNAs (miRNAs) are short (∼22 nucleotides) non-coding ribonucleic acid (RNA) molecules that negatively regulate the expression of protein-coding genes. Posttranscriptional silencing of target genes by miRNA is initiated by binding to the 3'-untranslated regions of target mRNAs, resulting in s...... of the hypothalamus and miRNAs have recently been shown to be important regulators of hypothalamic control functions. The aim of this review is to summarize some of the current knowledge regarding the expression and role of miRNAs in the hypothalamus....

  17. Long Non-coding RNAs Expression Profile in HepG2 Cells Reveals the Potential Role of Long Non-coding RNAs in the Cholesterol Metabolism

    Institute of Scientific and Technical Information of China (English)

    Gang Liu; Xinxin Zheng; Yanlu Xu; Jie Lu; Jingzhou Chen; Xiaohong Huang

    2015-01-01

    Background:Green tea has been shown to improve cholesterol metabolism in animal studies,but the molecular mechanisms underlying this function have not been fully understood.Long non-coding RNAs (lncRNAs) have recently emerged as a major class of regulatory molecules involved in a broad range of biological processes and complex diseases.Our aim was to identify important lncRNAs that might play an important role in contributing to the benefits of epigallocatechin-3-gallate (EGCG) on cholesterol metabolism.Methods:Microarrays was used to reveal the lncRNA and mRNA profiles in green tea polyphenol(-)-epigallocatechin gallate in cultured human liver (HepG2) hepatocytes treated with EGCG and bioinformatic analyses of the predicted target genes were performed to identify lncRNA-mRNA targeting relationships.RNA interference was used to investigate the role of lncRNAs in cholesterol metabolism.Results:The expression levels of 15 genes related to cholesterol metabolism and 285 lncRNAs were changed by EGCG treatment.Bioinformatic analysis found five matched lncRNA-mRNA pairs for five differentially expressed lncRNAs and four differentially expressed mRNA.In particular,the lncRNA4 T102202 and its potential targets mRNA-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) were identified.Using a real-time polymerase chain reaction technique,we confirmed that EGCG down-regulated mRNA expression level of the HMGCR and up-regulated expression ofAT102202.After AT102202 knockdown in HepG2,we observed that the level of HMGCR expression was significantly increased relative to the scrambled small interfering RNA control (P < 0.05).Conclusions:Our results indicated that EGCG improved cholesterol metabolism and meanwhile changed the lncRNAs expression profile in HepG2 cells.LncRNAs may play an important role in the cholesterol metabolism.

  18. Antibiotic drugs targeting bacterial RNAs

    Directory of Open Access Journals (Sweden)

    Weiling Hong

    2014-08-01

    Full Text Available RNAs have diverse structures that include bulges and internal loops able to form tertiary contacts or serve as ligand binding sites. The recent increase in structural and functional information related to RNAs has put them in the limelight as a drug target for small molecule therapy. In addition, the recognition of the marked difference between prokaryotic and eukaryotic rRNA has led to the development of antibiotics that specifically target bacterial rRNA, reduce protein translation and thereby inhibit bacterial growth. To facilitate the development of new antibiotics targeting RNA, we here review the literature concerning such antibiotics, mRNA, riboswitch and tRNA and the key methodologies used for their screening.

  19. An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs.

    Directory of Open Access Journals (Sweden)

    Rebecca A Mosher

    Full Text Available BACKGROUND: Small RNAs generated by RNA polymerase IV (Pol IV are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-siRNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus. RESULTS: Paternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases. CONCLUSIONS: Differential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2.

  20. Viral RNAs are unusually compact.

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    Ajaykumar Gopal

    Full Text Available A majority of viruses are composed of long single-stranded genomic RNA molecules encapsulated by protein shells with diameters of just a few tens of nanometers. We examine the extent to which these viral RNAs have evolved to be physically compact molecules to facilitate encapsulation. Measurements of equal-length viral, non-viral, coding and non-coding RNAs show viral RNAs to have among the smallest sizes in solution, i.e., the highest gel-electrophoretic mobilities and the smallest hydrodynamic radii. Using graph-theoretical analyses we demonstrate that their sizes correlate with the compactness of branching patterns in predicted secondary structure ensembles. The density of branching is determined by the number and relative positions of 3-helix junctions, and is highly sensitive to the presence of rare higher-order junctions with 4 or more helices. Compact branching arises from a preponderance of base pairing between nucleotides close to each other in the primary sequence. The density of branching represents a degree of freedom optimized by viral RNA genomes in response to the evolutionary pressure to be packaged reliably. Several families of viruses are analyzed to delineate the effects of capsid geometry, size and charge stabilization on the selective pressure for RNA compactness. Compact branching has important implications for RNA folding and viral assembly.

  1. [MicroRNAs and kidneys].

    Science.gov (United States)

    Stříteská, Jana; Nekvindová, Jana; Cerný, Vladimír; Palička, Vladimír

    2014-01-01

    MicroRNAs are short non-coding ribonucleic acid molecules that regulate gene expression at the post-transcriptional level thus affecting important physiological as well as pathophysiological processes in the organism, for example cell differentiation, proliferation, apoptosis, and metabolism. They are involved in pathogenesis of many diseases including cancer. Many microRNAs are tissue or organ-specific which implies their possible potential as biomarkers or maybe even therapeutical agents as documented by microRNA research interest rising exponentially during last years. Among all, microRNAs are important also for physiological function of the kidney and they are involved in various renal disorders. Today research is focused mainly on renal and urinary tract carcinogenesis, acute kidney injury, chronic renal diseases (polycystic kidney disease) or renal complications of systemic diseases such as diabetic or hypertension nephropathy and autoimmune kidney injury including acute allograft rejection after kidney transplantation. The review summarizes current information about microRNA effect on kidney development and function and also on the most common kidney diseases.

  2. miRNAs in brain development

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    Petri, Rebecca; Malmevik, Josephine; Fasching, Liana; Åkerblom, Malin; Jakobsson, Johan, E-mail: johan.jakobsson@med.lu.se

    2014-02-01

    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.

  3. Identification and Functional Prediction of Large Intergenic Noncoding RNAs (lincRNAs) in Rainbow Trout (Oncorhynchus mykiss)

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    Long noncoding RNAs (lncRNAs) have been recognized in recent years as key regulators of diverse cellular processes. Genome-wide large-scale projects have uncovered thousands of lncRNAs in many model organisms. Large intergenic noncoding RNAs (lincRNAs) are lncRNAs that are transcribed from intergeni...

  4. Characterization of human pseudogene-derived non-coding RNAs for functional potential.

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    Xingyi Guo

    Full Text Available Thousands of pseudogenes exist in the human genome and many are transcribed, but their functional potential remains elusive and understudied. To explore these issues systematically, we first developed a computational pipeline to identify transcribed pseudogenes from RNA-Seq data. Applying the pipeline to datasets from 16 distinct normal human tissues identified ∼ 3,000 pseudogenes that could produce non-coding RNAs in a manner of low abundance but high tissue specificity under normal physiological conditions. Cross-tissue comparison revealed that the transcriptional profiles of pseudogenes and their parent genes showed mostly positive correlations, suggesting that pseudogene transcription could have a positive effect on the expression of their parent genes, perhaps by functioning as competing endogenous RNAs (ceRNAs, as previously suggested and demonstrated with the PTEN pseudogene, PTENP1. Our analysis of the ENCODE project data also found many transcriptionally active pseudogenes in the GM12878 and K562 cell lines; moreover, it showed that many human pseudogenes produced small RNAs (sRNAs and some pseudogene-derived sRNAs, especially those from antisense strands, exhibited evidence of interfering with gene expression. Further integrated analysis of transcriptomics and epigenomics data, however, demonstrated that trimethylation of histone 3 at lysine 9 (H3K9me3, a posttranslational modification typically associated with gene repression and heterochromatin, was enriched at many transcribed pseudogenes in a transcription-level dependent manner in the two cell lines. The H3K9me3 enrichment was more prominent in pseudogenes that produced sRNAs at pseudogene loci and their adjacent regions, an observation further supported by the co-enrichment of SETDB1 (a H3K9 methyltransferase, suggesting that pseudogene sRNAs may have a role in regional chromatin repression. Taken together, our comprehensive and systematic characterization of pseudogene

  5. FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs.

    Science.gov (United States)

    Huang, Kun; Doyle, Francis; Wurz, Zachary E; Tenenbaum, Scott A; Hammond, Reza K; Caplan, Jeffrey L; Meyers, Blake C

    2017-08-21

    Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA-mRNA interactions combined with a fluorophore-binding sequence 'Spinach', a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Annotation of mammalian primary microRNAs

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

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    Miele E

    2012-07-01

    treatment regimens in a substantial way. These nanoparticles could be designed to surmount one or more of the barriers encountered by siRNA. Nanoparticle drug formulations afford the chance to improve drug bioavailability, exploiting superior tissue permeability, payload protection, and the “stealth” features of these entities. The main aims of this review are: to explain the siRNA mechanism with regard to potential applications in siRNA-based cancer therapy; to discuss the possible usefulness of nanoparticle-based delivery of certain molecules for overcoming present therapeutic limitations; to review the ongoing relevant clinical research with its pitfalls and promises; and to evaluate critically future perspectives and challenges in siRNA-based cancer therapy.Keywords: small interfering RNA, nanoparticles, cancer therapy, delivery strategies, biological barriers, clinical trials

  8. Use of cryopreserved cell aliquots in the high-throughput screening of small interfering RNA libraries.

    Science.gov (United States)

    Swearingen, Elissa A; Fajardo, Flordeliza; Wang, Xiangyun; Watson, J E Vivienne; Quon, Kim C; Kassner, Paul D

    2010-06-01

    Screening small interfering RNA (siRNA) libraries holds the potential to elucidate gene function as well as discover new targets for the therapeutic treatment of disease. Since the inception of siRNA as a discovery tool, there have been progressive improvements in siRNA design algorithms, the transfection reagents used to deliver them, and the assay formats used to monitor phenotypic changes. These changes have helped to improve the quality of the data emerging from siRNA screens. One variable that introduces inconsistency into high-throughput screening (HTS) of siRNA libraries is the state of the cells used in the assays. Multiple factors can contribute to differences in transfection efficiency as well as the basic cell biology, which can lead to differences in the genes identified in siRNA screens. The authors have developed a system using frozen cell aliquots to use in siRNA HTS, so that a major source of variability introduced into cell-based screens can be standardized. In addition, by transiently transfecting plasmids into cell lines and then freezing these cells down to use in siRNA transfection experiments, they have used this same technology to create new cell lines. This process of using aliquots of frozen cells is logistically advantageous in an HTS setting, as it reduces the time spent maintaining cell lines, as well as reducing possible downtime in screening due to lack of cells or poor cell health.

  9. Amber suppression in Escherichia coli by unusual mitochondria-like transfer RNAs.

    Science.gov (United States)

    Bourdeau, V; Steinberg, S V; Ferbeyre, G; Emond, R; Cermakian, N; Cedergren, R

    1998-02-17

    The "cloverleaf" base-pairing pattern was established as the structural paradigm of active tRNA species some 30 years ago. Nevertheless, this pattern does not accommodate the folding of certain mitochondrial tRNAs. For these recalcitrant tRNAs, we have proposed structures having from 5 to 10 base pairs in the anticodon stem rather than the canonical 6. The absence of these types of tRNAs in cytoplasmic translation systems, however, raises the possibility that they may not be bona fide alternate folding patterns for active tRNA molecules. For this reason, we have designed new tRNA genes based on our model of unusual mitochondrial tRNAs, having 7, 8, 9, and 10 base pairs in the anticodon stem with other modifications to the D-stem and connector regions. We show here that these synthetic genes produce tRNAs that actively suppress amber codons in vivo.

  10. Therapeutic targeting of microRNAs: current status and future challenges.

    Science.gov (United States)

    Li, Zhonghan; Rana, Tariq M

    2014-08-01

    MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that have crucial roles in regulating gene expression. Increasing evidence supports a role for miRNAs in many human diseases, including cancer and autoimmune disorders. The function of miRNAs can be efficiently and specifically inhibited by chemically modified antisense oligonucleotides, supporting their potential as targets for the development of novel therapies for several diseases. In this Review we summarize our current knowledge of the design and performance of chemically modified miRNA-targeting antisense oligonucleotides, discuss various in vivo delivery strategies and analyse ongoing challenges to ensure the specificity and efficacy of therapeutic oligonucleotides in vivo. Finally, we review current progress on the clinical development of miRNA-targeting therapeutics.

  11. Interferência em misturas de feijão Interference in bean mixtures

    Directory of Open Access Journals (Sweden)

    Janete Joanol da Silveira Mastrantonio

    2004-04-01

    Full Text Available O presente trabalho teve como objetivos examinar a magnitude dos efeitos de interferência em cultivares de feijão, quando em mistura, empregando um novo coeficiente (Coeficiente de Interferência e verificar a existência de correlações entre rendimento de grãos e seus componentes primários, para cada cultivar. Os experimentos foram conduzidos em Canguçu e Passo Fundo, RS, sendo os tratamentos compostos pelas cultivares Carioca, Guateian 6662 e Tayhú em estande puro e em duas misturas, Mis1 (equivalente a 240 mil plantas.ha-1, como em estande puro e Mis2 (120 mil plantas.ha-1. As variáveis avaliadas foram número de vagens por planta (NVP, número de sementes por vagem (NSV, peso de 100 sementes (PCS e rendimento de grãos. O delineamento experimental utilizado foi blocos casualizados com cinco repetições. Observou-se que Tayhú teve sua produtividade estimulada quando em mistura sendo este efeito bastante pronunciado sob a população de 240 mil plantas.ha-1; caracterizando a presença de efeitos positivos de interferência, sendo que Carioca e Guateian 6662 sofreram efeitos neutros quando em Mis1 e negativos quando em Mis2. Verificou-se que as correlações fenotípicas entre os componentes do rendimento dependem de cada cultivar e do ambiente em que se encontram, apresentando pouco valor em programas de melhoramento ou na predição do rendimento.This work had as main objectives 1 to examine the magnitude of interference effects on bean cultivars, under two population densities, through the use of a new coefficient (Coefficient of Interference; and 2 To verify possible correlations among grain yield and its primary components, for each cultivar. Trials were carried out in the locations of Canguçu and Passo Fundo, State of Rio Grande do Sul, Brazil, and the trataments were the cultivars Carioca, Guateian 6662 and Tayhú, under pure stand and under two mixed populations. Mixtures of cultivars were 240.000 (Mis1 and 120.000 (Mis2

  12. MicroRNAs in renal fibrosis

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    Arthur Chi-Kong Chung

    2015-02-01

    Full Text Available MicroRNAs (miRNAs are endogenous short noncoding RNAs that regulate most of important cellular processes by inhibiting gene expression through the post-transcriptional repression of their target mRNAs. . In kidneys, miRNAs have been associated in renal development, homeostasis, and physiological functions. Results from clinical and experimental animal studies demonstrate that miRNAs play essential roles in the pathogenesis of various renal diseases. Chronic kidney diseases (CKD is characterized by renal fibrosis. Transforming growth factor beta (TGF-β is recognized as a major mediator of renal fibrosis because it is able to stimulate the accumulation of extracellular matrix proteins to impair normal kidney function. Recently, emerging evidence demonstrate the relationship between TGF-β signaling and miRNAs expression during renal diseases. TGF-β regulates expression of several microRNAs, such as miR-21, miR-192, miR-200, miR-433, and miR-29. MiR-21, miR-192, and miR-433 which are positively induced by TGF-β signaling play a pathological role in kidney diseases. In contrast, members in both miR-29 and miR-200 families which are inhibited by TGF-β signaling protect kidneys from renal fibrosis by suppressing the deposition of extracellular matrix and preventing epithelial-to-mesenchymal transition, respectively. Clinically, the presence of miRNAs in blood and urine has been examined to be early biomarkers for detecting renal diseases. From experimental animal studies of CKD, targeting microRNAs also provides evidence about therapeutic potential of miRNAs during renal diseases. Now, it comes to the stage to examine the exact mechanisms of miRNAs during the initiation and progression of renal diseases. Therefore, determining the function of miRNAs in renal fibrosis may facilitate the development of both early diagnosis and treatment of renal diseases.

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

    Science.gov (United States)

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

    2013-06-01

    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.

  14. 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.

  15. Soft computing model for optimized siRNA design by identifying off target possibilities using artificial neural network model.

    Science.gov (United States)

    Murali, Reena; John, Philips George; Peter S, David

    2015-05-15

    The ability of small interfering RNA (siRNA) to do posttranscriptional gene regulation by knocking down targeted genes is an important research topic in functional genomics, biomedical research and in cancer therapeutics. Many tools had been developed to design exogenous siRNA with high experimental inhibition. Even though considerable amount of work has been done in designing exogenous siRNA, design of effective siRNA sequences is still a challenging work because the target mRNAs must be selected such that their corresponding siRNAs are likely to be efficient against that target and unlikely to accidentally silence other transcripts due to sequence similarity. In some cases, siRNAs may tolerate mismatches with the target mRNA, but knockdown of genes other than the intended target could make serious consequences. Hence to design siRNAs, two important concepts must be considered: the ability in knocking down target genes and the off target possibility on any nontarget genes. So before doing gene silencing by siRNAs, it is essential to analyze their off target effects in addition to their inhibition efficacy against a particular target. Only a few methods have been developed by considering both efficacy and off target possibility of siRNA against a gene. In this paper we present a new design of neural network model with whole stacking energy (ΔG) that enables to identify the efficacy and off target effect of siRNAs against target genes. The tool lists all siRNAs against a particular target with their inhibition efficacy and number of matches or sequence similarity with other genes in the database. We could achieve an excellent performance of Pearson Correlation Coefficient (R=0. 74) and Area Under Curve (AUC=0.906) when the threshold of whole stacking energy is ≥-34.6 kcal/mol. To the best of the author's knowledge, this is one of the best score while considering the "combined efficacy and off target possibility" of siRNA for silencing a gene. The proposed model

  16. microRNAs as neuroregulators, biomarkers and therapeutic agents in neurodegenerative diseases.

    Science.gov (United States)

    Basak, Indranil; Patil, Ketan S; Alves, Guido; Larsen, Jan Petter; Møller, Simon Geir

    2016-02-01

    The last decade has experienced the emergence of microRNAs as a key molecular tool for the diagnosis and prognosis of human diseases. Although the focus has mostly been on cancer, neurodegenerative diseases present an exciting, yet less explored, platform for microRNA research. Several studies have highlighted the significance of microRNAs in neurogenesis and neurodegeneration, and pre-clinical studies have shown the potential of microRNAs as biomarkers. Despite this, no bona fide microRNAs have been identified as true diagnostic or prognostic biomarkers for neurodegenerative disease. This is mainly due to the lack of precisely defined patient cohorts and the variability within and between individual cohorts. However, the discovery that microRNAs exist as stable molecules at detectable levels in body fluids has opened up new avenues for microRNAs as potential biomarker candidates. Furthermore, technological developments in microRNA biology have contributed to the possible design of microRNA-mediated disease intervention strategies. The combination of these advancements, with the availability of well-defined longitudinal patient cohort, promises to not only assist in developing invaluable diagnostic tools for clinicians, but also to increase our overall understanding of the underlying heterogeneity of neurodegenerative diseases. In this review, we present a comprehensive overview of the existing knowledge of microRNAs in neurodegeneration and provide a perspective of the applicability of microRNAs as a basis for future therapeutic intervention strategies.

  17. Genome-Wide Expression of MicroRNAs Is Regulated by DNA Methylation in Hepatocarcinogenesis

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    Jing Shen

    2015-01-01

    Full Text Available Background. Previous studies, including ours, have examined the regulation of microRNAs (miRNAs by DNA methylation, but whether this regulation occurs at a genome-wide level in hepatocellular carcinoma (HCC is unclear. Subjects/Methods. Using a two-phase study design, we conducted genome-wide screening for DNA methylation and miRNA expression to explore the potential role of methylation alterations in miRNAs regulation. Results. We found that expressions of 25 miRNAs were statistically significantly different between tumor and nontumor tissues and perfectly differentiated HCC tumor from nontumor. Six miRNAs were overexpressed, and 19 were repressed in tumors. Among 133 miRNAs with inverse correlations between methylation and expression, 8 miRNAs (6% showed statistically significant differences in expression between tumor and nontumor tissues. Six miRNAs were validated in 56 additional paired HCC tissues, and significant inverse correlations were observed for miR-125b and miR-199a, which is consistent with the inactive chromatin pattern found in HepG2 cells. Conclusion. These data suggest that the expressions of miR-125b and miR-199a are dramatically regulated by DNA hypermethylation that plays a key role in hepatocarcinogenesis.

  18. Characterization of rice black-streaked dwarf virus- and rice stripe virus-derived siRNAs in singly and doubly infected insect vector Laodelphax striatellus.

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    Junmin Li

    Full Text Available Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi which generates viral-derived small interfering RNAs (siRNAs. However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus was infected by Rice black-streaked dwarf virus (RBSDV (Reoviridae; Fijivirus, more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV, a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5'- and 3'-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.

  19. Interferência das plantas daninhas na cultura do tomate para processamento Weed interference in processing tomato crops

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    Adriano Stephan Nascente

    2004-09-01

    Full Text Available Para se alcançar eficiência no manejo cultural, é muito importante determinar o período crítico de interferência (PCI das plantas daninhas no cultivo das hortaliças. Avaliou-se neste trabalho a interferência das plantas daninhas na cultura do tomate para processamento, pelo transplantio de mudas, na Embrapa Hortaliças, em delineamento de blocos ao acaso com 3 repetições. Os tratamentos foram divididos em dois grupos, com períodos iniciais crescentes, denominados sem a interferência (no limpo e com interferência (no mato das plantas daninhas. No primeiro, a cultura de tomate permaneceu livre da interferência das plantas daninhas desde o transplantio das mudas até os seguintes períodos (dias do ciclo de desenvolvimento do tomate: 1-28, 1-35, 1-49, 1-63, 1-77, 1-91 (todo ciclo, colheita. Após estes períodos, as plantas daninhas cresceram livremente até o final do ciclo da cultura. No segundo grupo a cultura permaneceu com a interferência das plantas daninhas desde o preparo final do solo e transplantio das mudas até os mesmos períodos descritos para o primeiro grupo. Após estes períodos as plantas daninhas foram removidas manualmente até a colheita. De um total de 24, as espécies mais freqüentes e/ou de maior acúmulo de matéria seca foram: Bidens pilosa, Brachiaria plantaginea, Nicandra physaloides e Oxalis latifolia. As plantas daninhas causaram reduções na produção de tomate de até 75,5%, sendo que o PCI ocorreu no período do 33º ao 76º dia após a implantação da cultura.Weed control efficiency depends on the information about its interference on the crops. The interference of weed on tomato plants of a processing variety, transplanted directly into the field was evaluated. The field experiment was carried out, in a randomized block design with three replications, at Embrapa Hortaliças in Brasília, Brazil. Plots were laid out with increasing periods of time in the presence or absence of weeds. In the

  20. The Biogenesis of Nascent Circular RNAs

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    Yang Zhang

    2016-04-01

    Full Text Available Steady-state circular RNAs (circRNAs have been mapped to thousands of genomic loci in mammals. We studied circRNA processing using metabolic tagging of nascent RNAs with 4-thiouridine (4sU. Strikingly, the efficiency of circRNA processing from pre-mRNA is extremely low endogenously. Additional studies revealed that back-splicing outcomes correlate with fast RNA Polymerase II elongation rate and are tightly controlled by cis-elements in vivo. Additionally, prolonged 4sU labeling in cells shows that circRNAs are largely processed post-transcriptionally and that circRNAs are stable. Circular RNAs that are abundant at a steady-state level tend to accumulate. This is particularly true in cells, such as neurons, that have slow division rates. This study uncovers features of circRNA biogenesis by investigating the link between nascent circRNA processing and transcription.

  1. 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...... and cardiovascular diseases, and thus miRNAs have rapidly emerged as potential targets for therapeutics. LNAs (locked nucleic acids) comprise a class of bicyclic conformational analogues of RNA, which exhibit high binding affinity to complementary RNA molecules and high stability in blood and tissues in vivo. Recent...... reports on LNA-mediated miRNA silencing in rodents and primates support the potential of LNA-modified oligonucleotides in studying miRNA functions in vivo and in the future development of miRNA-based therapeutics....

  2. MicroRNAs in pancreas development.

    Science.gov (United States)

    Dumortier, O; Van Obberghen, E

    2012-10-01

    The development of the pancreas is a tightly regulated process involving extensive morphogenesis, proliferation and differentiation of the epithelium. The finely orchestrated control of gene expression plays a key role in this equilibrium by coordinating the expression of selected gene products at specific moments and in precise locations. MicroRNAs (miRNAs) are small non-coding RNAs that function in general as negative regulators of gene transcripts by interacting with the three prime untranslated regions (3'UTR) of target mRNAs. MiRNAs modulate the expression of numerous target genes that are involved in a variety of cellular systems. Hence the homeostatic control of miRNA biosynthesis and activity is important for the fine-tuning of many physiological processes such as cell differentiation, cell proliferation and organ development. In the present review, we will focus on the implication of these miRNAs on the development of the pancreas and more specifically on β-cells.

  3. MicroRNAs in the Hypothalamus

    DEFF Research Database (Denmark)

    Meister, Björn; Herzer, Silke; Silahtaroglu, Asli

    2013-01-01

    MicroRNAs (miRNAs) are short (∼22 nucleotides) non-coding ribonucleic acid (RNA) molecules that negatively regulate the expression of protein-coding genes. Posttranscriptional silencing of target genes by miRNA is initiated by binding to the 3'-untranslated regions of target mRNAs, resulting...... in specific cleavage and subsequent degradation of the mRNA or by translational repression resulting in specific inhibition of protein synthesis. An increasing amount of evidence shows that miRNAs control a large number of biological processes and there exists a direct link between miRNAs and disease. miRNA...... molecules are abundantly expressed in tissue-specific and regional patterns and have been suggested as potential biomarkers, disease modulators and drug targets. The central nervous system is a prominent site of miRNA expression. Within the brain, several miRNAs are expressed and/or enriched in the region...

  4. Promoter-targeted siRNAs induce gene silencing of simian immunodeficiency virus (SIV) infection in vitro.

    Science.gov (United States)

    Lim, Heidi G W; Suzuki, Kazuo; Cooper, David A; Kelleher, Anthony D

    2008-03-01

    RNA interference is a conserved process by which sequence-specific double-stranded RNA is converted into small interfering double-stranded RNAs (siRNAs) that can induce gene silencing via two pathways: post-transcriptional gene silencing and transcriptional gene silencing (TGS). We previously reported TGS of human immunodeficiency virus-1 (HIV-1) could be induced by siRNAs targeting regions within its 5'-long-terminal repeat (5'LTR) promoter region. Here we show that promoter-targeted siRNAs can also induce silencing of simian immunodeficiency virus (SIV) replication by similar mechanisms. Suppression of productive infection was achieved in two different cell lines: a CD4, CCR5, CXCR4 expressing HeLa cell line (MAGIC-5) and in a human lymphoid cell line (CEMx174). HpaII digestion demonstrated induction of methylation at a CpG site within the SIV promoter region following siRNA-induced suppression. Both 5-azacytidine (5-AzaC) and trichostatin A (TSA), inhibitors of DNA methyltransferases (DNMTs) and histone deacetylation, respectively, partially reversed the silencing effect. Furthermore, using chromatin immunoprecipitation (ChIP) assays we found enrichment in the region of the LTR of heterochromatin markers dimethylated histone 3 lysine 9 (H3K9) and trimethylated histone 3 lysine 27 (H3K27) in the siRNA silenced cultures. Together, these results strongly suggest certain siRNAs targeting the promoter region of SIV can effect viral silencing through the induction of epigenetic changes.

  5. Knockdown of cellular RNA helicase DDX3 by short hairpin RNAs suppresses HIV-1 viral replication without inducing apoptosis.

    Science.gov (United States)

    Ishaq, Musarat; Hu, Jiajie; Wu, Xiaoyun; Fu, Qiong; Yang, Yalin; Liu, Qingzhen; Guo, Deyin

    2008-07-01

    The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.

  6. Atypical RNAs in the coelacanth transcriptome.

    Science.gov (United States)

    Nitsche, Anne; Doose, Gero; Tafer, Hakim; Robinson, Mark; Saha, Nil Ratan; Gerdol, Marco; Canapa, Adriana; Hoffmann, Steve; Amemiya, Chris T; Stadler, Peter F

    2014-09-01

    Circular and apparently trans-spliced RNAs have recently been reported as abundant types of transcripts in mammalian transcriptome data. Both types of non-colinear RNAs are also abundant in RNA-seq of different tissue from both the African and the Indonesian coelacanth. We observe more than 8,000 lincRNAs with normal gene structure and several thousands of circularized and trans-spliced products, showing that such atypical RNAs form a substantial contribution to the transcriptome. Surprisingly, the majority of the circularizing and trans-connecting splice junctions are unique to atypical forms, that is, are not used in normal isoforms.

  7. Gene expression regulators--MicroRNAs

    Institute of Scientific and Technical Information of China (English)

    CHEN Fang; YIN Q. James

    2005-01-01

    A large class of non-coding RNAs found in small molecule RNAs are closely associated with the regulation of gene expression, which are called microRNA (miRNA). MiRNAs are coded in intergenic or intronic regions and can be formed into foldback hairpin RNAs. These transcripts are cleaved by Dicer, generating mature miRNAs that can silence their target genes in different modes of action. Now, research on small molecule RNAs has gotten breakthrough advance in biology. To discover miRNA genes and their target genes has become hot topics in RNA research. This review attempts to look back the history of miRNA discovery, to introduce the methods of screening miRNAs, to localize miRNA loci in genome, to seek miRNA target genes and the biological function, and to discuss the working mechanisms of miRNAs. Finally, we will discuss the potential important roles of miRNAs in modulating the genesis, development, growth, and differentiation of organisms. Thus, it can be predicted that a complete understanding of miRNA functions will bring us some new concepts, approaches and strategies for the study of living beings.

  8. MicroRNAs regulate osteogenesis and chondrogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Shiwu, E-mail: shiwudong@gmail.com [Laboratory of Biomechanics, Department of Anatomy, The Third Military Medical University, Chongqing (China); Yang, Bo; Guo, Hongfeng; Kang, Fei [Laboratory of Biomechanics, Department of Anatomy, The Third Military Medical University, Chongqing (China)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer To focus on the role of miRNAs in chondrogenesis and osteogenesis. Black-Right-Pointing-Pointer Involved in the regulation of miRNAs in osteoarthritis. Black-Right-Pointing-Pointer To speculate some therapeutic targets for bone diseases. -- Abstract: MicroRNAs (miRNAs) are a class of small molecules and non-coding single strand RNAs that regulate gene expression at the post-transcriptional level by binding to specific sequences within target genes. miRNAs have been recognized as important regulatory factors in organism development and disease expression. Some miRNAs regulate the proliferation and differentiation of osteoblasts, osteoclasts and chondrocytes, eventually influencing metabolism and bone formation. miRNAs are expected to provide potential gene therapy targets for the clinical treatment of metabolic bone diseases and bone injuries. Here, we review the recent research progress on the regulation of miRNAs in bone biology, with a particular focus on the miRNA-mediated control mechanisms of bone and cartilage formation.

  9. Progress and Current Challenges in Modeling Large RNAs.

    Science.gov (United States)

    Somarowthu, Srinivas

    2016-02-27

    Recent breakthroughs in next-generation sequencing technologies have led to the discovery of several classes of non-coding RNAs (ncRNAs). It is now apparent that RNA molecules are not only just carriers of genetic information but also key players in many cellular processes. While there has been a rapid increase in the number of ncRNA sequences deposited in various databases over the past decade, the biological functions of these ncRNAs are largely not well understood. Similar to proteins, RNA molecules carry out a function by forming specific three-dimensional structures. Understanding the function of a particular RNA therefore requires a detailed knowledge of its structure. However, determining experimental structures of RNA is extremely challenging. In fact, RNA-only structures represent just 1% of the total structures deposited in the PDB. Thus, computational methods that predict three-dimensional RNA structures are in high demand. Computational models can provide valuable insights into structure-function relationships in ncRNAs and can aid in the development of functional hypotheses and experimental designs. In recent years, a set of diverse RNA structure prediction tools have become available, which differ in computational time, input data and accuracy. This review discusses the recent progress and challenges in RNA structure prediction methods.

  10. TALEN-based knockout library for human microRNAs.

    Science.gov (United States)

    Kim, Young-Kook; Wee, Gabbine; Park, Joha; Kim, Jongkyu; Baek, Daehyun; Kim, Jin-Soo; Kim, V Narry

    2013-12-01

    Various technical tools have been developed to probe the functions of microRNAs (miRNAs), yet their application has been limited by low efficacy and specificity. To overcome the limitations, we used transcription activator-like effector nucleases (TALENs) to knock out human miRNA genes. We designed and produced a library of 540 pairs of TALENs for 274 miRNA loci, focusing on potentially important miRNAs. The knockout procedure takes only 2-4 weeks and can be applied to any cell type. As a case study, we generated knockout cells for two related miRNAs, miR-141 and miR-200c, which belong to the highly conserved miR-200 family. Interestingly, miR-141 and miR-200c, despite their overall similarity, suppress largely nonoverlapping groups of targets, thus suggesting that functional miRNA-target interaction requires strict seed-pairing. Our study illustrates the potency of TALEN technology and provides useful resources for miRNA research.

  11. Bioinformatics analysis suggests base modifications of tRNAs and miRNAs in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Jin Hailing

    2009-04-01

    Full Text Available Abstract Background Modifications of RNA bases have been found in some mRNAs and non-coding RNAs including rRNAs, tRNAs, and snRNAs, where modified bases are important for RNA function. Little is known about RNA base modifications in Arabidopsis thaliana. Results In the current work, we carried out a bioinformatics analysis of RNA base modifications in tRNAs and miRNAs using large numbers of cDNA sequences of small RNAs (sRNAs generated with the 454 technology and the massively parallel signature sequencing (MPSS method. We looked for sRNAs that map to the genome sequence with one-base mismatch (OMM, which indicate candidate modified nucleotides. We obtained 1,187 sites with possible RNA base modifications supported by both 454 and MPSS sequences. Seven hundred and three of these sites were within tRNA loci. Nucleotide substitutions were frequently located in the T arm (substitutions from A to U or G, upstream of the D arm (from G to C, U, or A, and downstream of the D arm (from G to U. The positions of major substitution sites corresponded with the following known RNA base modifications in tRNAs: N1-methyladenosine (m1A, N2-methylguanosine (m2G, and N2-N2-methylguanosine (m22G. Conclusion These results indicate that our bioinformatics method successfully detected modified nucleotides in tRNAs. Using this method, we also found 147 substitution sites in miRNA loci. As with tRNAs, substitutions from A to U or G and from G to C, U, or A were common, suggesting that base modifications might be similar in tRNAs and miRNAs. We suggest that miRNAs contain modified bases and such modifications might be important for miRNA maturation and/or function.

  12. Cloning and Identification of Novel MicroRNAs from Rat Hippocampus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    MicroRNAs (miRNAs) are small regulatory molecules post-transcriptionally suppressing mRNA activity. Many miRNAs in various organisms have been cloned but many unknown miRNAs remain to be identified. Here we describe the cloning of six new miRNAs from rat hippocampus. Among them, four were not found in the rat miRBase, but were identical to their human and/or mouse homolog, therefore they were designated as rno-miR-92b, rno-miR-146b, rno-let-7g, and rno-miR-551b. The other two were derived from the other arms of the known miRNA precursors of rno-miR-330 and rno-miR-384, and were not found in miRBase of all organisms. They were designated as rno-miR-330* and rno-miR-384*. The expression of these miRNAs was confirmed by RNA-tailing and primer-extension real-time reverse transcriptionpolymerase chain reaction. These six miRNAs were expressed at significantly higher levels in the hippocampus than in other tissues, including cerebral cortex, heart, liver, lung and kidney. miR-384* was 10 times more abundant than miR-384 in rat hippocampus, but little difference was found between miR-330* and miR-330 expression in the same tissue.

  13. 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.

  14. 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...

  15. Evaluation of the potential anti-viral activity of microRNAs in rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Bela-Ong, Dennis; Schyth, Brian Dall; Lorenzen, Niels

    2013-01-01

    have previously observed and validated that the fishspecific miRNAs, miR-462 and miR-731, were among the most highly expressed miRNAs in rainbow trout liver following Viral hemorrhagic septicemia virus (VHSV) infection. These miRNAs were also upregulated in the liver and muscle (vaccination site......) of fish vaccinated with a DNA vaccine encoding the glycoprotein gene of VHSV. Recent studies further suggest that the expression of these miRNAs is induced by interferons. In order to analyze if miRNA-462 and miRNA-731 have any antiviral effects, we designed inhibitory synthetic oligonucleotides called...... antagomiRs or anti-miRNAs. These antagomiRs were injected intraperitoneally into rainbow trout fingerlings followed by exposure of the fish to VHSV. Development of disease and levels of infection will be analysed and compared to data from fish treated with control miRNAs....

  16. MicroRNAs, macrocontrol : Regulation of miRNA processing

    NARCIS (Netherlands)

    Slezak-Prochazka, Izabella; Durmus, Selvi; Kroesen, Bart-Jan; van den Berg, Anke

    2010-01-01

    MicroRNAs (miRNAs) are a set of small, non-protein-coding RNAs that regulate gene expression at the post-transcriptional level. Maturation of miRNAs comprises several regulated steps resulting in similar to 22-nucleotide single-stranded mature miRNAs. Regulation of miRNA expression can occur both at

  17. Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in Escherichia coli.

    Science.gov (United States)

    Benhalevy, Daniel; Bochkareva, Elena S; Biran, Ido; Bibi, Eitan

    2015-01-01

    Are integral membrane protein-encoding mRNAs (MPRs) different from other mRNAs such as those encoding cytosolic mRNAs (CPRs)? This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner. MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices. To investigate this hypothesis, we designed DNA sequences encoding model untranslatable transcripts that mimic MPRs or CPRs. By utilizing in vitro-synthesized biotinylated RNAs mixed with Escherichia coli extracts, we identified a highly specific interaction that takes place between transcripts that mimic MPRs and the cold shock proteins CspE and CspC, which are normally expressed under physiological conditions. Co-purification studies with E. coli expressing 6His-tagged CspE or CspC confirmed that the specific interaction occurs in vivo not only with the model uracil-rich untranslatable transcripts but also with endogenous MPRs. Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

  18. Small RNAs in the genus Clostridium.

    Science.gov (United States)

    Chen, Yili; Indurthi, Dinesh C; Jones, Shawn W; Papoutsakis, Eleftherios T

    2011-01-25

    The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny.

  19. Extracellular RNAs: development as biomarkers of human disease

    Directory of Open Access Journals (Sweden)

    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. 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.

  1. Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by Polyethylenimine-Functionalized Graphene Oxide

    Science.gov (United States)

    Huang, Yuan-Pin; Hung, Chao-Ming; Hsu, Yi-Chiang; Zhong, Cai-Yan; Wang, Wan-Rou; Chang, Chi-Chang; Lee, Mon-Juan

    2016-05-01

    The carbon-based nanomaterial graphene can be chemically modified to associate with various molecules such as chemicals and biomolecules and developed as novel carriers for drug and gene delivery. In this study, a nonviral gene transfection reagent was produced by functionalizing graphene oxide (GO) with a polycationic polymer, polyethylenimine (PEI), to increase the biocompatibility of GO and to transfect small interfering RNA (siRNA) against C-X-C chemokine receptor type 4 (CXCR4), a biomarker associated with cancer metastasis, into invasive breast cancer cells. PEI-functionalized GO (PEI-GO) was a homogeneous aqueous solution that remained in suspension during storage at 4 °C for at least 6 months. The particle size of PEI-GO was 172 ± 4.58 and 188 ± 5.00 nm at 4 and 25 °C, respectively, and increased slightly to 262 ± 17.6 nm at 37 °C, but remained unaltered with time. Binding affinity of PEI-GO toward siRNA was assessed by electrophoretic mobility shift assay (EMSA), in which PEI-GO and siRNA were completely associated at a PEI-GO:siRNA weight ratio of 2:1 and above. The invasive breast cancer cell line, MDA-MB-231, was transfected with PEI-GO in complex with siRNAs against CXCR4 (siCXCR4). Suppression of the mRNA and protein expression of CXCR4 by the PEI-GO/siCXCR4 complex was confirmed by real-time PCR and western blot analysis. In addition, the metastatic potential of MDA-MB-231 cells was attenuated by the PEI-GO/siCXCR4 complex as demonstrated in wound healing assay. Our results suggest that PEI-GO is effective in the delivery of siRNA and may contribute to targeted gene therapy to suppress cancer metastasis.

  2. Cloned defective interfering influenza virus protects ferrets from pandemic 2009 influenza A virus and allows protective immunity to be established.

    Directory of Open Access Journals (Sweden)

    Nigel J Dimmock

    Full Text Available Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In addition there are periodic outbreaks in domestic poultry, horses, pigs, dogs, and cats. Infections of domestic birds can be fatal for the birds and their human contacts. Control in man operates through vaccines and antivirals, but both have their limitations. In the search for an alternative treatment we have focussed on defective interfering (DI influenza A virus. Such a DI virus is superficially indistinguishable from a normal virus but has a large deletion in one of the eight RNAs that make up the viral genome. Antiviral activity resides in the deleted RNA. We have cloned one such highly active DI RNA derived from segment 1 (244 DI virus and shown earlier that intranasal administration protects mice from lethal disease caused by a number of different influenza A viruses. A more cogent model of human influenza is the ferret. Here we found that intranasal treatment with a single dose of 2 or 0.2 µg 244 RNA delivered as A/PR/8/34 virus particles protected ferrets from disease caused by pandemic virus A/California/04/09 (A/Cal; H1N1. Specifically, 244 DI virus significantly reduced fever, weight loss, respiratory symptoms, and infectious load. 244 DI RNA, the active principle, was amplified in nasal washes following infection with A/Cal, consistent with its amelioration of clinical disease. Animals that were treated with 244 DI RNA cleared infectious and DI viruses without delay. Despite the attenuation of infection and disease by DI virus, ferrets formed high levels of A/Cal-specific serum haemagglutination-inhibiting antibodies and were solidly immune to rechallenge with A/Cal. Together with earlier data from mouse studies, we conclude that 244 DI virus is a highly effective antiviral with activity potentially against all influenza A subtypes.

  3. Efficient protection and transfection of small interfering RNA by cationic shell-crosslinked knedel-like nanoparticles.

    Science.gov (United States)

    Shen, Yuefei; Fang, Huafeng; Zhang, Ke; Shrestha, Ritu; Wooley, Karen L; Taylor, John-Stephen A

    2013-04-01

    Despite the great potential of small interfering RNA (siRNA) as a therapeutic agent, progress in this area has been hampered by a lack of efficient biocompatible transfection agents. Recently, cationic shell-crosslinked knedel-like nanoparticles (cSCKs) were found to possess lower cytotoxicity and better transfection ability for phosphorothioate ODNs and plasmid DNA than the commonly used cationic lipid-based agent Lipofectamine. To determine the usefulness of cSCKs for siRNA transfection, a small library of cSCKs with varying percentage of primary and tertiary amines was assessed for its ability to bind to siRNA, inhibit siRNA degradation in human serum, and to transfect HeLa and mouse macrophage cell lines. The silencing efficiency in HeLa cells was greatest with the cSCK with 100% primary amines (pa100) as determined by their viability following transfection with cytotoxic and non-cytotoxic siRNAs. cSCK-pa100 showed greater silencing efficiency than Lipofectamine 2000 in the HeLa cells, as well in 293T and human bronchial epithelial (HEK) cells, but was comparable in human bronchial epithelial (BEAS-2B) cells and human mammary epithelial (MCF10a) cells. cSCK-pa100 also showed greater silencing of iNOS expression than Lipofectamine 2000 in a mouse macrophage cell line, and provided greater protection from serum degradation, demonstrating its potential usefulness as an siRNA transfection agent. The siRNA silencing of iNOS at lower concentrations of siRNA could be enhanced by complexation with the fusogenic GALA peptide, which was shown to enhance endosomal escape following uptake.

  4. Multiple RNAs from the mouse carboxypeptidase M locus: functional RNAs or transcription noise?

    Directory of Open Access Journals (Sweden)

    Castilho Beatriz A

    2009-02-01

    functional elements on the genome. Furthermore, the transcription of other functional RNAs besides the protein coding RNA from a specific genomic locus imposes extra care when designing and interpreting experiments involving genetic manipulations or expression detection and quantification.

  5. Small RNAs derived from lncRNA RNase MRP have gene-silencing activity relevant to human cartilage–hair hypoplasia

    OpenAIRE

    2013-01-01

    Post-transcriptional processing of some long non-coding RNAs (lncRNAs) reveals that they are a source of miRNAs. We show that the 268-nt non-coding RNA component of mitochondrial RNA processing endoribonuclease, (RNase MRP), is the source of at least two short (∼20 nt) RNAs designated RMRP-S1 and RMRP-S2, which function as miRNAs. Point mutations in RNase MRP cause human cartilage–hair hypoplasia (CHH), and several disease-causing mutations map to RMRP-S1 and -S2. SHAPE chemical probing ident...

  6. Promoting workplace stair climbing: sometimes, not interfering is the best.

    Science.gov (United States)

    Åvitsland, Andreas; Solbraa, Ane Kristiansen; Riiser, Amund

    2017-01-01

    Stair climbing is a vigorous activity and can lead to several health benefits. Studies seeking to increase stair climbing in various public locations have shown positive effects, while results from similar studies conducted in the workplace are inconclusive. This study examined stair climbing in the workplace, and monitored effects from a single- and a combined intervention. Interventions were inspired by nudging, the libertarian method of influencing behavior. By quasi-experimental design, stair- and elevator traffic in two office buildings was monitored preceding-, during- and following interventions with stair leading footprints alone, and combined with stair-riser banners. Chi square tests were applied to determine differences between baseline and the subsequent periods. Web-based questionnaires were distributed after follow-up period. Elevators and stairs were used 45 237 times, of which 89.6% was stair use. Intervention site stair climbing at baseline (79.0%) was significantly reduced with footprints (-5.1%, p stair-riser banners (-5.7%, p stair climbing at the control site (94.2%) remained stable (p > 0.027). Stair climbing was significantly reduced during the intervention periods. Use of stair leading footprints alone, or combined with stair-riser banners in an attempt to influence stair climbing may be ineffective, or cause a negative reaction, when applied in a workplace with a pre-existing high amount of stair climbing.

  7. MicroRNAs in cardiac arrhythmia

    DEFF Research Database (Denmark)

    Hedley, Paula L; Carlsen, Anting L; Christiansen, Kasper M

    2014-01-01

    LQTS-causing mutations have been identified in 13 genes worldwide. Despite this, the genetic cause of 30-50% of LQTS is presently unknown. MicroRNAs (miRNAs) are small (∼ 22 nucleotides) noncoding RNAs which post-transcriptionally regulate gene expression by binding complementary sequences within...... messenger RNAs (mRNAs). The human genome encodes over 1800 miRNAs, which target about 60% of human genes. Consequently, miRNAs are likely to regulate many complex processes in the body, indeed aberrant expression of various miRNA species has been implicated in numerous disease states, including...... cardiovascular diseases. MiR-1 and MiR-133A are the most abundant miRNAs in the heart and have both been reported to regulate cardiac ion channels. We hypothesized that, as a consequence of their role in regulating cardiac ion channels, genetic variation in the genes which encode MiR-1 and MiR-133A might explain...

  8. Exploiting tRNAs to Boost Virulence

    Directory of Open Access Journals (Sweden)

    Suki Albers

    2016-01-01

    Full Text Available Transfer RNAs (tRNAs are powerful small RNA entities that are used to translate nucleotide language of genes into the amino acid language of proteins. Their near-uniform length and tertiary structure as well as their high nucleotide similarity and post-transcriptional modifications have made it difficult to characterize individual species quantitatively. However, due to the central role of the tRNA pool in protein biosynthesis as well as newly emerging roles played by tRNAs, their quantitative assessment yields important information, particularly relevant for virus research. Viruses which depend on the host protein expression machinery have evolved various strategies to optimize tRNA usage—either by adapting to the host codon usage or encoding their own tRNAs. Additionally, several viruses bear tRNA-like elements (TLE in the 5′- and 3′-UTR of their mRNAs. There are different hypotheses concerning the manner in which such structures boost viral protein expression. Furthermore, retroviruses use special tRNAs for packaging and initiating reverse transcription of their genetic material. Since there is a strong specificity of different viruses towards certain tRNAs, different strategies for recruitment are employed. Interestingly, modifications on tRNAs strongly impact their functionality in viruses. Here, we review those intersection points between virus and tRNA research and describe methods for assessing the tRNA pool in terms of concentration, aminoacylation and modification.

  9. Exploiting tRNAs to Boost Virulence

    Science.gov (United States)

    Albers, Suki; Czech, Andreas

    2016-01-01

    Transfer RNAs (tRNAs) are powerful small RNA entities that are used to translate nucleotide language of genes into the amino acid language of proteins. Their near-uniform length and tertiary structure as well as their high nucleotide similarity and post-transcriptional modifications have made it difficult to characterize individual species quantitatively. However, due to the central role of the tRNA pool in protein biosynthesis as well as newly emerging roles played by tRNAs, their quantitative assessment yields important information, particularly relevant for virus research. Viruses which depend on the host protein expression machinery have evolved various strategies to optimize tRNA usage—either by adapting to the host codon usage or encoding their own tRNAs. Additionally, several viruses bear tRNA-like elements (TLE) in the 5′- and 3′-UTR of their mRNAs. There are different hypotheses concerning the manner in which such structures boost viral protein expression. Furthermore, retroviruses use special tRNAs for packaging and initiating reverse transcription of their genetic material. Since there is a strong specificity of different viruses towards certain tRNAs, different strategies for recruitment are employed. Interestingly, modifications on tRNAs strongly impact their functionality in viruses. Here, we review those intersection points between virus and tRNA research and describe methods for assessing the tRNA pool in terms of concentration, aminoacylation and modification. PMID:26797637

  10. 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

    among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets...... of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites...

  11. Conversations between kingdoms: small RNAs.

    Science.gov (United States)

    Weiberg, Arne; Bellinger, Marschal; Jin, Hailing

    2015-04-01

    Humans, animals, and plants are constantly under attack from pathogens and pests, resulting in severe consequences on global human health and crop production. Small RNA (sRNA)-mediated RNA interference (RNAi) is a conserved regulatory mechanism that is involved in almost all eukaryotic cellular processes, including host immunity and pathogen virulence. Recent evidence supports the significant contribution of sRNAs and RNAi to the communication between hosts and some eukaryotic pathogens, pests, parasites, or symbiotic microorganisms. Mobile silencing signals—most likely sRNAs—are capable of translocating from the host to its interacting organism, and vice versa. In this review, we will provide an overview of sRNA communications between different kingdoms, with a primary focus on the advances in plant-pathogen interaction systems.

  12. Signal-to-noise analysis for detection sensitivity of small absorbing heterogeneity in turbid media with single-source and dual-interfering-source.

    Science.gov (United States)

    Chen, Y; Mu, C; Intes, X; Chance, B

    2001-08-13

    Previous studies have suggested that the phased-array detection can achieve high sensitivity in detecting and localizing inhomogeneities embedded in turbid media by illuminating with dual interfering sources. In this paper, we analyze the sensitivity of single-source and dual-interfering-source (phased array) systems with signal-to-noise ratio criteria. Analytical solutions are presented to investigate the sensitivity of detection using different degrees of absorption perturbation by varying the size and contrast of the object under similar configurations for single- and dual-source systems. The results suggest that dual-source configuration can provide higher detection sensitivity. The relation between the amplitude and phase signals for both systems is also analyzed using a vector model. The results can be helpful for optimizing the experimental design by combining the advantages of both single- and dual-source systems in object detection and localization.

  13. Transcriptional profiling of differentially expressed long non-coding RNAs in breast cancer

    Directory of Open Access Journals (Sweden)

    Lizhen Wang

    2015-12-01

    This study was specifically designed to identify the signatures relevant lncRNAs in breast cancer and characterize lncRNAs that modulate the phenotype. Here we provide detailed methods and analysis of microarray data, which is deposited in the Gene Expression Omnibus (GEO with the accession number GSE64790. The basic analysis as contained in the manuscript published in Oncotarget with the PMID 26078338. These data can be used to further elucidate the mechanisms of breast cancer.

  14. Dysregulated microRNAs in neurodegenerative disorders.

    Science.gov (United States)

    Lau, Pierre; de Strooper, Bart

    2010-09-01

    The complexity of the nervous system arises in part, from the large diversity of neural cell types that support the architecture of neuronal circuits. Recent studies have highlighted microRNAs as important players in regulating gene expression at the post-transcriptional level and therefore the phenotype of neural cells. A link between microRNAs and neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease is becoming increasingly evident. Here, we discuss microRNAs in neurodegeneration, from the fruit fly and mouse utilized as experimental models to dysregulated microRNAs in human neurodegenerative disorders. We propose that studying microRNAs and their mRNA targets in the context of neurodegeneration will significantly contribute to the identification of proteins important for neuronal function and might reveal underlying molecular networks that drive these diseases.

  15. Targeting microRNAs in obesity.

    Science.gov (United States)

    Xie, Huangming; Sun, Lei; Lodish, Harvey F

    2009-10-01

    Obesity is a serious health problem worldwide associated with an increased risk of life-threatening diseases such as type 2 diabetes, atherosclerosis, and certain types of cancer. Fundamental for the development of novel therapeutics for obesity and its associated metabolic syndromes is an understanding of the regulation of fat cell development. Recent computational and experimental studies have shown that microRNAs (miRNAs) play a role in metabolic tissue development, lipid metabolism and glucose homeostasis. In addition, many miRNAs are dysregulated in metabolic tissues from obese animals and humans, which potentially contributes to the pathogenesis of obesity-associated complications. In this review we summarize the current state of understanding of the roles of miRNAs in metabolic tissues under normal development and obese conditions, and discuss the potential use of miRNAs as therapeutic targets.

  16. Hidden layers of human small RNAs

    DEFF Research Database (Denmark)

    Kawaji, Hideya; Nakamura, Mari; Takahashi, Yukari;

    2008-01-01

    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...... 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...

  17. Optimal design and validation of antiviral siRNA for targeting hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Jie FU; Zhong-ming TANG; Xin GAO; Fan ZHAO; Hui ZHONG; Mao-rong WEN; Xiao SUN; Hai-feng SONG; Xiao-hong QIAN

    2008-01-01

    Aim: Optimal design of antiviral short-interfering RNA (siRNA) targeting highly divergent hepatitis B virus (HBV) was validated by quantitative structure-activity relationship (QSAR) analysis. Methods: The potency of 23 synthetic siRNAs targeting 23 sites throughout HBV pregenomic RNA were evaluated at 10 nmol/L by determining the inhibition on the expression of S/P/pregenomic mRNA and hepatitis B surface antigen (HBsAg) quantitatively in HepG2.2.15 cells. Genotype homology within HBV genomes was identified through plentiful computational analysis and the multiple linear regression analysis was made to validate the relationship between the functional siRNAs and primary characteristics. Based on the preliminary results, relationships between different determined endpoints [S/P mRNA, HBsAg, C/P mRNA, hepatitis B e antigen (HBeAg) and viral DNA load] and siRNA efficacy evaluation were investigated. Results: Genotype homology, open reading frame (ORF) S/E X and C had tight correlation with the ability of siRNAs on inhibiting the expression of S/P/Pregenomic mRNA and HBsAg (P<0.01), of which, ORF C was negatively correlated with the siRNA potency (P<0.05). Further study showed that siRNA potency evaluation was influenced by different determined endpoints. P-target siRNAs showed significant inhibition on the S mRNA and HBsAg expression. S-target siRNAs inhibited the expression of S mRNA and HBsAg strongly. X-target siRNAs played active roles in inhibiting all 5 determined endpoints. C-target siRNAs blocked the expression of C mRNA, HBeAg and viral DNA load significantly. Conclusion: The antiviral potency of siRNA was relevant to its primary characteristics and determined endpoints were important for siRNA efficacy evaluation for complex genome with overlapping ORF, which was helpful for siRNA optimal design.

  18. Computational models with thermodynamic and composition features improve siRNA design

    Directory of Open Access Journals (Sweden)

    Ogurtsov Aleksey Y

    2006-02-01

    Full Text Available Abstract Background Small interfering RNAs (siRNAs have become an important tool in cell and molecular biology. Reliable design of siRNA molecules is essential for the needs of large functional genomics projects. Results To improve the design of efficient siRNA molecules, we performed a comparative, thermodynamic and correlation analysis on a heterogeneous set of 653 siRNAs collected from the literature. We used this training set to select siRNA features and optimize computational models. We identified 18 parameters that correlate significantly with silencing efficiency. Some of these parameters characterize only the siRNA sequence, while others involve the whole mRNA. Most importantly, we derived an siRNA position-dependent consensus, and optimized the free-energy difference of the 5' and 3' terminal dinucleotides of the siRNA antisense strand. The position-dependent consensus is based on correlation and t-test analyses of the training set, and accounts for both significantly preferred and avoided nucleotides in all sequence positions. On the training set, the two parameters' correlation with silencing efficiency was 0.5 and 0.36, respectively. Among other features, a dinucleotide content index and the frequency of potential targets for siRNA in the mRNA added predictive power to our model (R = 0.55. We showed that our model is effective for predicting the efficiency of siRNAs at different concentrations. We optimized a neural network model on our training set using three parameters characterizing the siRNA sequence, and predicted efficiencies for the test siRNA dataset recently published by Novartis. On this validation set, the correlation coefficient between predicted and observed efficiency was 0.75. Using the same model, we performed a transcriptome-wide analysis of optimal siRNA targets for 22,600 human mRNAs. Conclusion We demonstrated that the properties of the siRNAs themselves are essential for efficient RNA interference. The 5' ends

  19. Genome-wide identification of non-coding RNAs interacted with microRNAs in soybean

    Directory of Open Access Journals (Sweden)

    Chuyu eYe

    2014-12-01

    Full Text Available A wide range of RNA species interacting with microRNAs (miRNAs form a complex gene regulation network and play vital roles in diverse biological processes. In this study, we performed a genome-wide identification of endogenous target mimics (eTMs for miRNAs and phased-siRNA-producing loci (PHAS in soybean with a focus on those involved in lipid metabolism. The results showed that a large number of eTMs and PHAS genes could be found in soybean. Additionally, we found that lipid metabolism related genes were potentially regulated by 28 miRNAs, and nine of them were potentially further regulated by a number of eTMs with expression evidence. Thirty-three miRNAs were found to trigger production of phasiRNAs from 49 PHAS genes, which were able to target lipid metabolism related genes. Degradome data supported miRNA- and/or phasiRNA-mediated cleavage of genes involved in lipid metabolism. Most eTMs for miRNAs involved in lipid metabolism and phasiRNAs targeting lipid metabolism related genes showed a tissue-specific expression pattern. Our bioinformatical evidences suggested that lipid metabolism in soybean is potentially regulated by a complex non-coding network, including miRNAs, eTMs and phasiRNAs, and the results extended our knowledge on functions of non-coding RNAs.

  20. The long non coding RNAs (lncRNAs : a new (player in the dark matter.

    Directory of Open Access Journals (Sweden)

    Thomas eDerrien

    2012-01-01

    Full Text Available The transcriptome of a cell is represented by a myriad of different RNA molecules with and without protein-coding capacities. In recent years, advances in sequencing technologies have allowed researchers to more fully appreciate the complexity of whole transcriptomes, showing that the vast majority of the genome is transcribed, producing a diverse population of non-protein coding RNAs (ncRNAs. Thus, the biological significance of non-coding RNAs (ncRNAs have been largely underestimated. Amongst these multiple classes of ncRNAs, the long non-coding RNAs (lncRNAs are apparently the most numerous and functionally diverse. A small but growing number of lncRNAs have been experimentally studied, and a view is emerging that these are key regulators of epigenetic gene regulation in mammalian cells. LncRNAs have already been implicated in human diseases such as cancer and neurodegeneration, highlighting the importance of this emergent field. In this article, we review the catalogues of annotated lncRNAs and the latest advances in our understanding of long non-coding RNAs.

  1. Interferência de plantas daninhas na cultura do sorgo cultivado em safrinha

    OpenAIRE

    Cabral,Paulo Henrique Ramos; Jakelaitis,Adriano; Cardoso,Isabella Sichierski; Araújo,Vinícius Tavares de; Pedrini,Evandro César Fernandes

    2013-01-01

    O sorgo apresenta grande importância na economia mundial, como fonte de energia para a alimentação humana e animal. No sudoeste Goiano, apesar de esta cultura apresentar alto potencial produtivo, a produtividade é baixa, fato atribuído, em grande parte, à interferência do manejo inadequado de plantas daninhas. Objetivou-se, nesta pesquisa, determinar os períodos de interferência de plantas daninhas sobre a cultura do sorgo cultivado em safrinha, no município de Rio Verde (GO). Os tratamentos ...

  2. Sum Capacity of Gaussian Interfering Multiple Access Channels in the Low Interference Regime

    CERN Document Server

    Jose, Jubin; Vishwanath, Sriram

    2011-01-01

    This paper establishes the sum capacity of Gaussian interfering multiple access channels (IF-MACs) in a low interference regime. IF-MACs studied consist of two MACs, with arbitrary number of users, interfering with each other. In the low interference regime, treating interference as noise along with successive decoding is shown to be sum rate optimal. The regime characterized in this paper solely depends on the minimum direct to indirect channel gain ratios, net interference to noise ratio and net signal to noise ratio associated with both MACs.

  3. Highly potent and specific siRNAs against E6 or E7 genes of HPV16- or HPV18-infected cervical cancers.

    Science.gov (United States)

    Chang, J T-C; Kuo, T-F; Chen, Y-J; Chiu, C-C; Lu, Y-C; Li, H-F; Shen, C-R; Cheng, A-J

    2010-12-01

    Infection with high-risk types (type 16 or type 18) of human papillomaviruses (HPVs) increases a patient's risk of cervical cancer. Given the importance of the cervix and the severe side effects resulting from traditional cancer therapies, this study aimed to achieve targeted inhibition of viral oncogenes in tumor cells using small interfering RNAs (siRNA). To accomplish this, we developed nine siRNAs against either the E6 or E7 genes of HPV-16 or HPV-18 in several combinations, yielding siRNAs targeting 16E6, 16E7, 18E6 and 18E7. We measured the effectiveness of the siRNAs by examining E6 or E7 mRNA expression after transfection of the siRNAs into HPV-positive CaSki (HPV-16) or HeLa (HPV-18) cell lines. We found that the HPV-siRNAs significantly reduced cell growth and colony formation in both cell lines. Flow cytometry analysis revealed a significant increase in apoptosis. The siRNAs had no effect on cell growth, colony formation or apoptosis in HPV-negative C33A cells, demonstrating a lack of off-target effects. In addition, an in vivo xenograft study showed that intra-tumoral injection of the siRNAs reduced tumor growth in BALB/c nude mice. In conclusion, we have developed highly specific and potent HPV-siRNAs that successfully suppress tumor growth and induce apoptosis in HPV-positive cervical cancer cells. siRNA treatment has potential for further development as an adjuvant therapy for cervical cancer.

  4. Spatially Controlled Delivery of siRNAs to Stem Cells in Implants Generated by Multi-Component Additive Manufacturing

    DEFF Research Database (Denmark)

    Andersen, Morten Østergaard; Le, Dang Quang Svend; Chen, Muwan

    2013-01-01

    Additive manufacturing is a promising technique in tissue engineering, as it enables truly individualized implants to be made to fit a particular defect. As previously shown, a feasible strategy to produce complex multicellular tissues is to deposit different small interfering RNA (siRNA) in porous...... implants that are subsequently sutured together. In this study, an additive manufacturing strategy to deposit carbohydrate hydrogels containing different siRNAs is applied into an implant, in a spatially controlled manner. When the obtained structures are seeded with mesenchymal stem (stromal) cells......, the selected siRNAs are delivered to the cells and induces specific and localized gene silencing. Here, it is demonstrated how to replicate part of a patient's spinal cord from a computed tomography scan, using an additive manufacturing technique to produce an implant with compartmentalized si...

  5. Therapeutic implications of microRNAs in human cancer

    OpenAIRE

    Michael Rossbach

    2011-01-01

    MicroRNAs (miRNAs) are a class of highly evolutionarily conserved non-coding RNAs (ncRNAs) that modulate gene expression. Several studies have shown that the expression of miRNAs is deregulated in human malignancies. For ncRNAs and miRNAs, such gene-profiling studies in tumorigenic tissues have identified significant signatures that are of both diagnostic and prognostic value. Addressing the functions of ncRNAs not only give insights into the molecular mechanisms that underlie complex genetic...

  6. MicroRNAs in neurodegenerative disorders.

    Science.gov (United States)

    Junn, Eunsung; Mouradian, M Maral

    2010-05-01

    MicroRNAs (miRNAs) are endogenous, small, noncoding RNAs regulating eukaryotic gene expression at the post-transcriptional level. During the last decade, considerable advances have been made in our understanding the biogenesis of miRNAs, the molecular mechanisms by which they regulate gene expression and their functional role in various physiological situations. miRNAs are abundant in the brain where they have crucial roles in development and synaptic plasticity. Accumulating evidence from postmortem brain analyses and animal model studies has begun to suggest that miRNA dysfunction contributes to neurodegenerative disorders. Here, we discuss several examples of investigations demonstrating the role of miRNAs in neurodegenerative disorders. As the expression of disease-causing genes is regulated by certain miRNA(s), changes in these miRNAs could lead to the accumulation of disease-causing proteins, and subsequently to neuronal dysfunction and death. Detailed understanding of these mechanisms can provide potential new therapeutic approaches to slow down or halt the progression of neurodegenerative diseases.

  7. Non-coding RNAs in cardiovascular ageing.

    Science.gov (United States)

    Gupta, Shashi Kumar; Piccoli, Maria Teresa; Thum, Thomas

    2014-09-01

    The increasing burden of ageing populations and their healthcare expenditure is a major challenge worldwide. Ageing is a complex disorder and can be defined as progressive decline in function with time leading to increased incidence of various cardiovascular, neurological and immunological diseases. The human genome comprises of many protein coding and even more non-coding RNA genes. MicroRNAs, a class of non-coding RNA, regulate the expression of multiple messenger RNAs post-transcriptionally and are reported to be involved in crucial aspects of cell biology encompassing ageing. Recently, several studies have reported the regulation of microRNAs with ageing and microRNAs like miR-34 have emerged as critical regulator of ageing extending from Caenorhabditis elegans to mammals. Here, we summarize the reported role of microRNAs as well as long noncoding RNAs (lncRNAs) in the process of ageing with a special emphasis on cardiovascular ageing. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Non-Coding RNAs in Hodgkin Lymphoma

    Directory of Open Access Journals (Sweden)

    Anna Cordeiro

    2017-05-01

    Full Text Available MicroRNAs (miRNAs, small non-coding RNAs that regulate gene expression by binding to the 3’-UTR of their target genes, can act as oncogenes or tumor suppressors. Recently, other types of non-coding RNAs—piwiRNAs and long non-coding RNAs—have also been identified. Hodgkin lymphoma (HL is a B cell origin disease characterized by the presence of only 1% of tumor cells, known as Hodgkin and Reed-Stenberg (HRS cells, which interact with the microenvironment to evade apoptosis. Several studies have reported specific miRNA signatures that can differentiate HL lymph nodes from reactive lymph nodes, identify histologic groups within classical HL, and distinguish HRS cells from germinal center B cells. Moreover, some signatures are associated with survival or response to chemotherapy. Most of the miRNAs in the signatures regulate genes related to apoptosis, cell cycle arrest, or signaling pathways. Here we review findings on miRNAs in HL, as well as on other non-coding RNAs.

  9. Small RNAs as potential platelet therapeutics.

    Science.gov (United States)

    Edelstein, Leonard C; Bray, Paul F

    2012-01-01

    MicroRNAs (miRNAs) are 21-23 nucleotide RNAs that regulate more than 60% of mammalian protein coding genes. miRNAs play critical roles in hematopoiesis and megakaryocyte function and development. Platelets, in addition to possessing functional miRNA processing machinery, have miRNA levels that have been correlated with platelet reactivity, and these miRNAs have been shown to target mRNAs that encode proteins that alter platelet function. There are potential uses of platelet miRNA as biomarkers and therapeutic agents. Due to the ability of platelets to release miRNA-containing microparticles at sites of activation, including angiogenic regions, tumors, and atherosclerotic plaques, there is the possibility of engineering platelets to deliver miRNA-based therapies to these sites. Cellpreferential expression of miRNAs could be exploited to restrict transgene expression in hematopoietic stem cell gene therapy to the desired lineage, including megakaryocytes and platelets. Finally, manipulation of gene expression in stored platelets may allow more effective platelet storage. Although much work remains to be done, there is great potential in miRNA-based platelet therapies.

  10. Long noncoding RNAs and Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Luo Q

    2016-06-01

    Full Text Available Qiong Luo,1,2 Yinghui Chen1,2 1Department of Neurology, Jinshan Hospital, 2Department of Neurology, Shanghai Medical College, Fudan University, Shanghai, People’s Republic of China Abstract: Long noncoding RNAs (lncRNAs are typically defined as transcripts longer than 200 nucleotides. lncRNAs can regulate gene expression at epigenetic, transcriptional, and posttranscriptional levels. Recent studies have shown that lncRNAs are involved in many neurological diseases such as epilepsy, neurodegenerative conditions, and genetic disorders. Alzheimer’s disease is a neurodegenerative disease, which accounts for >80% of dementia in elderly subjects. In this review, we will highlight recent studies investigating the role of lncRNAs in Alzheimer’s disease and focus on some specific lncRNAs that may underlie Alzheimer’s disease pathophysiology and therefore could be potential therapeutic targets. Keywords: lncRNA, Alzheimer’s disease, ncRNAs, amyloid β peptide, BACE1, BC200, BACE1-AS

  11. microRNAs and ceRNAs: RNA networks in pathogenesis of cancer

    Institute of Scientific and Technical Information of China (English)

    Xiangqian Su; Jiadi Xing; Zaozao Wang; Lei Chen; Ming Cui; Beihai Jiang

    2013-01-01

    microRNAs (miRNAs) are a class of endogenous,single-stranded non-coding RNAs of 20-23 nucleotides in length,functioning as negative regulators of gene expression at the post-transcriptional level.The dysregulation of miRNAs has been demonstrated to play critical roles in tumorigenesis,either through inhibiting tumor suppressor genes or activating oncogenes inappropriately.Besides their promising clinical applications in cancer diagnosis and treatment,recent studies have uncovered that miRNAs could act as a regulatory language,through which messenger RNAs,transcribed pseudogenes,and long noncoding RNAs crosstalk with each other and form a novel regulatory network.RNA transcripts involved in this network have been termed as competing endogenous RNAs (ceRNAs),since they influence each other's level by competing for the same pool of miRNAs through miRNA response elements (MREs) on their target transcripts.The discovery of miRNA-ceRNA network not only provides the possibility of an additional level of post-transcriptional regulation,but also dictates a reassessment of the existing regulatory pathways involved in cancer initiation and progression.

  12. Removal of interfering substances in samples prepared for two-dimensional (2-D) electrophoresis.

    Science.gov (United States)

    Berkelman, Tom

    2008-01-01

    Biological samples may contain contaminants that interfere with analysis by two-dimensional (2-D) electrophoresis. Lysates or biological fluids are complex mixtures that contain a wide variety of nonprotein substances in addition to the proteins to be analyzed. These substances often interfere with the resolution of the electrophoretic separation or the visualization of the result. Macromolecules (e.g., polysaccharides and DNA) can interfere with electrophoretic separation by clogging gel pores. Small ionic molecules can impair isoelectric focusing (IEF) separation by rendering the sample too conductive. Other substances (e.g., phenolics and lipids) can bind to proteins, influencing their electrophoretic properties or solubility. In many cases, measures to remove interfering substances can result in significantly clearer 2-D patterns with more visible spots and better resolution. It should be borne in mind, however, that analysis of samples by 2-D electrophoresis is usually most successful and informative when performed with minimally processed samples, so it is important that any steps taken to remove interfering substance be appropriate to the sample and only performed when necessary. Procedures for the removal of interfering substances therefore represent a compromise between removing nonprotein contaminants, and minimizing interference with the integrity and relative abundances of the sample proteins. This chapter presents a number of illustrative examples of optimized sample preparation methods in which specific interfering substances are removed by a variety of different strategies.

  13. 7 CFR 56.72 - Interfering with a grader or employee of the AMS.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Interfering with a grader or employee of the AMS. 56... a grader or employee of the AMS. Any interference with or obstruction or any attempted interference or obstruction of or assault upon any graders, licensees, or employees of the AMS in the...

  14. Complex dynamics of defective interfering baculoviruses during serial passage in insect cells

    NARCIS (Netherlands)

    Zwart, M.P.; Pijlman, G.P.; Sardanyes, J.; Duarte, J.; Januario, C.; Elena, S.F.

    2013-01-01

    Defective interfering (DI) viruses are thought to cause oscillations in virus levels, known as the ‘Von Magnus effect’. Interference by DI viruses has been proposed to underlie these dynamics, although experimental tests of this idea have not been forthcoming. For the baculoviruses, insect viruses c

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    INTRODUCTION: Development of drugs based on RNA interference by small interfering RNA (siRNA) has been progressing slowly due to a number of challenges associated with the in vivo behavior of siRNA. A central problem is controlling siRNA delivery to specific cell types. Here, we review existing...

  16. The Host RNAs in Retroviral Particles

    Directory of Open Access Journals (Sweden)

    Alice Telesnitsky

    2016-08-01

    Full Text Available As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA, some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations. These RNAs’ packaging determinants differ from the viral genome’s, and several of the abundantly packaged host ncRNAs serve cells as the scaffolds of ribonucleoprotein particles. Because virion assembly is equally efficient whether or not genomic RNA is available, yet RNA appears critical to the structural integrity of retroviral particles, it seems possible that the selectively encapsidated host ncRNAs might play roles in assembly. Indeed, some host ncRNAs appear to act during replication, as some transfer RNA (tRNA species may contribute to nuclear import of human immunodeficiency virus 1 (HIV-1 reverse transcription complexes, and other tRNA interactions with the viral Gag protein aid correct trafficking to plasma membrane assembly sites. However, despite high conservation of packaging for certain host RNAs, replication roles for most of these selectively encapsidated RNAs—if any—have remained elusive.

  17. Species specific inhibition of viral replication using dicer substrate siRNAs (DsiRNAs) targeting the viral nucleoprotein of the fish pathogenic rhabdovirus viral hemorrhagic septicemia virus (VHSV).

    Science.gov (United States)

    Bohle, Harry; Lorenzen, Niels; Schyth, Brian Dall

    2011-06-01

    Gene knock down by the use of small interfering RNAs (siRNAs) is widely used as a method for reducing the expression of specific genes in eukaryotic cells via the RNA interference pathway. But, the effectivity of siRNA induced gene knock down in cells from fish has in several studies been questioned and the specificity seems to be a general problem in cells originating from both lower and higher vertebrates. Here we show that we are able to reduce the level of viral gene expression and replication specifically in fish cells in vitro. We do so by using 27/25-mer DsiRNAs acting as substrates for dicer for the generation of siRNAs targeting the nucleoprotein N gene of viral hemorrhagic septicemia virus (VHSV). This rhabdovirus infects salmonid fish and is responsible for large yearly losses in aquaculture production. Specificity of the DsiRNA is assured in two ways: first, by using the conventional method of testing a control DsiRNA which should not target the gene of interest. Second, by assuring that replication of a heterologous virus of the same genus as the target virus was not inhibited by the DsiRNA. Target controls are, as we have previously highlighted, essential for verification of the specificity of siRNA-induced interference with virus multiplication, but they are still not in general use.

  18. Noncoding RNAs: Emerging Players in Muscular Dystrophies

    Directory of Open Access Journals (Sweden)

    Germana Falcone

    2014-01-01

    Full Text Available The fascinating world of noncoding RNAs has recently come to light, thanks to the development of powerful sequencing technologies, revealing a variety of RNA molecules playing important regulatory functions in most, if not all, cellular processes. Many noncoding RNAs have been implicated in regulatory networks that are determinant for skeletal muscle differentiation and disease. In this review, we outline the noncoding RNAs involved in physiological mechanisms of myogenesis and those that appear dysregulated in muscle dystrophies, also discussing their potential use as disease biomarkers and therapeutic targets.

  19. MicroRNAs: Potential biomarkers in cancer

    OpenAIRE

    George, G. P.; Mittal, Rama Devi

    2010-01-01

    microRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs, also known as micromanagers of gene expression. Polymorphisms in the miRNA pathway (miR-polymorphisms) are emerging as powerful tools to study the biology of a disease and have the potential to be used in disease prognosis and diagnosis. Advancements in the miRNA field also indicate a clear involvement of deregulated miRNA gene signatures in cancers, and several polymorphisms in pre-miRNA, miRNA binding sites or targets hav...

  20. Gene silencing by chemically modified siRNAs.

    Science.gov (United States)

    Engels, Joachim W

    2013-03-25

    RNA interference (RNAi) has not only already risen as a gold standard for validating gene function in basic science studies, but also holds great promise as a new therapeutic paradigm. Advantages of RNAi-based therapeutics include relatively fast initial screening and the ability to target proteins not yet addressable by traditional drug design strategies. In this review we describe the development of chemically modified small inhibiting siRNAs and their application as potential therapeutics during the past decade. Focus is on proper siRNA design, choice of chemical modification and how to circumvent immunogenicity as well as off-target effects.

  1. MicroRNAs in Heart Failure, Cardiac Transplantation, and Myocardial Recovery: Biomarkers with Therapeutic Potential.

    Science.gov (United States)

    Shah, Palak; Bristow, Michael R; Port, J David

    2017-09-22

    Heart failure is increasing in prevalence with a lack of recently developed therapies that produce major beneficial effects on its associated mortality. MicroRNAs are small non-coding RNA molecules that regulate gene expression, are differentially regulated in heart failure, and are found in the circulation serving as a biomarker of heart failure. Data suggests that microRNAs may be used to detect allograft rejection in cardiac transplantation and may predict the degree of myocardial recovery in patients with a left ventricular assist device or treated with beta-blocker therapy. Given their role in regulating cellular function, microRNAs are an intriguing target for oligonucleotide therapeutics, designed to mimic or antagonize (antagomir) their biological effects. We review the current state of microRNAs as biomarkers of heart failure and associated conditions, the mechanisms by which microRNAs control cellular function, and how specific microRNAs may be targeted with novel therapeutics designed to treat heart failure.

  2. Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing.

    Science.gov (United States)

    Liang, Gang; Zhang, Huimin; Lou, Dengji; Yu, Diqiu

    2016-02-19

    The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers' ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.

  3. 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

    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.

  4. MicroRNAs in mantle cell lymphoma

    DEFF Research Database (Denmark)

    Husby, Simon; Geisler, Christian; Grønbæk, Kirsten

    2013-01-01

    Mantle cell lymphoma (MCL) is a rare and aggressive subtype of non-Hodgkin lymphoma. New treatment modalities, including intensive induction regimens with immunochemotherapy and autologous stem cell transplant, have improved survival. However, many patients still relapse, and there is a need...... for novel therapeutic strategies. Recent progress has been made in the understanding of the role of microRNAs (miRNAs) in MCL. Comparisons of tumor samples from patients with MCL with their normal counterparts (naive B-cells) have identified differentially expressed miRNAs with roles in cellular growth...... and survival pathways, as demonstrated in various biological model systems. In addition, MCL clinico-pathological and prognostic subtypes can be identified using individual miRNAs or miRNA classifiers. miRNA based therapies have now shown efficacy in animal models, and many efforts are currently being made...

  5. Non-Coding RNAs in Muscle Dystrophies

    Directory of Open Access Journals (Sweden)

    Alessandra Ferlini

    2013-09-01

    Full Text Available ncRNAs are the most recently identified class of regulatory RNAs with vital functions in gene expression regulation and cell development. Among the variety of roles they play, their involvement in human diseases has opened new avenues of research towards the discovery and development of novel therapeutic approaches. Important data come from the field of hereditary muscle dystrophies, like Duchenne muscle dystrophy and Myotonic dystrophies, rare diseases affecting 1 in 7000–15,000 newborns and is characterized by severe to mild muscle weakness associated with cardiac involvement. Novel therapeutic approaches are now ongoing for these diseases, also based on splicing modulation. In this review we provide an overview about ncRNAs and their behavior in muscular dystrophy and explore their links with diagnosis, prognosis and treatments, highlighting the role of regulatory RNAs in these pathologies.

  6. 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.

  7. New miRNAs cloned from neuroblastoma

    Directory of Open Access Journals (Sweden)

    Glatting Karl-Heinz

    2008-01-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are a novel class of gene expression regulators implicated in cancer biology. Neuroblastoma (NB is an embryonal tumour consisting of neural crest-derived undifferentiated cells and is characterised by variable clinical courses ranging from spontaneous regression to therapy-resistant progression. Recent advances identified a subset of miRNAs with putative function in NB biology. However, the full repertoire of miRNAs expressed in NBs is not available. Results We describe miRNA profiles of 13 NB specimens and 2 NB cell lines as determined by miRNA cloning. A total of 3153 sequences were sequenced and analysed by a miRNA prediction tool (miRpredict. Our library covered 27% miRNAs known to date. 39 reads corresponding to 25 individual sequences were classified as novel miRNAs, including miRNA* species of 10 known miRNAs. Expression of 5 new miRNA* forms and 8 individual sequences was supported by Northern blotting. Most of the novel miRNA genes are not related to each other and do not share homology with the annotated sequences in the public miRNA database, but they are conserved within mammals or have close homologues in primates genomes. Conclusion We provide evidence for 29 new miRNA and miRNA-like sequences (24 novel sequences and 5 miRNAs discovered initially in other species. Some of these newly identified sequences reside within frequently altered chromosomal regions in NB tumours and may play a role in NB biology.

  8. RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.

    Science.gov (United States)

    Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

    2016-01-01

    Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

  9. Long noncoding RNAs(lncRNAs) and the molecular hallmarks of aging.

    Science.gov (United States)

    Grammatikakis, Ioannis; Panda, Amaresh C; Abdelmohsen, Kotb; Gorospe, Myriam

    2014-12-01

    During aging, progressive deleterious changes increase the risk of disease and death. Prominent molecular hallmarks of aging are genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, cellular senescence, stem cell exhaustion, and altered intercellular communication. Long noncoding RNAs (lncRNAs) play important roles in a wide range of biological processes, including age-related diseases like cancer, cardiovascular pathologies, and neurodegenerative disorders. Evidence is emerging that lncRNAs influence the molecular processes that underlie age-associated phenotypes. Here, we review our current understanding of lncRNAs that control the development of aging traits.

  10. Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs.

    Science.gov (United States)

    Zinoviev, Alexandra; Hellen, Christopher U T; Pestova, Tatyana V

    2015-03-19

    Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long open reading frames (ORFs) on cellular mRNAs, reinitiation occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG, designated "termination upstream ribosomal binding site" (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2(∗). To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes and diminishes dependence on eukaryotic initiation factor 3 (eIF3) of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. High throughput sequencing of small RNAs in Potato Leaves

    Science.gov (United States)

    Understanding the role of small RNAs (sRNAs) in gene regulation, function and development is a rapidly evolving field. sRNAs result from transcript degradation and to regulatory micro RNA (miRNA) and small inhibitory RNA (siRNA) classes involved in gene regulation. The sRNAs from potato leaves were...

  12. Insights in microRNAs biology.

    Science.gov (United States)

    Gargalionis, Antonios N; Basdra, Efthimia K

    2013-01-01

    MicroRNAs (miRNAs) are small, non-coding RNAs, that function as post-transcriptional regulators of gene expression. Recent studies now predict that numerous miRNA molecules regulate a large proportion of the human transcriptome, thus creating a whole new research field that utilizes their potential impact on gene expression in favor of diagnosis, prognosis and drug development. MiRNAs are generated from transcription of respective genes into primary structures that usually follow a two-step maturation process in the cell nucleus and cytoplasm. Active miRNA folds downregulate protein expression either via direct repression of targeted messenger RNA (mRNA) or mRNA cleavage. They are critical factors that control human development and organogenesis and reemerge as key-molecules that profoundly influence adult cells and tissues under stress-responsive conditions. Therefore, several miRNAs exhibit dysregulated functions in almost all aspects of human pathology such as cancer, cardiovascular diseases, metabolic disorders, genetic and neurodegenerative diseases, forming tissue-specific molecular profiles that further define salient pathologic features. The present article offers an overview on miRNAs biogenesis and functional processes, major aspects of their participation in human development and milestones regarding their contribution in human diseases. Furthermore, their utility as extracellular biomarkers and the rationale behind miRNA inhibition or miRNA delivery are being discussed.

  13. microRNAs and Neurodegenerative Diseases.

    Science.gov (United States)

    Qiu, Lifeng; Tan, Eng King; Zeng, Li

    2015-01-01

    microRNAs (miRNAs) are small, noncoding RNA molecules that through imperfect base-pairing with complementary sequences of target mRNA molecules, typically cleave target mRNA, causing subsequent degradation or translation inhibition. Although an increasing number of studies have identified misregulated miRNAs in the neurodegenerative diseases (NDDs) Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, which suggests that alterations in the miRNA regulatory pathway could contribute to disease pathogenesis, the molecular mechanisms underlying the pathological implications of misregulated miRNA expression and the regulation of the key genes involved in NDDs remain largely unknown. In this chapter, we provide evidence of the function and regulation of miRNAs and their association with the neurological events in NDDs. This will help improve our understanding of how miRNAs govern the biological functions of key pathogenic genes in these diseases, which potentially regulate several pathways involved in the progression of neurodegeneration. Additionally, given the growing interest in the therapeutic potential of miRNAs, we discuss current clinical challenges to developing miRNA-based therapeutics for NDDs.

  14. MicroRNAs as Haematopoiesis Regulators

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    Ram Babu Undi

    2013-01-01

    Full Text Available The production of different types of blood cells including their formation, development, and differentiation is collectively known as haematopoiesis. Blood cells are divided into three lineages erythriod (erythrocytes, lymphoid (B and T cells, and myeloid (granulocytes, megakaryocytes, and macrophages. Haematopoiesis is a complex process regulated by several mechanisms including microRNAs (miRNAs. miRNAs are small RNAs which regulate the expression of a number of genes involved in commitment and differentiation of hematopoietic stem cells. Evidence shows that miRNAs play an important role in haematopoiesis; for example, myeloid and erythroid differentiation is blocked by the overexpression of miR-15a. miR-221, miR-222, and miR-24 inhibit the erythropoiesis, whereas miR-150 plays a role in B and T cell differentiation. miR-146 and miR-10a are downregulated in megakaryopoiesis. Aberrant expression of miRNAs was observed in hematological malignancies including chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myelomas, and B cell lymphomas. In this review we have focused on discussing the role of miRNA in haematopoiesis.

  15. 36 CFR 261.3 - Interfering with a Forest officer, volunteer, or human resource program enrollee or giving false...

    Science.gov (United States)

    2010-07-01

    ... officer, volunteer, or human resource program enrollee or giving false report to a Forest officer. 261.3... General Prohibitions § 261.3 Interfering with a Forest officer, volunteer, or human resource program..., intimidating, or intentionally interfering with any Forest officer, volunteer, or human resource program...

  16. Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target GADD45B To Protect Infected Cells from Cell Cycle Arrest and Apoptosis.

    Science.gov (United States)

    Liu, Xiaoyan; Happel, Christine; Ziegelbauer, Joseph M

    2017-02-01

    Kaposi's sarcoma is one of the most common malignancies in HIV-infected individuals. The responsible agent, Kaposi's sarcoma-associated herpesvirus (KSHV; HHV8), expresses multiple microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. After infection in primary endothelial cells with KSHV, growth arrest DNA damage-inducible gene 45 beta (GADD45B) is one of the most repressed genes using genomic expression profiling. GADD45B was also repressed in mRNA expression profiling experiments when KSHV miRNAs were introduced to uninfected cells. We hypothesized that KSHV miRNAs target human GADD45B to protect cells from consequences of DNA damage, which can be triggered by viral infection. Expression of GADD45B protein is induced by the p53 activator, Nutlin-3, and KSHV miRNA-K9 inhibits this induction. In addition, Nutlin-3 increased apoptosis and cell cycle arrest based on flow cytometry assays. KSHV miR-K9 protected primary endothelial cells from apoptosis and cell cycle arrest following Nutlin-3 treatment. Similar protective phenotypes were seen for targeting GADD45B with short interfering RNAs (siRNAs), as with miR-K9. KSHV miR-K9 also decreased the protein levels of cleaved caspase-3, cleaved caspase-7, and cleaved poly(ADP-ribose) polymerase (PARP). In B lymphocytes latently infected with KSHV, specific inhibitors of KSHV miR-K9 led to increased GADD45B expression and apoptosis, indicating that miR-K9 is important for reducing apoptosis in infected cells. Furthermore, ectopic expression of GADD45B in KSHV-infected cells promoted apoptosis. Together, these results identify a new miRNA target and demonstrate that KSHV miRNAs are important for protecting infected cells from DNA damage responses.

  17. Update on non-canonical microRNAs

    OpenAIRE

    2014-01-01

    Non-canonical microRNAs are a recently-discovered subset of microRNAs. They structurally and functionally resemble canonical miRNAs, but were found to follow distinct maturation pathways, typically bypassing one or more steps of the classic canonical biogenesis pathway. Non-canonical miRNAs were found to have diverse origins, including introns, snoRNAs, endogenous shRNAs and tRNAs. Our knowledge about their functions remains relatively primitive; however, many interesting discoveries have tak...

  18. Analysis of antisense expression by whole genome tiling microarrays and siRNAs suggests mis-annotation of Arabidopsis orphan protein-coding genes.

    Directory of Open Access Journals (Sweden)

    Casey R Richardson

    Full Text Available BACKGROUND: MicroRNAs (miRNAs and trans-acting small-interfering RNAs (tasi-RNAs are small (20-22 nt long RNAs (smRNAs generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery. PRINCIPAL FINDINGS: We explored rice (Oryza sativa sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis 'orphan' hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the "ancient" (deeply conserved class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for "new" rapidly-evolving MIRNA genes. CONCLUSIONS: Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non

  19. Small RNA Sequencing Based Identification of MiRNAs in Daphnia magna.

    Science.gov (United States)

    Ünlü, Ercan Selçuk; Gordon, Donna M; Telli, Murat

    2015-01-01

    Small RNA molecules are short, non-coding RNAs identified for their crucial role in post-transcriptional regulation. A well-studied example includes miRNAs (microRNAs) which have been identified in several model organisms including the freshwater flea and planktonic crustacean Daphnia. A model for epigenetic-based studies with an available genome database, the identification of miRNAs and their potential role in regulating Daphnia gene expression has only recently garnered interest. Computational-based work using Daphnia pulex, has indicated the existence of 45 miRNAs, 14 of which have been experimentally verified. To extend this study, we took a sequencing approach towards identifying miRNAs present in a small RNA library isolated from Daphnia magna. Using Perl codes designed for comparative genomic analysis, 815,699 reads were obtained from 4 million raw reads and run against a database file of known miRNA sequences. Using this approach, we have identified 205 putative mature miRNA sequences belonging to 188 distinct miRNA families. Data from this study provides critical information necessary to begin an investigation into a role for these transcripts in the epigenetic regulation of Daphnia magna.

  20. Functional Roles of microRNAs in Agronomically Important Plants-Potential as Targets for Crop Improvement and Protection.

    Science.gov (United States)

    Djami-Tchatchou, Arnaud T; Sanan-Mishra, Neeti; Ntushelo, Khayalethu; Dubery, Ian A

    2017-01-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression, mainly through cleavage and/or translation inhibition of the target mRNAs during or after transcription. miRNAs play important roles by regulating a multitude of biological processes in plants which include maintenance of genome integrity, development, metabolism, and adaptive responses toward environmental stresses. The increasing population of the world and their food demands requires focused efforts for the improvement of crop plants to ensure sustainable food production. Manipulation of mRNA transcript abundance via miRNA control provides a unique strategy for modulating differential plant gene expression and miRNAs are thus emerging as the next generation targets for genetic engineering for improvement of the agronomic properties of crops. However, a deeper understanding of its potential and the mechanisms involved will facilitate the design of suitable strategies to obtain the desirable traits with minimum trade-offs in the modified crops. In this regard, this review highlights the diverse roles of conserved and newly identified miRNAs in various food and industrial crops and recent advances made in the uses of miRNAs to improve plants of agronomically importance so as to significantly enhance crop yields and increase tolerance to various environmental stress agents of biotic-or abiotic origin.

  1. Removing interfering clutter associated with radar pulses that an airborne radar receives from a radar transponder

    Science.gov (United States)

    Ormesher, Richard C.; Axline, Robert M.

    2008-12-02

    Interfering clutter in radar pulses received by an airborne radar system from a radar transponder can be suppressed by developing a representation of the incoming echo-voltage time-series that permits the clutter associated with predetermined parts of the time-series to be estimated. These estimates can be used to estimate and suppress the clutter associated with other parts of the time-series.

  2. Interaction between cationic agents and small interfering RNA and DNA molecules

    Science.gov (United States)

    Unksov, I. N.; Slita, A. V.; Petrova, A. V.; Pereviazko, I.; Bakulev, V. M.; Rolich, V. I.; Bondarenko, A. B.; Kasyanenko, N. A.

    2016-11-01

    Azobenzene containing surfactant AzoTAB was used for investigation of binding in cationic- agent + nucleic acid in NaCl salt aqueous solutions. Two nucleic acids, macromolecular DNA and small interfering RNA, were examined upon the interaction with the surfactant. For DNA the interaction was studied using spectral methods and the methods of viscometry and flow birefringence measurement. For siRNA the possibility of surfactant-based delivery was checked in vitro.

  3. Identification of Four New agr Quorum Sensing-Interfering Cyclodepsipeptides from a Marine Photobacterium

    DEFF Research Database (Denmark)

    Kjærulff, Louise; Nielsen, Anita; Månsson, Maria

    2013-01-01

    , including the known pyrrothine antibiotic holomycin and a wide range of peptides, from diketopiperazines to cyclodepsipeptides of 500–900 Da. Purification of components from the pellet fraction led to the isolation and structure elucidation of four new cyclodepsipeptides, ngercheumicin F, G, H, and I....... The ngercheumicins interfered with expression of virulence genes known to be controlled by the agr quorum sensing system of Staphylococcus aureus, although to a lesser extent than the previously described solonamides from the same strain of Photobacterium....

  4. Períodos de interferência de plantas daninhas na cultura do girassol

    Directory of Open Access Journals (Sweden)

    Remon Ribeiro da Silva

    2013-01-01

    Full Text Available Objetivou-se neste trabalho determinar os períodos de interferência de plantas daninhas na cultura do girassol. Foram realizados dois experimentos com o girassol híbrido Helio 250, sendo um conduzido no verão e o outro, no inverno. Para ambos, o delineamento experimental utilizado foi em blocos casualizados, com quatro repetições, e cada unidade experimental foi constituída de cinco linhas de plantas com 6,0 m de comprimento, espaçadas por 0,7 m. No experimento de verão foram avaliados oito períodos de convivência e controle de plantas daninhas: 10, 20, 30, 40, 50, 60, 70 e 88 dias após a emergência (dae. No experimento de inverno, os períodos foram: 7, 14, 21, 28, 38, 48, 58 e 82 dae. Avaliaram-se a massa da matéria seca e a área foliar das plantas de girassol, produtividade, massa de mil aquênios e massa da matéria seca de plantas daninhas. Estimaram-se os períodos anteriores à interferência (PAI, períodos críticos de prevenção à interferência (PCPI e períodos totais de prevenção à interferência (PTPI. Considerando-se a produtividade de aquênios no verão, o PAI foi de 16 dae da cultura, o PTPI, de 39 dae e o PCPI abrangeu 23 dias do ciclo da cultura. No inverno, o PAI foi de 12 dae, o PTPI estendeu-se até os 34 dae, resultando em um PCPI de 22 dias.

  5. Thiophenone Attenuates Enteropathogenic Escherichia coli O103:H2 Virulence by Interfering with AI-2 Signaling.

    Science.gov (United States)

    Witsø, Ingun Lund; Valen Rukke, Håkon; Benneche, Tore; Aamdal Scheie, Anne

    2016-01-01

    Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli.

  6. The Influence of Interfering Substances on the Antimicrobial Activity of Selected Quaternary Ammonium Compounds

    Directory of Open Access Journals (Sweden)

    Paula A. Araújo

    2013-01-01

    Full Text Available Standard cleaning processes may not remove all the soiling typically found in food industry, such as carbohydrates, fats, or proteins. Contaminants have a high impact in disinfection as their presence may reduce the activity of disinfectants. The influence of alginic acid, bovine serum albumin, yeast extract, and humic acids was assessed on the antimicrobial activities of benzalkonium chloride and cetyltrimethyl ammonium bromide against Bacillus cereus vegetative cells and Pseudomonas fluorescens. The bacteria (single and consortium were exposed to surfactants (single and combined in the absence and presence of potential disinfection interfering substances. The antimicrobial effects of the surfactants were assessed based on the bacterial respiratory activity measured by oxygen uptake rate due to glucose oxidation. The tested surfactants were efficient against both bacteria (single and consortium with minimum bactericidal concentrations ranging from 3 to 35 mg·L−1. The strongest effect was caused by humic acids that severely quenched antimicrobial action, increasing the minimum bactericidal concentration of the surfactants on P. fluorescens and the consortium. The inclusion of the other interfering substances resulted in mild interferences in the antibacterial activity. This study clearly demonstrates that humic acids should be considered as an antimicrobial interfering substance in the development of disinfection strategies.

  7. The Influence of Interfering Substances on the Antimicrobial Activity of Selected Quaternary Ammonium Compounds.

    Science.gov (United States)

    Araújo, Paula A; Lemos, Madalena; Mergulhão, Filipe; Melo, Luís; Simões, Manuel

    2013-01-01

    Standard cleaning processes may not remove all the soiling typically found in food industry, such as carbohydrates, fats, or proteins. Contaminants have a high impact in disinfection as their presence may reduce the activity of disinfectants. The influence of alginic acid, bovine serum albumin, yeast extract, and humic acids was assessed on the antimicrobial activities of benzalkonium chloride and cetyltrimethyl ammonium bromide against Bacillus cereus vegetative cells and Pseudomonas fluorescens. The bacteria (single and consortium) were exposed to surfactants (single and combined) in the absence and presence of potential disinfection interfering substances. The antimicrobial effects of the surfactants were assessed based on the bacterial respiratory activity measured by oxygen uptake rate due to glucose oxidation. The tested surfactants were efficient against both bacteria (single and consortium) with minimum bactericidal concentrations ranging from 3 to 35 mg·L(-1). The strongest effect was caused by humic acids that severely quenched antimicrobial action, increasing the minimum bactericidal concentration of the surfactants on P. fluorescens and the consortium. The inclusion of the other interfering substances resulted in mild interferences in the antibacterial activity. This study clearly demonstrates that humic acids should be considered as an antimicrobial interfering substance in the development of disinfection strategies.

  8. Progress and Prospects of Long Noncoding RNAs (lncRNAs in Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Chen Li

    2015-05-01

    Full Text Available Hepatocellular carcinoma (HCC is one of the most frequently occurring cancers with poor prognosis, and novel diagnostic or prognostic biomarkers and therapeutic targets for HCC are urgently required. With the advance of high-resolution microarrays and massively parallel sequencing technology, lncRNAs are suggested to play critical roles in the tumorigenesis and development of human HCC. To date, dysregulation of many HCC-related lncRNAs such as HULC, HOTAIR, MALAT1, and H19 have been identified. From transcriptional “noise” to indispensable elements, lncRNAs may re-write the central dogma. Also, lncRNAs found in body fluids have demonstrated their utility as fluid-based noninvasive markers for clinical use and as therapeutic targets for HCC. Even though several lncRNAs have been characterized, the underlying mechanisms of their contribution to HCC remain unknown, and many important questions about lncRNAs need resolving. A better understanding of the molecular mechanism in HCC-related lncRNAs will provide a rationale for novel effective lncRNA-based targeted therapies. In this review, we highlight the emerging roles of lncRNAs in HCC, and discuss their potential clinical applications as biomarkers for the diagnosis, prognosis, monitoring and treatment of HCC.

  9. Identification and characterization of novel microRNAs from Schistosoma japonicum.

    Directory of Open Access Journals (Sweden)

    Xiangyang Xue

    Full Text Available BACKGROUND: Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs. METHODOLOGY AND RESULTS: We have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18-26 nt RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host. CONCLUSIONS: Authentic miRNAs were identified for the first time in S. japonicum, including a new schistosome

  10. Potentials of Long Noncoding RNAs (LncRNAs in Sarcoma: From Biomarkers to Therapeutic Targets

    Directory of Open Access Journals (Sweden)

    Li Min

    2017-03-01

    Full Text Available Sarcoma includes some of the most heterogeneous tumors, which make the diagnosis, prognosis and treatment of these rare yet diverse neoplasms especially challenging. Long noncoding RNAs (lncRNAs are important regulators of cancer initiation and progression, which implies their potential as neoteric prognostic and diagnostic markers in cancer, including sarcoma. A relationship between lncRNAs and sarcoma pathogenesis and progression is emerging. Recent studies demonstrate that lncRNAs influence sarcoma cell proliferation, metastasis, and drug resistance. Additionally, lncRNA expression profiles are predictive of sarcoma prognosis. In this review, we summarize contemporary advances in the research of lncRNA biogenesis and functions in sarcoma. We also highlight the potential for lncRNAs to become innovative diagnostic and prognostic biomarkers as well as therapeutic targets in sarcoma.

  11. MicroRNAs in Cardiometabolic Diseases

    Directory of Open Access Journals (Sweden)

    Anna Meiliana

    2013-08-01

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are ~22-nucleotide noncoding RNAs with critical functions in multiple physiological and pathological processes. An explosion of reports on the discovery and characterization of different miRNA species and their involvement in almost every aspect of cardiac biology and diseases has established an exciting new dimension in gene regulation networks for cardiac development and pathogenesis. CONTENT: Alterations in the metabolic control of lipid and glucose homeostasis predispose an individual to develop cardiometabolic diseases, such as type 2 diabetes mellitus and atherosclerosis. Work over the last years has suggested that miRNAs play an important role in regulating these physiological processes. Besides a cell-specific transcription factor profile, cell-specific miRNA-regulated gene expression is integral to cell fate and activation decisions. Thus, the cell types involved in atherosclerosis, vascular disease, and its myocardial sequelae may be differentially regulated by distinct miRNAs, thereby controlling highly complex processes, for example, smooth muscle cell phenotype and inflammatory responses of endothelial cells or macrophages. The recent advancements in using miRNAs as circulating biomarkers or therapeutic modalities, will hopefully be able to provide a strong basis for future research to further expand our insights into miRNA function in cardiovascular biology. SUMMARY: MiRNAs are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. They are potent modulators of diverse biological processes and pathologies. Recent findings demonstrated the importance of miRNAs in the vasculature and the orchestration of lipid metabolism and glucose homeostasis. MiRNA networks represent an additional layer of regulation for gene expression that absorbs perturbations and ensures the robustness of biological systems. A detailed understanding of the molecular and cellular mechanisms of mi

  12. The regulatory epicenter of miRNAs

    Indian Academy of Sciences (India)

    Ashwani Jha; Mrigaya Mehra; Ravi Shankar

    2011-09-01

    miRNAs are small non-coding RNAs with average length of ∼21 bp. miRNA formation seems to be dependent upon multiple factors besides Drosha and Dicer, in a tissue/stage-specific manner, with interplay of several specific binding factors. In the present study, we have investigated transcription factor binding sites in and around the genomic sequences of precursor miRNAs and RNA-binding protein (RBP) sites in miRNA precursor sequences, analysed and tested in comprehensive manner. Here, we report that miRNA precursor regions are positionally enriched for binding of transcription factors as well as RBPs around the 3′ end of mature miRNA region in 5′ arm. The pattern and distribution of such regulatory sites appears to be a characteristic of precursor miRNA sequences when compared with non-miRNA sequences as negative dataset and tested statistically. When compared with 1 kb upstreamregions, a sudden sharp peak for binding sites arises in the enriched zone near the mature miRNA region. An expression-data-based correlation analysis was performed between such miRNAs and their corresponding transcription factors and RBPs for this region. Some specific groups of binding factors and associated miRNAs were identified. We also identified some of the overrepresented transcription factors and associated miRNAs with high expression correlation values which could be useful in cancer-related studies. The highly correlated groups were found to host experimentally validated composite regulatory modules, in which Lmo2-GATA1 appeared as the predominant one. For many of RBP–miRNAs associations, co-expression similarity was also evident among the associated miRNA common to given RBPs, supporting the Regulon model, suggesting a common role and common control of these miRNAs by the associated RBPs. Based on our findings, we propose that the observed characteristic distribution of regulatory sites in precursor miRNA sequence regions could be critical inmiRNA transcription, processing

  13. Non coding RNAs in aortic aneurysmal disease

    Directory of Open Access Journals (Sweden)

    Aparna eDuggirala

    2015-04-01

    Full Text Available An aneurysm is a local dilatation of a vessel wall which is >50% its original diameter. Withinthe spectrum of cardiovascular diseases, aortic aneurysms are among the most challenging to treat. Most patients present acutely after aneurysm rupture or dissection from a previous asymptomatic condition and are managed by open surgical or endovascular repair. In addition, patients may harbour concurrent disease contraindicating surgical intervention. Collectively, these factors have driven the search for alternative methods of identifying, monitoring and treating aortic aneurisms using less invasive approaches. Non-coding RNA (ncRNAs are emerging as new fundamental regulators of gene expression. The small microRNAs have opened the field of ncRNAs capturing the attention of basic and clinical scientists for their potential to become new therapeutic targets and clinical biomarkers for aortic aneurysm. More recently, long ncRNAs (lncRNAs have started to be actively investigated, leading to first exciting reports, which further suggest their important and yet largely unexplored contribution to vascular physiology and disease.

  14. Silencing human cancer: identification and uses of microRNAs.

    Science.gov (United States)

    Nicolas, Francisco E; Lopez-Gomollon, Sara; Lopez-Martinez, Alfonso F; Dalmay, Tamas

    2011-01-01

    MicroRNAs (miRNAs) are a new class of negative regulators that repress gene expression by pairing with their target messenger RNAs (mRNAs). There are hundreds of miRNAs coded in the human genome and thousands of target mRNAs participating in a wide variety of physiological processes such as development and cell identity. It is therefore not surprising that several recent reports involved deregulated miRNAs in the complex mechanism of human carcinogenesis, and proposed them as new key regulators to correct the unbalanced expression of oncogenes and tumour suppressor genes exhibited in cancer cells. This review summarises most of the recent patents related to the use of miRNA signatures in cancer diagnosis and prognosis, the detection and profiling of miRNAs from tumour samples and the identification of oncogenes and tumour suppressor genes targeted by miRNAs, as well as new cancer therapies based on miRNA modulators.

  15. piRNAs and Their Functions in the Brain

    Science.gov (United States)

    Zuo, Lingjun; Wang, Zhiren; Tan, Yunlong; Chen, Xiangning; Luo, Xingguang

    2016-01-01

    Piwi-interacting RNAs (piRNAs) are the non-coding RNAs with 24–32 nucleotides (nt). They exhibit stark differences in length, expression pattern, abundance, and genomic organization when compared to micro-RNAs (miRNAs). There are hundreds of thousands unique piRNA sequences in each species. Numerous piRNAs have been identified and deposited in public databases. Since the piRNAs were originally discovered and well-studied in the germline, a few other studies have reported the presence of piRNAs in somatic cells including neurons. This paper reviewed the common features, biogenesis, functions, and distributions of piRNAs and summarized their specific functions in the brain. This review may provide new insights and research direction for brain disorders. PMID:27512315

  16. Circular RNAs in cancer: an emerging key player

    Directory of Open Access Journals (Sweden)

    Yeping Dong

    2017-01-01

    Full Text Available Abstract Circular RNAs (circRNAs are a class of endogendous RNAs that form a covalently closed continuous loop and exist extensively in mammalian cells. Majority of circRNAs are conserved across species and often show tissue/developmental stage-specific expression. CircRNAs were first thought to be the result of splicing error; however, subsequent research shows that circRNAs can function as microRNA (miRNA sponges and regulate splicing and transcription. Emerging evidence shows that circRNAs possess closely associated with human diseases, especially cancers, and may serve as better biomarkers. After miRNA and long noncoding RNA (lncRNA, circRNAs are becoming a new hotspot in the field of RNA of cancer. Here, we review biogenesis and metabolism of circRNAs, their functions, and potential roles in cancer.

  17. Role of Exosomal Noncoding RNAs in Lung Carcinogenesis

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    Tao Sun

    2015-01-01

    Full Text Available Lung cancer is the major cause of cancer death worldwide. Novel, recently discovered classes of noncoding RNAs (ncRNAs have diverse functional and regulatory activities and increasing evidence suggests crucial roles for deregulated ncRNAs in the onset and progression of cancer, including lung cancer. Exosomes are small extracellular membrane vesicles of endocytic origin that are released by many cells and are found in most body fluids. Tumor-derived exosomes mediate tumorigenesis by facilitating tumor growth and metastasis. MicroRNAs (miRNAs are a subclass of ncRNAs that are present in exosomes. miRNAs are taken up by neighboring or distant cells and modulate various functions of recipient cells. Here, we review exosome-derived ncRNAs with a focus on miRNAs and their role in lung cancer biology.

  18. Efficient delivery of small interfering RNA into injured spinal cords in rats by photomechanical waves

    Science.gov (United States)

    Ando, Takahiro; Sato, Shunichi; Toyooka, Terushige; Kobayashi, Hiroaki; Nawashiro, Hiroshi; Ashida, Hiroshi; Obara, Minoru

    2011-03-01

    In the central nervous system, lack of axonal regeneration leads to permanent functional disabilities. In spinal cord injury (SCI), the over-expressions of intermediate filament (IF) proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are mainly involved in glial scar formation; these proteins work as both physical and biochemical barriers to axonal regeneration. Thus, silencing of these IF proteins would be an attractive strategy to treat SCI. In this study, we first attempted to deliver fluorescent probe-labeled siRNAs into injured spinal cords in rats by applying photomechanical waves (PMWs) to examine the capability of PMWs as a tool for siRNA delivery. Intense fluorescence from siRNAs was observed in much broader regions in the spinal cords with PMW application when compared with those with siRNA injection alone. Based on this result, we delivered siRNAs for GFAP and vimentin into injured spinal tissues in rats by applying PMWs. The treatment resulted in efficient silencing of the proteins at five days after SCI and a decrease of the cavity area in the injured tissue at three weeks after SCI when compared with those with siRNA injection alone. These results demonstrate the capability of PMWs for efficient delivery of siRNAs into injured spinal cords and treatment of SCIs.

  19. 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.

  20. miRNAs modified by dietary lipids in Caco-2 cells. A microarray screening

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    Lidia Daimiel

    2015-09-01

    Full Text Available We performed a screening of miRNAs regulated by dietary lipids in a cellular model of enterocytes, Caco-2 cells. Our aim was to describe new lipid-modified miRNAs with an implication in lipid homeostasis and cardiovascular disease [1,2]. For that purpose, we treated differentiated Caco-2 cells with micelles containing the assayed lipids (cholesterol, conjugated linoleic acid and docosahexaenoic acid and the screening of miRNAs was carried out by microarray using the μParaflo®Microfluidic Biochip Technology of LC Sciences (Huston, TX, USA. Experimental design, microarray description and raw data have been made available in the GEO database with the reference number of GSE59153. Here we described in detail the experimental design and methods used to obtain the relative expression data.

  1. MicroRNAs in biliary diseases

    Institute of Scientific and Technical Information of China (English)

    Patricia Munoz-Garrido; Maite García-Fernández de Barrena; Elizabeth Hijona; Miguel Carracedo; José J G Marín; Luis Bujanda; Jesús M Banales

    2012-01-01

    Cholangiopathies are a group of diseases primarily or secondarily affecting bile duct cells,and result in cholangiocyte proliferation,regression,and/or transformation.Their etiopathogenesis may be associated with a broad variety of causes of different nature,which includes genetic,neoplastic,immune-associated,infectious,vascular,and drug-induced alterations,or being idiopathic.miRNAs,small non-coding endogenous RNAs that post-transcriptionally regulate gene expression,have been associated with pathophysiological processes in different organs and cell types,and are postulated as potential targets for diagnosis and therapy.In the current manuscript,knowledge regarding the role of miRNAs in the development and/or progression of cholangiopathies has been reviewed and the most relevant findings in this promising field of hepatology have been highlighted.

  2. MicroRNAs, epigenetics and disease

    DEFF Research Database (Denmark)

    Silahtaroglu, Asli; Stenvang, Jan

    2010-01-01

    Epigenetics is defined as the heritable chances that affect gene expression without changing the DNA sequence. Epigenetic regulation of gene expression can be through different mechanisms such as DNA methylation, histone modifications and nucleosome positioning. MicroRNAs are short RNA molecules...... which do not code for a protein but have a role in post-transcriptional silencing of multiple target genes by binding to their 3' UTRs (untranslated regions). Both epigenetic mechanisms, such as DNA methylation and histone modifications, and the microRNAs are crucial for normal differentiation...... diseases. In the present chapter we will mainly focus on microRNAs and methylation and their implications in human disease, mainly in cancer....

  3. Extracting viral RNAs from plant protoplasts.

    Science.gov (United States)

    Fabian, Marc R; Andrew White, K

    2007-08-01

    The analysis of viral RNA is a fundamental aspect of plant RNA virus research. Studies that focus on viral RNAs often involve virus infections of plant protoplasts (see UNITS 16D.1-16D.4). Protoplast offer the advantage of simultaneous initiation of infections, which allows for superior temporal and quantitative analyses of viral RNAs. The efficient isolation of intact viral RNA is key to any such investigations. This unit describes two basic protocols for extracting viral RNAs from plant protoplasts. An approach for preparing double-stranded viral RNA from total RNA pools is also provided. The viral RNA prepared by using these techniques can be used for further analyses such as primer extension, reverse transcription-PCR, and northern blotting.

  4. LncRNAs H19 and HULC, activated by oxidative stress, promote cell migration and invasion in cholangiocarcinoma through a ceRNA manner

    Directory of Open Access Journals (Sweden)

    Wen-Tao Wang

    2016-11-01

    Full Text Available Abstract Background Long non-coding RNAs (lncRNAs are known to play important roles in different cell contexts, including cancers. However, little is known about lncRNAs in cholangiocarcinoma (CCA, a cholangiocyte malignancy with poor prognosis, associated with chronic inflammation and damage to the biliary epithelium. The aim of the study is to identify if any lncRNA might associate with inflammation or oxidative stress in CCA and regulate the disease progression. Methods In this study, RNA-seqs datasets were used to identify aberrantly expressed lncRNAs. Small interfering RNA and overexpressed plasmids were used to modulate the expression of lncRNAs, and luciferase target assay RNA immunoprecipitation (RIP was performed to explore the mechanism of miRNA-lncRNA sponging. Results We firstly analyzed five available RNA-seqs datasets to investigate aberrantly expressed lncRNAs which might associate with inflammation or oxidative stress. We identified that two lncRNAs, H19 and HULC, were differentially expressed among all the samples under the treatment of hypoxic or inflammatory factors, and they were shown to be stimulated by short-term oxidative stress responses to H2O2 and glucose oxidase in CCA cell lines. Further studies revealed that these two lncRNAs promoted cholangiocyte migration and invasion via the inflammation pathway. H19 and HULC functioned as competing endogenous RNAs (ceRNAs by sponging let-7a/let-7b and miR-372/miR-373, respectively, which activate pivotal inflammation cytokine IL-6 and chemokine receptor CXCR4. Conclusions Our study revealed that H19 and HULC, up-regulated by oxidative stress, regulate CCA cell migration and invasion by targeting IL-6 and CXCR4 via ceRNA patterns of sponging let-7a/let-7b and miR-372/miR-373, respectively. The results suggest that these lncRNAs might be the chief culprits of CCA pathogenesis and progression. The study provides new insight into the mechanism linking lncRNA function with CCA and

  5. Do microRNAs induced by Viral Hemorrhagic Septicemia virus in rainbow trout (Oncorhynchus mykiss) possess anti-viral activity?

    DEFF Research Database (Denmark)

    Bela-Ong, Dennis; Schyth, Brian Dall; Lorenzen, Niels

    2013-01-01

    Microribonucleic acids (miRNAs) are small (18-22 nucleotides) endogenous RNAs that potently regulate the deadenylation, translation, and decay of a wide spectrum of target mRNAs. Their discovery adds a new layer to the mechanisms of control of gene expression, impacting a broad range of biological......RNAs were also up regulated in the liver and muscle (vaccination site) of fish vaccinated with a DNA vaccine expressing the VHSV glycoprotein gene. Recent studies further indicate that the expression of these miRNAs is induced by interferons. In order to analyze if miRNA-462 and miRNA-731 have any anti......-viral effects, we designed inhibitory synthetic oligonucleotides called antagomiRs or anti-miRNAs. These saline-formulated 2’-O-methylated Locked Nucleic Acid (LNA)-based antagomiRs were injected intraperitoneally into rainbow trout fingerlings followed by exposure of the fish to VHSV. Development of disease...

  6. Frontotemporal Lobar Degeneration and microRNAs

    Directory of Open Access Journals (Sweden)

    Paola ePiscopo

    2016-02-01

    Full Text Available Frontotemporal lobar degeneration (FTLD includes a spectrum of disorders characterized by changes of personality and social behaviour and, often, a gradual and progressive language dysfunction. In the last years, several efforts have been fulfilled in identifying both genetic mutations and pathological proteins associated with FTLD. The molecular bases undergoing the onset and progression of the disease remain still unknown. Recent literature prompts an involvement of RNA metabolism in FTLD, particularly miRNAs. Dysregulation of miRNAs in several disorders, including neurodegenerative diseases, and increasing importance of circulating miRNAs in different pathologies has suggested to implement the study of their possible application as biological markers and new therapeutic targets; moreover, miRNA-based therapy is becoming a powerful tool to deepen the function of a gene, the mechanism of a disease, and validate therapeutic targets. Regarding FTLD, different studies showed that miRNAs are playing an important role. For example, several reports have evaluated miRNA regulation of the progranulin gene suggesting that it is under their control, as described for miR-29b, miR-107 and miR-659. More recently, it has been demonstrated that TMEM106B gene, which protein is elevated in FTLD-TDP brains, is repressed by miR-132/212 cluster; this post-transcriptional mechanism increases intracellular levels of progranulin, affecting its pathways. These findings if confirmed could suggest that these microRNAs have a role as potential targets for some related-FTLD genes. In this review, we focus on the emerging roles of the miRNAs in the pathogenesis of FTLD.

  7. Quantitative aspects of gene regulation by small RNAs

    Science.gov (United States)

    Mehta, Pankaj

    2007-03-01

    Small, non-coding RNAs (sRNAs) play an important role as genetic regulators in both prokaryotes and eukaryotes. Many sRNAs act through base-pairing interaction with target messenger RNAs (mRNAs) to regulate transcription, translation, and mRNA stability. sRNAs represent a novel form of genetic regulation distinct from more thoroughly studied protein regulators. This talk addresses quantitative aspectsof sRNA-mediated genetic regulation, focusing on noise, tunability, and feedback. In particular, we compare and contrast sRNA and protein regulators in an attempt to understand the compartive advantages of each form of regulation.

  8. Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis

    DEFF Research Database (Denmark)

    Pauli, Andrea; Valen, Eivind; Lin, Michael F.;

    2012-01-01

    vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time series of RNA-Seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast...... overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to introns. Subsets of lncRNAs carry chromatin signatures characteristic...

  9. Micromanagement of the immune system by microRNAs.

    Science.gov (United States)

    Lodish, Harvey F; Zhou, Beiyan; Liu, Gwen; Chen, Chang-Zheng

    2008-02-01

    MicroRNAs (miRNAs) are an abundant class of evolutionarily conserved small non-coding RNAs that are thought to control gene expression by targeting mRNAs for degradation or translational repression. Emerging evidence suggests that miRNA-mediated gene regulation represents a fundamental layer of genetic programmes at the post-transcriptional level and has diverse functional roles in animals. Here, we provide an overview of the mechanisms by which miRNAs regulate gene expression, with specific focus on the role of miRNAs in regulating the development of immune cells and in modulating innate and adaptive immune responses.

  10. Research of the relationship between radiotherapy and microRNAs

    Institute of Scientific and Technical Information of China (English)

    Jiao Yang; Ximing Xu; Yanrong Hao

    2012-01-01

    MicroRNAs (miRNAs) are endogenous short non-coding RNAs, and play a pivotal role in regulating a variety of cellular processes, including proliferation and apoptosis, both of which are cellular responses to radiation treatment. In response to radiation, multiple miRNAs show altered expression, which act as oncogenes or tumor suppressors. Recent evidence has also shown that some miRNAs have radiotherapy sensitization or radiation resistance role in malignant tumors. This review focuses on analysis of these characteristics and mechanisms of miRNAs, and will provide some insight into the therapeutic application of radiation.

  11. Small RNAs controlling outer membrane porins

    DEFF Research Database (Denmark)

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

    2007-01-01

    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...... 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....

  12. Transcription start site associated RNAs (TSSaRNAs are ubiquitous in all domains of life.

    Directory of Open Access Journals (Sweden)

    Livia S Zaramela

    Full Text Available A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq and a primary-transcript enriched (dRNA-seq strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome. Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

  13. Long non coding RNAs (lncRNAs are dysregulated in Malignant Pleural Mesothelioma (MPM.

    Directory of Open Access Journals (Sweden)

    Casey M Wright

    Full Text Available Malignant Pleural Mesothelioma (MPM is an aggressive cancer that is often diagnosed at an advanced stage and is characterized by a long latency period (20-40 years between initial exposure and diagnosis and prior exposure to asbestos. Currently accurate diagnosis of MPM is difficult due to the lack of sensitive biomarkers and despite minor improvements in treatment, median survival rates do not exceed 12 months. Accumulating evidence suggests that aberrant expression of long non-coding RNAs (lncRNAs play an important functional role in cancer biology. LncRNAs are a class of recently discovered non-protein coding RNAs >200 nucleotides in length with a role in regulating transcription. Here we used NCode long noncoding microarrays to identify differentially expressed lncRNAs potentially involved in MPM pathogenesis. High priority candidate lncRNAs were selected on the basis of statistical (P3-fold difference. Expression levels of 9 candidate lncRNAs were technically validated using RT-qPCR, and biologically validated in three independent test sets: (1 57 archived MPM tissues obtained from extrapleural pneumonectomy patients, (2 15 cryopreserved MPM and 3 benign pleura, and (3 an extended panel of 10 MPM cell lines. RT-qPCR analysis demonstrated consistent up-regulation of these lncRNAs in independent datasets. ROC curve analysis showed that two candidates were able to separate benign pleura and MPM with high sensitivity and specificity, and were associated with nodal metastases and survival following induction chemotherapy. These results suggest that lncRNAs have potential to serve as biomarkers in MPM.

  14. Identification of CRISPR and riboswitch related RNAs among novel noncoding RNAs of the euryarchaeon Pyrococcus abyssi

    Directory of Open Access Journals (Sweden)

    Carpousis Agamemnon J

    2011-06-01

    Full Text Available Abstract Background Noncoding RNA (ncRNA has been recognized as an important regulator of gene expression networks in Bacteria and Eucaryota. Little is known about ncRNA in thermococcal archaea except for the eukaryotic-like C/D and H/ACA modification guide RNAs. Results Using a combination of in silico and experimental approaches, we identified and characterized novel P. abyssi ncRNAs transcribed from 12 intergenic regions, ten of which are conserved throughout the Thermococcales. Several of them accumulate in the late-exponential phase of growth. Analysis of the genomic context and sequence conservation amongst related thermococcal species revealed two novel P. abyssi ncRNA families. The CRISPR family is comprised of crRNAs expressed from two of the four P. abyssi CRISPR cassettes. The 5'UTR derived family includes four conserved ncRNAs, two of which have features similar to known bacterial riboswitches. Several of the novel ncRNAs have sequence similarities to orphan OrfB transposase elements. Based on RNA secondary structure predictions and experimental results, we show that three of the twelve ncRNAs include Kink-turn RNA motifs, arguing for a biological role of these ncRNAs in the cell. Furthermore, our results show that several of the ncRNAs are subjected to processing events by enzymes that remain to be identified and characterized. Conclusions This work proposes a revised annotation of CRISPR loci in P. abyssi and expands our knowledge of ncRNAs in the Thermococcales, thus providing a starting point for studies needed to elucidate their biological function.

  15. Screening candidate microRNAs (miRNAs) in different lambskin hair follicles in Hu sheep.

    Science.gov (United States)

    Gao, Wen; Sun, Wei; Yin, Jinfeng; Lv, Xiaoyang; Bao, Jianjun; Yu, Jiarui; Wang, Lihong; Jin, Chengyan; Hu, Liang

    2017-01-01

    Hu sheep lambskin is a unique white lambskin from China that exhibits three types of flower patterns, including small waves, medium waves, and large waves, with small waves considered the best quality. However, our understanding of the molecular mechanism underlying flower pattern formation in Hu sheep lambskin is limited. The aim of the present study was to further explore the relevance between candidate microRNAs (miRNAs) and developmental characteristics of hair follicles and screen miRNAs for later functional validation. Herein, we employed Illumina Hiseq 2500 to identify differentially expressed miRNAs in hair follicles of different flower patterns with small, medium, and large waves to construct a comprehensive sequence database on the mechanism of hair follicle development. Paraffin sections of lambskin tissue were prepared to assess the structure of different hair follicles. Expression levels of candidate miRNAs in different flower patterns were analyzed by relative quantitation using real-time PCR, combined with histological observation and micro-observation technologies, and the correlation between expression levels of candidate miRNAs and histological properties of hair follicles was analyzed by using SPSS 17.0. A total of 522 differentially expressed miRNAs were identified, and RNA-seq analysis detected 7,266 target genes in different groups of flower patterns. Gene ontological analysis indicated these target genes were mainly involved in cell proliferation, differentiation, growth, apoptosis, and ion transport, and 14 miRNAs, including miR-143, miR-10a, and let-7 were screened as candidate miRNAs in Hu sheep hair follicle growth and development. In the same field of vision, variance analysis showed that the number of secondary follicles in small waves was significantly larger than that in large and medium waves (Phair follicles, highly significant differences in miRNA-143 expression levels between large and small waves were observed (Phair follicle

  16. Functional profiling of precursor MicroRNAs identifies MicroRNAs essential for glioma proliferation.

    Directory of Open Access Journals (Sweden)

    Saija Haapa-Paananen

    Full Text Available Cancer initiation and progression involve microRNAs that can function like tumor suppressors and oncogenes. The functional significance of most miRNAs is currently unknown. To determine systematically which microRNAs are essential for glioma growth, we screened a precursor microRNA library in three human glioblastoma and one astroglial cell line model systems. The most prominent and consistent cell proliferation-reducing hits were validated in secondary screening with an additional apoptosis endpoint. The functional screening data were integrated in the miRNA expression data to find underexpressed true functional tumor suppressor miRNAs. In addition, we used miRNA-target gene predictions and combined siRNA functional screening data to find the most probable miRNA-target gene pairs with a similar functional effect on proliferation. Nine novel functional miRNAs (hsa-miR-129, -136, -145, -155, -181b, -342-5p, -342-3p, -376a/b in GBM cell lines were validated for their importance in glioma cell growth, and similar effects for six target genes (ROCK1, RHOA, MET, CSF1R, EIF2AK1, FGF7 of these miRNAs were shown functionally. The clinical significance of the functional hits was validated in miRNA expression data from the TCGA glioblastoma multiforme (GBM tumor cohort. Five tumor suppressor miRNAs (hsa-miR-136, -145, -342, -129, -376a showed significant underexpression in clinical GBM tumor samples from the TCGA GBM cohort further supporting the role of these miRNAs in vivo. Most importantly, higher hsa-miR-145 expression in GBM tumors yielded significantly better survival (p<0.005 in a subset of patients thus validating it as a genuine tumor suppressor miRNA. This systematic functional profiling provides important new knowledge about functionally relevant miRNAs in GBM biology and may offer new targets for treating glioma.

  17. MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant

    Science.gov (United States)

    Lu, Cheng; Kulkarni, Karthik; Souret, Frédéric F.; MuthuValliappan, Ramesh; Tej, Shivakundan Singh; Poethig, R. Scott; Henderson, Ian R.; Jacobsen, Steven E.; Wang, Wenzhong; Green, Pamela J.; Meyers, Blake C.

    2006-01-01

    The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is ∼155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1–7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs. PMID:16954541

  18. Detection of piRNAs in whitespotted bamboo shark liver.

    Science.gov (United States)

    Yang, Lingrong; Ge, Yinghua; Cheng, Dandan; Nie, Zuoming; Lv, Zhengbing

    2016-09-15

    Piwi-interacting RNAs (piRNAs) are 26 to 31-nt small non-coding RNAs that have been reported mostly in germ-line cells and cancer cells. However, the presence of piRNAs in the whitespotted bamboo shark liver has not yet been reported. In a previous study of microRNAs in shark liver, some piRNAs were detected from small RNAs sequenced by Solexa technology. A total of 4857 piRNAs were predicted and found in shark liver. We further selected 17 piRNAs with high and significantly differential expression between normal and regenerative liver tissues for subsequent verification by Northern blotting. Ten piRNAs were further identified, and six of these were matched to known piRNAs in piRNABank. The actual expression of six known and four novel piRNAs was validated by qRT-PCR. In addition, a total of 401 target genes of the 10 piRNAs were predicted by miRanda. Through GO and pathway function analyses, only five piRNAs could be annotated with eighteen GO annotations. The results indicated that the identified piRNAs are involved in many important biological responses, including immune inflammation, cell-specific differentiation and development, and angiogenesis. This manuscript provides the first identification of piRNAs in the liver of whitespotted bamboo shark using Solexa technology as well as further elucidation of the regulatory role of piRNAs in whitespotted bamboo shark liver. These findings may provide a useful resource and may facilitate the development of therapeutic strategies against liver damage.

  19. Reduced expression of brain-enriched microRNAs in glioblastomas permits targeted regulation of a cell death gene.

    Directory of Open Access Journals (Sweden)

    Rebecca L Skalsky

    Full Text Available Glioblastoma is a highly aggressive malignant tumor involving glial cells in the human brain. We used high-throughput sequencing to comprehensively profile the small RNAs expressed in glioblastoma and non-tumor brain tissues. MicroRNAs (miRNAs made up the large majority of small RNAs, and we identified over 400 different cellular pre-miRNAs. No known viral miRNAs were detected in any of the samples analyzed. Cluster analysis revealed several miRNAs that were significantly down-regulated in glioblastomas, including miR-128, miR-124, miR-7, miR-139, miR-95, and miR-873. Post-transcriptional editing was observed for several miRNAs, including the miR-376 family, miR-411, miR-381, and miR-379. Using the deep sequencing information, we designed a lentiviral vector expressing a cell suicide gene, the herpes simplex virus thymidine kinase (HSV-TK gene, under the regulation of a miRNA, miR-128, that was found to be enriched in non-tumor brain tissue yet down-regulated in glioblastomas, Glioblastoma cells transduced with this vector were selectively killed when cultured in the presence of ganciclovir. Using an in vitro model to recapitulate expression of brain-enriched miRNAs, we demonstrated that neuronally differentiated SH-SY5Y cells transduced with the miRNA-regulated HSV-TK vector are protected from killing by expression of endogenous miR-128. Together, these results provide an in-depth analysis of miRNA dysregulation in glioblastoma and demonstrate the potential utility of these data in the design of miRNA-regulated therapies for the treatment of brain cancers.

  20. Competition at the Wireless Sensor Network MAC Layer: Low Power Probing interfering with X-MAC

    Science.gov (United States)

    Zacharias, Sven; Newe, Thomas

    2011-08-01

    Wireless Sensor Networks (WSNs) combine sensors with computer networks and enable very dense, in-situ and live measurements of data over a large area. Since this emerging technology has the potential to be embedded almost everywhere for numberless applications, interference between different networks can become a serious issue. For most WSNs, it is assumed today that the network medium access is non-competitive. On the basis of X-MAC interfered by Low Power Probing, this paper shows the danger and the effects of different sensor networks communicating on a single wireless channel of the 2.4 GHz band, which is used by the IEEE 802.15.4 standard.

  1. Heterophilic antibodies interfering with radioimmunoassay. A false-positive pregnancy test

    Energy Technology Data Exchange (ETDEWEB)

    Vladutiu, A.O.; Sulewski, J.M.; Pudlak, K.A.; Stull, C.G.

    1982-11-19

    A young woman with amenorrhea had a consistently positive pregnancy test result (serum radioimmunoassay measurement of ..beta..-human chorionic gonadotropin hormone). No fetal or placental tissue was found after uterine curettage and exploratory laparotomy. The false-positive pregnancy test result was due to heterophilic antibovine and antigoat antibodies in the patient's serum. These antibodies interfered with radioimmunoassays using goat antibodies. This case shows that serum heterophilic antibodies can interfere with immunoassays and result in unnecessary diagnostic procedures and/or unnecessary treatment.

  2. Two-Pronged Approach to Overcome Spectroscopically Interfering Organic Compounds with Isotopic Water Analysis

    Science.gov (United States)

    Saad, Nabil; Hsiao, Gregor; Chapellet-Volpini, London; Vu, Danthu

    2013-04-01

    The ability to measure the stable isotopes of hydrogen (dD) and oxygen (d18O) has become much more accessible with the advent of Cavity Ring-Down Spectroscopy (CRDS) laser optical devices. These small and inexpensive analyzers have led to a significant increase in the acquisition of data from a variety of studies in the fields of groundwater, watershed, and other water source applications. However for some samples, such as those linked to fracking, mining, and other activities where higher than normal concentrations of organic materials are to be found, optical spectroscopy may require an adaptation from current methodologies in order to ensure data confidence. That is because CRDS is able to measure all the components within a spectral region - which will include the spectral characteristics of the isotopologues of water as well as the available features from interfering organic molecules. Although, at the first level, the information from the organic material provides spectral overlaps that can perturb the isotopic ratios, a more thorough review shows that these features are a source of information that will be inherently useful. This presentation will examine the approaches developed within the past year to allow for more accurate analyses of such samples by optical methods. The first approach uses an advanced spectroscopic model to flag the presence of organic material in the sample. Signals from known interfering compounds (i.e., alcohols, ketones, aldehydes, short-chain hydrocarbons, etc.) are incorporated into the overall fit of the measured spectra used to calculate the concentration of the individual isotopes. The second approach uses physical treatment of the sample to break down the organic molecules into non-interfering species. The vaporized liquid or solid sample travels through a cartridge packed with an oxidation catalyst. The interfering organic molecules will undergo high temperature oxidation using O2 present in the air carrier gas stream prior

  3. Modelo de interferência aerodinâmica entre jato e empenagem

    OpenAIRE

    Juan Bautista Villa Wanderley

    1990-01-01

    Neste trabalho apresenta-se uma metodologia para a estimativa dos efeitos de interferência aerodinâmica sobre empenagens devido a jatos turbulentos, subsônicos e aquecidos. Primeiramente emprega-se um método de diferenças finitas para resolver numericamente as equações de Reynolds com as aproximações de camada limite. As equações de reynolds são resolvidas simultaneamente com a equação diferencial parcial oriundado modelo (K) da energia cinética turbulenta desenvolvido por Prandtl. Desta form...

  4. Neutralization Interfering Antibodies: A “Novel” Example of Humoral Immune Dysfunction Facilitating Viral Escape?

    Directory of Open Access Journals (Sweden)

    Roberto Burioni

    2012-09-01

    Full Text Available The immune response against some viral pathogens, in particular those causing chronic infections, is often ineffective notwithstanding a robust humoral neutralizing response. Several evasion mechanisms capable of subverting the activity of neutralizing antibodies (nAbs have been described. Among them, the elicitation of non-neutralizing and interfering Abs has been hypothesized. Recently, this evasion mechanism has acquired an increasing interest given its possible impact on novel nAb-based antiviral therapeutic and prophylactic approaches. In this review, we illustrate the mechanisms of Ab-mediated interference and the viral pathogens described in literature as able to adopt this “novel” evasion strategy.

  5. Small RNAs tell big stories in Whistler.

    Science.gov (United States)

    Seila, Amy C; Sharp, Phillip A

    2008-06-01

    The Keystone Symposium on RNAi, microRNA and non-coding RNA convened on March 25-30 at Whistler Resort in Whistler, British Columbia, Canada. Researchers with backgrounds in different biochemical disciplines came together to exchange ideas on short RNAs and their roles in a host of biological processes.

  6. MicroRNAs in pancreatic ductal adenocarcinoma

    Institute of Scientific and Technical Information of China (English)

    Jong Y Park; James Helm; Domenico Coppola; Donghwa Kim; Mokenge Malafa; Seung Joon Kim

    2011-01-01

    Ductal adenocarcinoma of the pancreas is a lethal cancer for which the only chance of long-term survival belongs to the patient with localized disease in whom a potentially curative resection can be done.Therefore,biomarkers for early detection and new therapeutic strategies are urgently needed.miRNAs are a recently discovered class of small endogenous non-coding RNAs of about 22 nucleotides that have gained attention for their role in downregulation of mRNA expression at the posttranscriptional level.miRNAs regulate proteins involved in critical cellular processes such as differentiation,proliferation,and apoptosis.Evidence suggests that deregulated miRNA expression is involved in carcinogenesis at many sites,including the pancreas.Aberrant expression of miRNAs may upregulate the expression of oncogenes or downregulate the expression of tumor suppressor genes,as well as play a role in other mechanisms of carcinogenesis.The purpose of this review is to summarize our knowledge of deregulated miRNA expression in pancreatic cancer and discuss the implication for potential translation of this knowledge into clinical practice.

  7. Short stories on zebrafish long noncoding RNAs.

    Science.gov (United States)

    Haque, Shadabul; Kaushik, Kriti; Leonard, Vincent Elvin; Kapoor, Shruti; Sivadas, Ambily; Joshi, Adita; Scaria, Vinod; Sivasubbu, Sridhar

    2014-12-01

    The recent re-annotation of the transcriptome of human and other model organisms, using next-generation sequencing approaches, has unravelled a hitherto unknown repertoire of transcripts that do not have a potential to code for proteins. These transcripts have been largely classified into an amorphous class popularly known as long noncoding RNAs (lncRNA). This discovery of lncRNAs in human and other model systems have added a new layer to the understanding of gene regulation at the transcriptional and post-transcriptional levels. In recent years, three independent studies have discovered a number of lncRNAs expressed in different stages of zebrafish development and adult tissues using a high-throughput RNA sequencing approach, significantly adding to the repertoire of genes known in zebrafish. A subset of these transcripts also shows distinct and specific spatiotemporal patterns of gene expression, pointing to a tight regulatory control and potential functional roles in development, organogenesis, and/ or homeostasis. This review provides an overview of the lncRNAs in zebrafish and discusses how their discovery could provide new insights into understanding biology, explaining mutant phenotypes, and helping in potentially modeling disease processes.

  8. Staufen2 Regulates Neuronal Target RNAs

    Directory of Open Access Journals (Sweden)

    Jacki E. Heraud-Farlow

    2013-12-01

    Full Text Available RNA-binding proteins play crucial roles in directing RNA translation to neuronal synapses. Staufen2 (Stau2 has been implicated in both dendritic RNA localization and synaptic plasticity in mammalian neurons. Here, we report the identification of functionally relevant Stau2 target mRNAs in neurons. The majority of Stau2-copurifying mRNAs expressed in the hippocampus are present in neuronal processes, further implicating Stau2 in dendritic mRNA regulation. Stau2 targets are enriched for secondary structures similar to those identified in the 3′ UTRs of Drosophila Staufen targets. Next, we show that Stau2 regulates steady-state levels of many neuronal RNAs and that its targets are predominantly downregulated in Stau2-deficient neurons. Detailed analysis confirms that Stau2 stabilizes the expression of one synaptic signaling component, the regulator of G protein signaling 4 (Rgs4 mRNA, via its 3′ UTR. This study defines the global impact of Stau2 on mRNAs in neurons, revealing a role in stabilization of the levels of synaptic targets.

  9. MicroRNAs horizon in retinoblastoma.

    Directory of Open Access Journals (Sweden)

    Mojgan Mirakholi

    2013-12-01

    Full Text Available In the retinoblastoma research, it is of great interest to identify molecular markers associated with the genetics of tumorigenesis. microRNAs (miRNAs are small non-coding RNA molecules that play a regulatory role in many crucial cellular pathways such as differentiation, cell cycle progression, and apoptosis. A body of evidences showed dysregulation of miRNAs in tumor biology and many diseases. They potentially play a significant role in tumorigenesis processes and have been the subject of research in many types of cancers including retinal tumorigenesis. miRNA expression profiling was found to be associated with tumor development, progression and treatment. These associations demonstrate the putative applications of miRNAs in monitoring of different aspect of tumors consisting diagnostic, prognostic and therapeutic. Herein, we review the current literature concerning to the study of miRNA target recognition, function to tumorigenesis and treatment in retinoblastoma. Identification the specific miRNA biomarkers associated with retinoblastoma cancer may help to establish new therapeutic approaches for salvage affected eyes in patients.

  10. Circulating microRNAs in Cardiovascular Diseases.

    Science.gov (United States)

    Orlicka-Płocka, Marta; Gurda, Dorota; Fedoruk-Wyszomirska, Agnieszka; Smolarek, Iwona; Wyszko, Eliza

    2016-01-01

    Cardiovascular Diseases (CD) are currently one of the most common causes of death. Because heart related deaths occur on such an enormous scale this phenomenon is referred to as an epidemic. Chronic and acute injury of the heart could be an effect of cardiac remodeling, which is a result of molecular, cellular and interstitial changes, influenced by hemodynamic load or neurohormonal activation (Cohn et al., 2000). These small deviations in cardiac activity and morphology may lead to an enormous negative effect. Despite a significant progress, knowledge of standard risk factors for cardiovascular diseases has become less and less effective, which is why predicting and seeking an appropriate treatment is very challenging. As a result, there is a growing interest in finding new markers of the CD. MicroRNAs (miRNAs), are short, non-coding RNAs responsible for regulation of gene expression at the post-transcriptional level. Among them that have the greatest potential are microRNA molecules that circulate in the blood plasma or serum, that are related to direct activation of signaling pathways, implicated in the aging process and thus for the development of cardiovascular disease. This paper is a summary of the current state of knowledge on miRNAs, their biogenesis and potential role as biomarkers to diagnose heart disease.

  11. Small-interfering RNA (siRNA)-based functional micro- and nanostructures for efficient and selective gene silencing.

    Science.gov (United States)

    Lee, Soo Hyeon; Chung, Bong Hyun; Park, Tae Gwan; Nam, Yoon Sung; Mok, Hyejung

    2012-07-17

    Because of RNA's ability to encode structure and functional information, researchers have fabricated diverse geometric structures from this polymer at the micro- and nanoscale. With their tunable structures, rigidity, and biocompatibility, novel two-dimensional and three-dimensional RNA structures can serve as a fundamental platform for biomedical applications, including engineered tissues, biosensors, and drug delivery vehicles. The discovery of the potential of small-interfering RNA (siRNA) has underscored the applications of RNA-based micro- and nanostructures in medicine. Small-interfering RNA (siRNA), synthetic double-stranded RNA consisting of approximately 21 base pairs, suppresses problematic target genes in a sequence-specific manner via inherent RNA interference (RNAi) processing. As a result, siRNA offers a potential strategy for treatment of many human diseases. However, due to inefficient delivery to cells and off-target effects, the clinical application of therapeutic siRNA has been very challenging. To address these issues, researchers have studied a variety of nanocarrier systems for siRNA delivery. In this Account, we describe several strategies for efficient siRNA delivery and selective gene silencing. We took advantage of facile chemical conjugation and complementary hybridization to design novel siRNA-based micro- and nanostructures. Using chemical crosslinkers and hydrophobic/hydrophilic polymers at the end of siRNA, we produced various RNA-based structures, including siRNA block copolymers, micelles, linear siRNA homopolymers, and microhydrogels. Because of their increased charge density and flexibility compared with conventional siRNA, these micro- and nanostructures can form polyelectrolyte complexes with poorly charged and biocompatible cationic carriers that are both more condensed and more homogenous than the complexes formed in other carrier systems. In addition, the fabricated siRNA-based structures are linked by cleavable disulfide

  12. Identification of novel sRNAs in mycobacterial species.

    Directory of Open Access Journals (Sweden)

    Chen-Hsun Tsai

    Full Text Available Bacterial small RNAs (sRNAs are short transcripts that typically do not encode proteins and often act as regulators of gene expression through a variety of mechanisms. Regulatory sRNAs have been identified in many species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Here, we use a computational algorithm to predict sRNA candidates in the mycobacterial species M. smegmatis and M. bovis BCG and confirmed the expression of many sRNAs using Northern blotting. Thus, we have identified 17 and 23 novel sRNAs in M. smegmatis and M. bovis BCG, respectively. We have also applied a high-throughput technique (Deep-RACE to map the 5' and 3' ends of many of these sRNAs and identified potential regulators of sRNAs by analysis of existing ChIP-seq datasets. The sRNAs identified in this work likely contribute to the unique biology of mycobacteria.

  13. The mitochondrial genome encodes abundant small noncoding RNAs

    Institute of Scientific and Technical Information of China (English)

    Seungil Ro; Hsiu-Yen Ma; Chanjae Park; Nicole Ortogero; Rui Song; Grant W Hennig; Huili Zheng

    2013-01-01

    Small noncoding RNAs identified thus far are all encoded by the nuclear genome.Here,we report that the murine and human mitochondriai genomes encode thousands of small noncoding RNAs,which are predominantly derived from the sense transcripts of the mitochondrial genes (host genes),and we termed these small RNAs mitochondrial genome-encoded small RNAs (mitosRNAs).DICER inactivation affected,but did not completely abolish mitosRNA production.MitosRNAs appear to be products of currently unidentified mitochondrial ribonucleases.Overexpression of mitosRNAs enhanced expression levels of their host genes in vitro,and dysregulated mitosRNA expression was generally associated with aberrant mitochondrial gene expression in vivo.Our data demonstrate that in addition to 37 known mitochondrial genes,the mammalian mitochondrial genome also encodes abundant mitosRNAs,which may play an important regulatory role in the control of mitochondrial gene expression in the cell.

  14. Long Non-coding RNAs In Cancer Progression

    Directory of Open Access Journals (Sweden)

    Keiko eTano

    2012-10-01

    Full Text Available Recent large-scale transcriptome analyses have revealed that transcription is spread throughout the mammalian genomes, yielding large numbers of transcripts, including long non-coding (lnc RNAs with little or no protein-coding capacity. Dozens of lncRNAs have been identified as biologically significant. In many cases, lncRNAs act as key molecules in the regulation of processes such as chromatin remodeling, transcription and post-transcriptional processing. Several lncRNAs (e.g., MALAT1, HOTAIR and ANRIL are associated with human diseases, including cancer. Those lncRNAs associated with cancer are often aberrantly expressed. Although the underlying molecular mechanisms by which lncRNAs regulate cancer development are unclear, recent studies have revealed that such aberrant expression of lncRNAs affects the progression of cancers. In this review, we highlight recent findings regarding the roles of lncRNAs in cancer biology.

  15. Could MicroRNAs be Regulators of Gout Pathogenesis?

    Directory of Open Access Journals (Sweden)

    Yangang Wang

    2015-07-01

    Full Text Available MicroRNAs (miRNAs are a class of noncoding RNAs that mainly negatively regulate gene expression. miRNAs have important roles in many diseases, including inflammatory diseases. Gout is a common arthritis caused by deposition of monosodium urate crystals within joints. Recent studies suggested that miRNAs may be involved in the development of inflammatory arthritis, including acute gouty arthritis. In the present review, we systemically discuss relevant publications in order to provide a better understanding on the possible role of miRNAs in gout. miRNAs may act as regulators of gout pathogenesis via several pathways. Targeting miRNAs may be a promisingstrategy in the treatment of gout.

  16. Rewiring two-component signal transduction with small RNAs.

    Science.gov (United States)

    Göpel, Yvonne; Görke, Boris

    2012-04-01

    Bacterial two-component systems (TCSs) and small regulatory RNAs (sRNAs) form densely interconnected networks that integrate and transduce information from the environment into fine-tuned changes of gene expression. Many TCSs control target genes indirectly through regulation of sRNAs, which in turn regulate gene expression by base-pairing with mRNAs or targeting a protein. Conversely, sRNAs may control TCS synthesis, thereby recruiting the TCS regulon to other regulatory networks. Several TCSs control expression of multiple homologous sRNAs providing the regulatory networks with further flexibility. These sRNAs act redundantly, additively or hierarchically on targets. The regulatory speed of sRNAs and their unique features in gene regulation make them ideal players extending the flexibility, dynamic range or timing of TCS signaling. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Long Non-coding RNAs in the Cytoplasm

    Institute of Scientific and Technical Information of China (English)

    Farooq Rashid; Abdullah Shah; Ge Shan

    2016-01-01

    An enormous amount of long non-coding RNAs (lncRNAs) transcribed from eukaryotic genome are important regulators in different aspects of cellular events. Cytoplasm is the residence and the site of action for many lncRNAs. The cytoplasmic lncRNAs play indispensable roles with multiple molecular mechanisms in animal and human cells. In this review, we mainly talk about functions and the underlying mechanisms of lncRNAs in the cytoplasm. We highlight relatively well-studied examples of cytoplasmic lncRNAs for their roles in modulating mRNA stability, regulating mRNA translation, serving as competing endogenous RNAs, functioning as precursors of microRNAs, and mediating protein modifications. We also elaborate the perspectives of cytoplasmic lncRNA studies.

  18. Identification of Cassava MicroRNAs under Abiotic Stress

    National Research Council Canada - National Science Library

    Ballén-Taborda, Carolina; Plata, Germán; Ayling, Sarah; Rodríguez-Zapata, Fausto; Becerra Lopez-Lavalle, Luis Augusto; Duitama, Jorge; Tohme, Joe

    2013-01-01

      The study of microRNAs (miRNAs) in plants has gained significant attention in recent years due to their regulatory role during development and in response to biotic and abiotic stresses. Although cassava...

  19. Dos nuevos microRNAs (miRNAs) identificados en ovario bovino

    OpenAIRE

    LA Muñoz-Bañales; Sánchez-Ramírez, B.; E. González-Rodríguez; Moreno-Brito, V; C González-Horta; ME Burrola-Barraza

    2014-01-01

    Para entender los mecanismos de maduración, fertilización y desarrollo embrionario en el ganado bovino, es necesario la identificación y caracterización de microRNAs (miRNAs) involucrados en la regulación del crecimiento del ovocito y la transición ovocito-embrión. Con el objetivo de identificar nuevos miRNAs, con una posible función dentro del proceso de maduración del ovocito y de embriogénesis temprana, se realizó una librería genómica con miRNAs provenientes de ovarios de vacas de rastro....

  20. Differential expression of microRNAs with progression of gestation and inflammation in the human chorioamniotic membranes

    Science.gov (United States)

    Montenegro, Daniel; Romero, Roberto; Pineles, Beth L.; Tarca, Adi L.; Kim, Yeon Mee; Draghici, Sorin; Kusanovic, Juan Pedro; Kim, Jung-Sun; Erez, Offer; Mazaki-Tovi, Shali; Hassan, Sonia; Espinoza, Jimmy; Kim, Chong Jai

    2010-01-01

    Objective The aim of this study was to identify differential expression of microRNAs (miRNAs) in chorioamniotic membranes with advancing gestation, labor, and inflammation. Study design Expression profiles of 157 miRNAs in the chorioamniotic membranes were obtained from patients in the following groups: 1) term not in labor (n=10); 2) term in labor (n=10); 3) preterm labor with histologic chorioamnionitis (n=9); and 4) without histologic chorioamnionitis (n=10). Results More than 95% of the miRNAs screened were expressed. Gestational age-dependent changes in expression were observed for 13 miRNAs. No differences in miRNA expression were observed between women without labor and women in labor. Membranes with chorioamnionitis displayed increased expression of miR-223 and miR-338. Gene Ontology analysis of genes targeted by differentially expressed miRNAs revealed enrichment for specific biological process categories. Conclusion Chorioamniotic membranes with advancing gestational age and chorioamnionitis are associated with the differential expression of a subset of miRNAs. PMID:17826424

  1. Identification of Novel and Conserved microRNAs in Homalodisca vitripennis, the Glassy-Winged Sharpshooter by Expression Profiling.

    Directory of Open Access Journals (Sweden)

    Raja Sekhar Nandety

    Full Text Available The glassy-winged sharpshooter (GWSS Homalodisca vitripennis (Hemiptera: Cicadellidae, is a xylem-feeding leafhopper and an important vector of the bacterium Xylella fastidiosa; the causal agent of Pierce's disease of grapevines. MicroRNAs are a class of small RNAs that play an important role in the functional development of various organisms including insects. In H. vitripennis, we identified microRNAs using high-throughput deep sequencing of adults followed by computational and manual annotation. A total of 14 novel microRNAs that are not found in the miRBase were identified from adult H. vitripennis. Conserved microRNAs were also found in our datasets. By comparison to our previously determined transcriptome sequence of H. vitripennis, we identified the potential targets of the microRNAs in the transcriptome. This microRNA profile information not only provides a more nuanced understanding of the biological and physiological mechanisms that govern gene expression in H. vitripennis, but may also lead to the identification of novel mechanisms for biorationally designed management strategies through the use of microRNAs.

  2. MicroRNAs and drug addiction

    Directory of Open Access Journals (Sweden)

    Paul J Kenny

    2013-05-01

    Full Text Available Drug addiction is considered a disorder of neuroplasticity in brain reward and cognition systems resulting from aberrant activation of gene expression programs in response to prolonged drug consumption. Noncoding RNAs are key regulators of almost all aspects of cellular physiology. MicroRNAs (miRNAs are small (~21–23 nucleotides noncoding RNA transcripts that regulate gene expression at the post-transcriptional level. Recently, microRNAs were shown to play key roles in the drug-induced remodeling of brain reward systems that likely drives the emergence of addiction. Here, we review evidence suggesting that one particular miRNA, miR-212, plays a particularly prominent role in vulnerability to cocaine addiction. We review evidence showing that miR-212 expression is increased in the dorsal striatum of rats that show compulsive-like cocaine-taking behaviors. Increases in miR-212 expression appear to protect against cocaine addiction, as virus-mediated striatal miR-212 over-expression decreases cocaine consumption in rats. Conversely, disruption of striatal miR-212 signaling using an antisense oligonucleotide increases cocaine intake. We also review data that identify two mechanisms by which miR-212 may regulate cocaine intake. First, miR-212 has been shown to amplify striatal CREB signaling through a mechanism involving activation of Raf1 kinase. Second, miR-212 was also shown to regulate cocaine intake by repressing striatal expression of methyl CpG binding protein 2 (MeCP2, consequently decreasing protein levels of brain-derived neurotrophic factor (BDNF. The concerted actions of miR-212 on striatal CREB and MeCP2/BDNF activity greatly attenuate the motivational effects of cocaine. These findings highlight the unique role for miRNAs in simultaneously controlling multiple signaling cascades implicated in addiction.

  3. Decoding the function of nuclear long non-coding RNAs.

    Science.gov (United States)

    Chen, Ling-Ling; Carmichael, Gordon G

    2010-06-01

    Long non-coding RNAs (lncRNAs) are mRNA-like, non-protein-coding RNAs that are pervasively transcribed throughout eukaryotic genomes. Rather than silently accumulating in the nucleus, many of these are now known or suspected to play important roles in nuclear architecture or in the regulation of gene expression. In this review, we highlight some recent progress in how lncRNAs regulate these important nuclear processes at the molecular level. Copyright 2010 Elsevier Ltd. All rights reserved.

  4. Utility of MicroRNAs and siRNAs in Cervical Carcinogenesis

    Science.gov (United States)

    Díaz-González, Sacnite del Mar; Benítez-Boijseauneau, Odelia; Gómez-Cerón, Claudia; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Peralta-Zaragoza, Oscar

    2015-01-01

    MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3′-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer. PMID:25874209

  5. Current Insights into Long Non-Coding RNAs (LncRNAs) in Prostate Cancer

    Science.gov (United States)

    Smolle, Maria A.; Bauernhofer, Thomas; Pummer, Karl; Calin, George A.; Pichler, Martin

    2017-01-01

    The importance of long non-coding RNAs (lncRNAs) in the pathogenesis of various malignancies has been uncovered over the last few years. Their dysregulation often contributes to or is a result of tumour progression. In prostate cancer, the most common malignancy in men, lncRNAs can promote castration resistance, cell proliferation, invasion, and metastatic spread. Expression patterns of lncRNAs often change during tumour progression; their expression levels may constantly rise (e.g., HOX transcript antisense RNA, HOTAIR), or steadily decrease (e.g., downregulated RNA in cancer, DRAIC). In prostate cancer, lncRNAs likewise have diagnostic (e.g., prostate cancer antigen 3, PCA3), prognostic (e.g., second chromosome locus associated with prostate-1, SChLAP1), and predictive (e.g., metastasis-associated lung adenocarcinoma transcript-1, MALAT-1) functions. Considering their dynamic role in prostate cancer, lncRNAs may also serve as therapeutic targets, helping to prevent development of castration resistance, maintain stable disease, and prohibit metastatic spread. PMID:28241429

  6. Current Insights into Long Non-Coding RNAs (LncRNAs in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Maria A. Smolle

    2017-02-01

    Full Text Available The importance of long non-coding RNAs (lncRNAs in the pathogenesis of various malignancies has been uncovered over the last few years. Their dysregulation often contributes to or is a result of tumour progression. In prostate cancer, the most common malignancy in men, lncRNAs can promote castration resistance, cell proliferation, invasion, and metastatic spread. Expression patterns of lncRNAs often change during tumour progression; their expression levels may constantly rise (e.g., HOX transcript antisense RNA, HOTAIR, or steadily decrease (e.g., downregulated RNA in cancer, DRAIC. In prostate cancer, lncRNAs likewise have diagnostic (e.g., prostate cancer antigen 3, PCA3, prognostic (e.g., second chromosome locus associated with prostate-1, SChLAP1, and predictive (e.g., metastasis-associated lung adenocarcinoma transcript-1, MALAT-1 functions. Considering their dynamic role in prostate cancer, lncRNAs may also serve as therapeutic targets, helping to prevent development of castration resistance, maintain stable disease, and prohibit metastatic spread.

  7. Utility of MicroRNAs and siRNAs in Cervical Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Sacnite del Mar Díaz-González

    2015-01-01

    Full Text Available MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3′-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer.

  8. Circulating microRNAs as biomarkers of adult Crohn's disease

    DEFF Research Database (Denmark)

    Jensen, Michael D; Andersen, Rikke F; Christensen, Henry

    2015-01-01

    OBJECTIVE: Previous studies have found a differential expression of microRNAs (miRNAs) in the blood of patients with Crohn's disease (CD) compared with healthy controls. The aim of this study was to identify circulating miRNAs expressed in CD and assess their performance as biomarkers in patients...

  9. Brain expressed microRNAs implicated in schizophrenia etiology

    DEFF Research Database (Denmark)

    Hansen, Thomas; Olsen, Line; Lindow, Morten;

    2007-01-01

    Protein encoding genes have long been the major targets for research in schizophrenia genetics. However, with the identification of regulatory microRNAs (miRNAs) as important in brain development and function, miRNAs genes have emerged as candidates for schizophrenia-associated genetic factors...

  10. eRNAs lure NELF from paused polymerases.

    Science.gov (United States)

    Plosky, Brian S

    2014-10-02

    RNAs transcribed from enhancers (eRNAs) have been linked to enhancer function. In this issue of Molecular Cell, Schaukowitch et al. (2014) show that upon activation, eRNAs can bind NELF and are necessary for its transient removal from promoters to release paused RNA polymerase II and drive expression of immediate-early genes in neurons.

  11. Identification of Bacterial Small RNAs by RNA Sequencing

    DEFF Research Database (Denmark)

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

    2014-01-01

    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 seque...

  12. The expression profile of microRNAs in mouse embryos.

    Science.gov (United States)

    Mineno, Junichi; Okamoto, Sachiko; Ando, Tatsuya; Sato, Masahiro; Chono, Hideto; Izu, Hiroyuki; Takayama, Masanori; Asada, Kiyozo; Mirochnitchenko, Oleg; Inouye, Masayori; Kato, Ikunoshin

    2006-01-01

    MicroRNAs (miRNAs), which are non-coding RNAs 18-25 nt in length, regulate a variety of biological processes, including vertebrate development. To identify new species of miRNA and to simultaneously obtain a comprehensive quantitative profile of small RNA expression in mouse embryos, we used the massively parallel signature sequencing technology that potentially identifies virtually all of the small RNAs in a sample. This approach allowed us to detect a total of 390 miRNAs, including 195 known miRNAs covering approximately 80% of previously registered mouse miRNAs as well as 195 new miRNAs, which are so far unknown in mouse. Some of these miRNAs showed temporal expression profiles during prenatal development (E9.5, E10.5 and E11.5). Several miRNAs were positioned in polycistron clusters, including one particular large transcription unit consisting of 16 known and 23 new miRNAs. Our results indicate existence of a significant number of new miRNAs expressed at specific stages of mammalian embryonic development and which were not detected by earlier methods.

  13. Noncoding RNAs in cancer and cancer stem cells

    Institute of Scientific and Technical Information of China (English)

    Tianzhi Huang; Angel Alvarez; Bo Hu; Shi-Yuan Cheng

    2013-01-01

    In recent years, it has become increasingly apparent that noncoding RNAs (ncRNA) are of crucial importance for human cancer. The functional relevance of ncRNAs is particularly evident for microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). miRNAs are endogenously expressed small RNA sequences that act as post-transcriptional regulators of gene expression and have been extensively studied for their roles in cancers, whereas lncRNAs are emerging as important players in the cancer paradigm in recent years. These noncoding genes are often aberrantly expressed in a variety of human cancers. However, the biological functions of most ncRNAs remain largely unknown. Recently, evidence has begun to accumulate describing how ncRNAs are dysregulated in cancer and cancer stem cells, a subset of cancer cells harboring self-renewal and differentiation capacities. These studies provide insight into the functional roles that ncRNAs play in tumor initiation, progression, and resistance to therapies, and they suggest ncRNAs as attractive therapeutic targets and potential y useful diagnostic tools.

  14. Regulatory RNAs in prokaryotes: here, there and everywhere.

    Science.gov (United States)

    Narberhaus, Franz; Vogel, Jörg

    2009-10-01

    A recent meeting on 'Regulatory RNAs in prokaryotes' reflected the growing interest in this research topic. Almost 200 scientists met to discuss the identification, structure, function and mechanistic details of regulatory RNAs in bacteria and archaea. The topics included small regulatory RNAs, riboswitches, RNA thermosensors and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) elements.

  15. Bioavailability of transgenic microRNAs in genetically modified plants

    Science.gov (United States)

    Transgenic expression of small RNAs is a prevalent approach in agrobiotechnology for the global enhancement of plant foods. Meanwhile, emerging studies have, on the one hand, emphasized the potential of transgenic microRNAs (miRNAs) as novel dietary therapeutics and, on the other, suggested potentia...

  16. A bootstrap based analysis pipeline for efficient classification of phylogenetically related animal miRNAs

    Directory of Open Access Journals (Sweden)

    Gu Xun

    2007-03-01

    Full Text Available Abstract Background Phylogenetically related miRNAs (miRNA families convey important information of the function and evolution of miRNAs. Due to the special sequence features of miRNAs, pair-wise sequence identity between miRNA precursors alone is often inadequate for unequivocally judging the phylogenetic relationships between miRNAs. Most of the current methods for miRNA classification rely heavily on manual inspection and lack measurements of the reliability of the results. Results In this study, we designed an analysis pipeline (the Phylogeny-Bootstrap-Cluster (PBC pipeline to identify miRNA families based on branch stability in the bootstrap trees derived from overlapping genome-wide miRNA sequence sets. We tested the PBC analysis pipeline with the miRNAs from six animal species, H. sapiens, M. musculus, G. gallus, D. rerio, D. melanogaster, and C. elegans. The resulting classification was compared with the miRNA families defined in miRBase. The two classifications were largely consistent. Conclusion The PBC analysis pipeline is an efficient method for classifying large numbers of heterogeneous miRNA sequences. It requires minimum human involvement and provides measurements of the reliability of the classification results.

  17. The Role of microRNAs in the Biology of Rare Diseases

    Directory of Open Access Journals (Sweden)

    Domenica Taruscio

    2011-10-01

    Full Text Available Rare diseases (RD are characterized by low prevalence and affect not more than five individuals per 10,000 in the European population; they are a large and heterogeneous group of disorders including more than 7,000 conditions and often involve all organs and tissues, with several clinical subtypes within the same disease. Very often information concerning either diagnosis and/or prognosis on many RD is insufficient. microRNAs are a class of small non-coding RNAs that regulate gene expression at the posttranscriptional level by either degrading or blocking translation of messenger RNA targets. Recently, microRNA expression patterns of body fluids underscored their potential as noninvasive biomarkers for various diseases. The role of microRNAs as potential biomarkers has become particularly attractive. The identification of disease-related microRNAs is essential for understanding the pathogenesis of diseases at the molecular level, and is critical for designing specific molecular tools for diagnosis, treatment and prevention. Computational analysis of microRNA-disease associations is an important complementary means for prioritizing microRNAs for further experimental examination. In this article, we explored the added value of miRs as biomarkers in a selected panel of RD hitting different tissues/systems at different life stages, but sharing the need of better biomarkers for diagnostic and prognostic purposes.

  18. Iduna Protects the Brain from Glutamate Excitotoxicity and Stroke by Interfering with Parthanatos

    Science.gov (United States)

    Andrabi, Shaida A.; Kang, Ho Chul; Haince, Jean-François; Lee, Yun-Il; Zhang, Jian; Chi, Zhikai; West, Andrew B.; Koehler, Raymond C.; Poirier, Guy G.; Dawson, Ted M.; Dawson, Valina L.

    2013-01-01

    Glutamate acting on N-methyl-D-aspartate (NMDA) receptors plays an important role in neurodegenerative diseases and neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a novel NMDA receptor-induced survival gene that is neuroprotective against glutamate NMDA receptor mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer induced cell death (parthanatos). Iduna’s protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer binding protein and mutations at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion (MCAO)-induced stroke in mice. These results define Iduna as the first endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling offers a new therapeutic strategy for the treatment of neurologic disorders. PMID:21602803

  19. An investigation into failure analysis of interfering part of a steam turbine journal bearing

    Directory of Open Access Journals (Sweden)

    M. Mehdizadeh

    2014-10-01

    Full Text Available Journal bearings as so sensitive parts of steam turbines are very susceptible to failure through different mechanisms of wear, fatigue and crush during service conditions. Failure occurring through these mechanisms lead to turbine completely shut down as a result of interfering in working conditions of bearing different parts. In this research, failed interfered part of a journal bearing related to a 320,000 kW steam turbine was examined. Failure analysis investigations were performed by utilizing of stereographic, optical microscopy, scanning electron microscopy (SEM, energy-dispersive X-ray spectroscopy (EDS analysis and hardness test. Surface crush, large amounts of surface cracks, no noticeable changes of failed surface chemical composition and microstructure with significant hardness improvement were the main obtained results. The studies were revealed that the bearing part loosing and inappropriate clearance can produce relative displacements under cyclic gradient loading. This condition was detrimental for the service life of turbine journal bearing via failure through fretting fatigue mechanism.

  20. Effect of Atmospheric Interfering Absorption on Measurement of BTX by DOAS

    Institute of Scientific and Technical Information of China (English)

    Fu-min Peng; Pin-hua Xie; Hai-yang Li; Ying-hua Zhang; Jun-de Wang; Wen-qing Liu

    2008-01-01

    It was reported on the elimination of interfering absorption of BTX. the absorption of O2 includes different absorption bands, which change differently when the partial pressure of oxygen is varied. These cause the nonlinear absorption of O2 and the observed band shape to vary with the column density of O2. The absorption ratios of molecular absorption in each of the Herzberg bands and dimer absorptions, as well as the contribution to the correction error of molecular absorption, are studied based on the characteristic of these absorption bands. The optimized way to eliminate the interfering absorption is obtained in the end and the effectiveness of using interpolation proposed by Volkamer et al. to remove 02 absorption is proved again. As to O3 and SO2, the effect of the thermal effect of characteristic spectra on the elimination error of their absorption is studied. Solutions to these problems are discussed and demonstrated together with methods to optimize the interpolation of spectra. As a sample application, differential optical absorption spectroscopy (DOAS) measurements of BTX are carried out. Results show a low detection limit and the good correlation with point instruments are achieved. All these prove the feasibility of using spectral interpolation to improve the accuracy of DOAS measurements of aromatic hydrocarbons for practical purposes.

  1. Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    DEFF Research Database (Denmark)

    Balcells, Ingrid; Cirera Salicio, Susanna; Busk, Peter K.

    2011-01-01

    be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological...... samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision...... settings. RESULTS: We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could...

  2. Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

    DEFF Research Database (Denmark)

    Balcells, Ingrid; Cirera, Susanna; Busk, Peter Kamp

    2011-01-01

    be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological...... settings. Results We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could...... samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision...

  3. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    Science.gov (United States)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  4. Expression profiles of miRNAs in human pancreatic cancer cell lines%胰腺癌细胞系中miRNAs的表达分析

    Institute of Scientific and Technical Information of China (English)

    Shineng Zhang; Haijun Zuo; Zhong Yu; Fengting Huang; Wa Zhong

    2009-01-01

    Objective:To analyze initially the differences of miRNAs expression profiles in human pancreatic cencer cell lines by microarray technique.Methods:A total of 743 probes were designed according to lhe known miRNAs sequences of human,mice,and rats.miRNAs microarray was manufactured and its credibility was verified.Total RNAs were extracted and miRNAs were separated from human pancreatic cancer cell lines(SW1990,Capan-2.BxPC-3,Aspc-1,and Panc 1)and immortal human pancreatic duct epithelial cell line H6C7.They were labeled with T4 RNA ligase.then were hybridized with microarray.Through array scan and analysis,miRNAs expression profiles in pancreatic cancer were obtained.The results were verified by Northern blotting and RT-PCR.Results:A totsl of 63 miRNAs related to pancreatic cancer were found to be differentially expressed in 5 pancreatic cancer cell lines.including 25 down-regulated and 38 up-regulated miRNAs.Expres- sions of mir-21 and let-7 were also confirmed.Conclusion:The results suggested that miRNAs expression profiles could befound in pancreatic cancer cells.

  5. Períodos de interferência de plantas daninhas na cultura do sorgo Weed interference periods in sorghum crop

    Directory of Open Access Journals (Sweden)

    A.C.P. Rodrigues

    2010-01-01

    Full Text Available Objetivou-se com este trabalho estimar os períodos de interferência das plantas daninhas na cultura do sorgo granífero BRS 310. O experimento foi realizado no município de Botucatu/SP, em delineamento experimental de blocos ao acaso, com quatro repetições. Os tratamentos constituíram-se de oito períodos de controle, nos quais a cultura foi mantida livre da comunidade de plantas daninhas, e de oito períodos de convivência, nos quais a cultura foi mantida na presença da comunidade infestante. Os períodos foram: 7, 14, 21, 28, 35, 42, 49 e 56 dias após a semeadura (DAS, além de uma testemunha mantida livre da interferência das plantas daninhas e outra mantida na presença destas. Foram identificadas 11 famílias e 17 espécies de plantas daninhas, destacando-se as famílias Asteraceae e Poaceae, com o maior número de espécies. Para determinação dos períodos de interferência, os dados de produtividade de grãos, comprimento da panícula e massa seca da parte aérea das plantas de sorgo obtidos foram ajustados ao modelo de regressão não linear: y = a/[1+(x/bc]. Considerando como aceitável perda de 5% para a produtividade de grãos, o comprimento da panícula e a massa seca da parte aérea das plantas de sorgo, determinou-se o período anterior à interferência (PAI de 42, 72 e 15 DAS e o período total de prevenção à interferência (PTPI de 26, 17 e 12 DAS, respectivamente. Não houve período crítico de prevenção da interferência (PCPI para os parâmetros avaliados.The aim of this work was to estimate weed control periods and weed coexistence periods in sorghum 'BRS 310' crop. The experiment was carried out in Botucatu, São Paulo, Brazil. The experimental design was a randomized complete block with four replications and the treatments consisted of eight control periods, in which the crop was maintained weed- free and of eight coexistence periods, in which the crop was maintained in the presence of the weeds. The

  6. Computational identification of conserved microRNAs and their targets from expression sequence tags of blueberry (Vaccinium corybosum).

    Science.gov (United States)

    Li, Xuyan; Hou, Yanming; Zhang, Li; Zhang, Wenhao; Quan, Chen; Cui, Yuhai; Bian, Shaomin

    2014-06-06

    MicroRNAs (miRNAs) are a class of endogenous, approximately 21nt in length, non-coding RNA, which mediate the expression of target genes primarily at post-transcriptional levels. miRNAs play critical roles in almost all plant cellular and metabolic processes. Although numerous miRNAs have been identified in the plant kingdom, the miRNAs in blueberry, which is an economically important small fruit crop, still remain totally unknown. In this study, we reported a computational identification of miRNAs and their targets in blueberry. By conducting an EST-based comparative genomics approach, 9 potential vco-miRNAs were discovered from 22,402 blueberry ESTs according to a series of filtering criteria, designated as vco-miR156-5p, vco-miR156-3p, vco-miR1436, vco-miR1522, vco-miR4495, vco-miR5120, vco-miR5658, vco-miR5783, and vco-miR5986. Based on sequence complementarity between miRNA and its target transcript, 34 target ESTs from blueberry and 70 targets from other species were identified for the vco-miRNAs. The targets were found to be involved in transcription, RNA splicing and binding, DNA duplication, signal transduction, transport and trafficking, stress response, as well as synthesis and metabolic process. These findings will greatly contribute to future research in regard to functions and regulatory mechanisms of blueberry miRNAs.

  7. Novel and conserved microRNAs in soybean floral whorls.

    Science.gov (United States)

    Kulcheski, F R; Molina, L G; da Fonseca, G C; de Morais, G L; de Oliveira, L F V; Margis, R

    2016-01-10

    MicroRNAs (miRNAs) correspond to a class of endogenous small non-coding RNAs (19-24 nt) that regulates the gene expression, through mRNA target cleavage or translation inhibition. In plants, miRNAs have been shown to play pivotal roles in a wide variety of metabolic and biological processes like plant growth, development, and response to biotic and abiotic stress. Soybean is one of the most important crops worldwide, due to the production of oil and its high protein content. The reproductive phase is considered the most important for soybean yield, which is mainly intended to produce the grains. The identification of miRNAs is not yet saturated in soybean, and there are no studies linking them to the different floral organs. In this study, three different mature soybean floral whorls were used in the construction of sRNA libraries. The sequencing of petal, carpel and stamen libraries generated a total of 10,165,661 sequences. Subsequent analyses identified 200 miRNAs sequences, among which, 41 were novel miRNAs, 80 were conserved soybean miRNAs, 31 were new antisense conserved soybean miRNAs and 46 were soybean miRNAs isoforms. We also found a new miRNA conserved in other plant species, and finally one miRNA-sibling of a soybean conserved miRNA. Conserved and novel miRNAs were evaluated by RT-qPCR. We observed a differential expression across the three whorls for six miRNAs. Computational predicted targets for miRNAs analyzed by RT-qPCR were identified and present functions related to reproductive process in plants. In summary, the increased accumulation of specific and novel miRNAs in different whorls indicates that miRNAs are an important part of the regulatory network in soybean flower.

  8. Self containment, a property of modular RNA structures, distinguishes microRNAs.

    Directory of Open Access Journals (Sweden)

    Miler T Lee

    Full Text Available RNA molecules will tend to adopt a folded conformation through the pairing of bases on a single strand; the resulting so-called secondary structure is critical to the function of many types of RNA. The secondary structure of a particular substring of functional RNA may depend on its surrounding sequence. Yet, some RNAs such as microRNAs retain their specific structures during biogenesis, which involves extraction of the substructure from a larger structural context, while other functional RNAs may be composed of a fusion of independent substructures. Such observations raise the question of whether particular functional RNA substructures may be selected for invariance of secondary structure to their surrounding nucleotide context. We define the property of self containment to be the tendency for an RNA sequence to robustly adopt the same optimal secondary structure regardless of whether it exists in isolation or is a substring of a longer sequence of arbitrary nucleotide content. We measured degree of self containment using a scoring method we call the self-containment index and found that miRNA stem loops exhibit high self containment, consistent with the requirement for structural invariance imposed by the miRNA biogenesis pathway, while most other structured RNAs do not. Further analysis revealed a trend toward higher self containment among clustered and conserved miRNAs, suggesting that high self containment may be a characteristic of novel miRNAs acquiring new genomic contexts. We found that miRNAs display significantly enhanced self containment compared to other functional RNAs, but we also found a trend toward natural selection for self containment in most functional RNA classes. We suggest that self containment arises out of selection for robustness against perturbations, invariance during biogenesis, and modular composition of structural function. Analysis of self containment will be important for both annotation and design of functional

  9. 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

  10. Salivary MicroRNAs and Oral Cancer Detection

    OpenAIRE

    Yoshizawa, Janice M.; Wong, David T.W.

    2013-01-01

    MicroRNAs (miRNAs) in human saliva have recently become an emerging field in saliva research for diagnostics applications and its potential role in biological implications. miRNAs are short noncoding RNA molecules that play important roles in regulating a variety of cellular processes. Dysregulation of miRNAs are known to be associated with many diseases. miRNAs were found present in the saliva of OSCC patients and could serve as potential biomarkers for oral cancer detection. Understanding t...

  11. Non-coding RNAs in primary liver cancer

    Directory of Open Access Journals (Sweden)

    Michele eGhidini

    2015-06-01

    Full Text Available Hepatocellular carcinoma (HCC is a primary malignancy of the liver with poor prognosis and limited therapeutic options. Over the past few years, many studies have evaluated the role of non-coding RNAs (ncRNAs in hepatocarcinogenesis and tumour progression. ncRNAs were shown to have diagnostic, prognostic and therapeutic potential in HCC. In this manuscript, we review the latest major discoveries concerning microRNAs and long ncRNAs in HCC pathogenesis, and discuss the potentials and the limitations for their use in clinical practice.

  12. MicroRNAs of parasites: current status and future perspectives.

    Science.gov (United States)

    Liu, Quan; Tuo, Wenbin; Gao, Hongwei; Zhu, Xing-Quan

    2010-08-01

    MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs regulating gene expression in eukaryotes at the post-transcriptional level. The complex life cycles of parasites may require the ability to respond to environmental and developmental signals through miRNA-mediated gene expression. Over the past 17 years, thousands of miRNAs have been identified in the nematode Caenorhabditis elegans and other parasites. Here, we review the current status and potential functions of miRNAs in protozoan, helminths, and arthropods, and propose some perspectives for future studies.

  13. miRNAs as biomarkers of atrial fibrillation.

    Science.gov (United States)

    Gomes da Silva, Ananília Medeiros; Silbiger, Vivian Nogueira

    2014-12-01

    Atrial fibrillation (AF) is a highly prevalent arrhythmia with pronounced morbidity and mortality. Genetics analysis has established electrophysiological substrates, which determine individual vulnerability to AF occurrence and maintenance. MicroRNAs (miRNAs) found in virtually all organisms function as negative regulators of protein-coding genes. Several studies have suggested a role for miRNAs in the regulation of cardiac excitability and arrhythmogenesis. This review is based on 18 studies conducted between 2009 and 2013 to investigate the association of miRNAs with AF. miRNAs are discussed here as candidate biomarkers for AF in blood and cardiac tissues and as potential targets for AF therapy.

  14. Genome organization and characteristics of soybean microRNAs

    Directory of Open Access Journals (Sweden)

    Turner Marie

    2012-05-01

    Full Text Available Abstract Background microRNAs (miRNAs are key regulators of gene expression and play important roles in many aspects of plant biology. The role(s of miRNAs in nitrogen-fixing root nodules of leguminous plants such as soybean is not well understood. We examined a library of small RNAs from Bradyrhizobium japonicum-inoculated soybean roots and identified novel miRNAs. In order to enhance our understanding of miRNA evolution, diversification and function, we classified all known soybean miRNAs based on their phylogenetic conservation (conserved, legume- and soybean-specific miRNAs and examined their genome organization, family characteristics and target diversity. We predicted targets of these miRNAs and experimentally validated several of them. We also examined organ-specific expression of selected miRNAs and their targets. Results We identified 120 previously unknown miRNA genes from soybean including 5 novel miRNA families. In the soybean genome, genes encoding miRNAs are primarily intergenic and a small percentage were intragenic or less than 1000 bp from a protein-coding gene, suggesting potential co-regulation between the miRNA and its parent gene. Difference in number and orientation of tandemly duplicated miRNA genes between orthologous genomic loci indicated continuous evolution and diversification. Conserved miRNA families are often larger in size and produce less diverse mature miRNAs than legume- and soybean-specific families. In addition, the majority of conserved and legume-specific miRNA families produce 21 nt long mature miRNAs with distinct nucleotide distribution and regulate a more conserved set of target mRNAs compared to soybean-specific families. A set of nodule-specific target mRNAs and their cognate regulatory miRNAs had inverse expression between root and nodule tissues suggesting that spatial restriction of target gene transcripts by miRNAs might govern nodule-specific gene expression in soybean. Conclusions Genome

  15. Identification of Conserved Cotton MicroRNAs and Their Targets

    Institute of Scientific and Technical Information of China (English)

    ZHANG Bao-hong; WANG Qing-lian

    2008-01-01

    @@ No study has been performed on identifying microRNAs (miRNAs) and their targets in cotton although cotton is one of the most important fiber and economic crops around the world.In this study,we found 30 potential cotton miRNAs using a comparative genomic approach based on genomic survey sequence analysis and miRNA secondary structure.These cotton miRNAs belong to 22 miRNA families.Expressed sequence tag (EST) analysis indicated that the predicted miRNAs were expressed in cotton plants.

  16. Identification and Function of MicroRNAs Encoded by Herpesviruses

    Institute of Scientific and Technical Information of China (English)

    Zhi-qing Bai; Xiu-fen Lei; Lin-ding Wang; Shou-jiang Gao

    2008-01-01

    MicroRNAs (miRNAs) play important roles in eukaryotes,plants and some viruses.It is increasingly clear that miRNAs-encoded by viruses can affect the viral life cycle and host physiology.Viral miRNAs could repress the innate and adaptive host immunity,modulate cellular signaling pathways,and regulate the expression of cellular and viral genes.These functions facilitate viral acute and persistent infections,and have profound effects on the host cell survival and disease progression.Here,we discuss the miRNAs encoded by herpesviruses,and their regulatory roles involved in virus-host interactions.

  17. Ribonucleases, antisense RNAs and the control of bacterial plasmids.

    Science.gov (United States)

    Saramago, Margarida; Bárria, Cátia; Arraiano, Cecília M; Domingues, Susana

    2015-03-01

    In the last decade regulatory RNAs have emerged as powerful tools to regulate the expression of genes both in prokaryotes and in eukaryotes. RNases, by degrading these RNA molecules, control the right amount of regulatory RNAs, which is fundamental for an accurate regulation of gene expression in the cell. Remarkably the first antisense RNAs identified were plasmid-encoded and their detailed study was crucial for the understanding of prokaryotic antisense RNAs. In this review we highlight the role of RNases in the precise modulation of antisense RNAs that control plasmid replication, maintenance and transfer.

  18. Design

    DEFF Research Database (Denmark)

    Volf, Mette

    This publication is unique in its demystification and operationalization of the complex and elusive nature of the design process. The publication portrays the designer’s daily work and the creative process, which the designer is a part of. Apart from displaying the designer’s work methods...... and design parameters, the publication shows examples from renowned Danish design firms. Through these examples the reader gets an insight into the designer’s reality....

  19. Effect of 5' and 3' terminal sequences, overall length, and coding capacity on the accumulation of defective RNAs associated with broad bean mottle bromovirus in planta.

    Science.gov (United States)

    Pogany, J; Romero, J; Bujarski, J J

    1997-02-17

    Broad bean mottle bromovirus (BBMV) was shown to accumulate RNA2-derived defective interfering (DI) RNAs [Romero et al., Virology 194, 576-584 (1993); Pogany et al., Virology 212, 574-586 (1995)]. In this work, we utilize three sets of BBMV RNA2-derived artificial DI RNA constructs to determine factors that affect the accumulation of defective RNAs in planta. One set of deletion constructs was used to localize sequences required for efficient accumulation within the 5' 883 nt and the 3' 387 nt of the DI RNAs. The second set had a gradually increasing size of 3' nested deletions to determine the minimal length required for efficient DI RNA accumulation. The smallest DI RNA still accumulating in plants was found to be 1712 nt long. The third set consisted of frameshift mutants which showed that at least 64.4% of BBMV DI RNA sequences must have the 5' portion of the 2a open reading frame to ensure efficient accumulation. The importance of these factors in the selection of DI RNAs is discussed.

  20. Transposon defense by endo-siRNAs, piRNAs and somatic pilRNAs in Drosophila: contributions of Loqs-PD and R2D2.

    Directory of Open Access Journals (Sweden)

    Milijana Mirkovic-Hösle

    Full Text Available Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing activities that target transposons: endogenous siRNAs (esiRNAs or endo-siRNAs and Piwi-interacting small RNAs (piRNAs. The biogenesis of endo-siRNAs involves the Dicer-2 co-factors Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2, which primarily helps to direct siRNAs into the RNA interference effector Ago2. Nonetheless, loss of either protein is not sufficient to produce a phenotype comparable with a dcr-2 mutation. We provide further deep sequencing evidence supporting the notion that R2D2 and Loqs-PD have partially overlapping function. Certain transposons display a preference for either dsRBD-protein during production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway.

  1. Circulating microRNAs in breast cancer

    DEFF Research Database (Denmark)

    Hamam, Rimi; Hamam, Dana; Alsaleh, Khalid A.

    2017-01-01

    in the past several years has highlighted the potential use of peripheral blood circulating nucleic acids such as DNA, mRNA and micro (mi)RNA in breast cancer diagnosis, prognosis and for monitoring response to anticancer therapy. Among these, circulating miRNA is increasingly recognized as a promising...... biomarker, given the ease with which miRNAs can be isolated and their structural stability under different conditions of sample processing and isolation. In this review, we provide current state-of-the-art of miRNA biogenesis, function and discuss the advantages, limitations, as well as pitfalls of using...... circulating miRNAs as diagnostic, prognostic or predictive biomarkers in breast cancer management....

  2. MicroRNAs, epigenetics and disease

    DEFF Research Database (Denmark)

    Silahtaroglu, Asli; Stenvang, Jan

    2010-01-01

    Epigenetics is defined as the heritable chances that affect gene expression without changing the DNA sequence. Epigenetic regulation of gene expression can be through different mechanisms such as DNA methylation, histone modifications and nucleosome positioning. MicroRNAs are short RNA molecules...... which do not code for a protein but have a role in post-transcriptional silencing of multiple target genes by binding to their 3' UTRs (untranslated regions). Both epigenetic mechanisms, such as DNA methylation and histone modifications, and the microRNAs are crucial for normal differentiation......, development and maintenance of tissue-specific gene expression. These mechanisms also explain how cells with the same DNA content can differentiate into cells with different functions. Changes in epigenetic processes can lead to changes in gene function, cancer formation and progression, as well as other...

  3. microRNAs in CNS disorders

    DEFF Research Database (Denmark)

    Kocerha, Jannet; Kauppinen, Sakari; Wahlestedt, Claes

    2009-01-01

    In recent years, there has been a shift in the conventional paradigms for transcriptional and translational regulation as extensive sequencing efforts have yielded new insights into the landscape of the human genome and transcriptome. Hundreds of non-coding regulatory RNA molecules called microRNAs...... (miRNAs) have been identified in the mammalian central nervous system (CNS) and are reported to mediate pivotal roles in many aspects of neuronal functions. Disruption of miRNA-based post-transcriptional regulation has been implicated in a range of CNS disorders as one miRNA is predicted to impact...... the expression of numerous downstream mRNA targets. The intricate molecular networks mediated by an miRNA form a robust mechanism for rapid and potent responses to cellular events throughout the development of the human brain. Recent studies have identified a molecular and ultimately pathogenic role for a subset...

  4. Multiple Export Mechanisms for mRNAs

    Science.gov (United States)

    Delaleau, Mildred; Borden, Katherine L. B.

    2015-01-01

    Nuclear mRNA export plays an important role in gene expression. We describe the mechanisms of mRNA export including the importance of mRNP assembly, docking with the nuclear basket of the nuclear pore complex (NPC), transit through the central channel of the NPC and cytoplasmic release. We describe multiple mechanisms of mRNA export including NXF1 and CRM1 mediated pathways. Selective groups of mRNAs can be preferentially transported in order to respond to cellular stimuli. RNAs can be selected based on the presence of specific cis-acting RNA elements and binding of specific adaptor proteins. The role that dysregulation of this process plays in human disease is also discussed. PMID:26343730

  5. Triple combination of siRNAs targeting TGFβ1, TGFβR2, and CTGF enhances reduction of collagen I and smooth muscle actin in corneal fibroblasts.

    Science.gov (United States)

    Sriram, Sriniwas; Robinson, Paulette; Pi, Liya; Lewin, Alfred S; Schultz, Gregory

    2013-12-17

    Transforming growth factor β1 (TGFβ1), TGFβ receptor (TGFβR2), and connective tissue growth factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin, and kidney. We developed an antifibrotic treatment targeting these three critical scarring genes by using a combination of small interfering RNAs (siRNAs) and assessed its effect on downstream scarring genes, collagen I, and α smooth muscle actin (SMA). Up to six individual siRNAs for each of the three target gene mRNAs were transfected into cultures of rabbit corneal fibroblasts at concentrations from 15 to 90 nM. The knockdown of target gene proteins was measured by ELISA, and the two most effective siRNAs were tested in dual combinations. Knockdown percentages of both individual and dual siRNA combinations were analyzed for synergy by using combination index to predict "effective" and "ineffective" triple siRNA combinations. Effects of both triple siRNA combinations on target and downstream mRNAs were measured by using quantitative RT-PCR, and levels of SMA protein were assessed by immunohistochemistry. Single and dual siRNA combinations produced a wide range of protein knockdown of target genes (5%-80%). The effective triple siRNA combination significantly reduced mRNA levels of target genes (>80%) and downstream scarring genes (>85%), and of SMA protein (>95%), and significantly reduced cell migration without reducing cell viability. Simultaneous targeting of TGFβ1, TGFβR2, and CTGF genes by effective triple siRNA combination produced high knockdown of target and downstream scarring genes without cell toxicity, which may have clinical applications in reducing corneal fibrosis and scarring in other tissues.

  6. Euchromatic transposon insertions trigger production of novel Pi- and endo-siRNAs at the target sites in the drosophila germline.

    Directory of Open Access Journals (Sweden)

    Sergey Shpiz

    2014-02-01

    Full Text Available The control of transposable element (TE activity in germ cells provides genome integrity over generations. A distinct small RNA-mediated pathway utilizing Piwi-interacting RNAs (piRNAs suppresses TE expression in gonads of metazoans. In the fly, primary piRNAs derive from so-called piRNA clusters, which are enriched in damaged repeated sequences. These piRNAs launch a cycle of TE and piRNA cluster transcript cleavages resulting in the amplification of piRNA and TE silencing. Using genome-wide comparison of TE insertions and ovarian small RNA libraries from two Drosophila strains, we found that individual TEs inserted into euchromatic loci form novel dual-stranded piRNA clusters. Formation of the piRNA-generating loci by active individual TEs provides a more potent silencing response to the TE expansion. Like all piRNA clusters, individual TEs are also capable of triggering the production of endogenous small interfering (endo-si RNAs. Small RNA production by individual TEs spreads into the flanking genomic regions including coding cellular genes. We show that formation of TE-associated small RNA clusters can down-regulate expression of nearby genes in ovaries. Integration of TEs into the 3' untranslated region of actively transcribed genes induces piRNA production towards the 3'-end of transcripts, causing the appearance of genic piRNA clusters, a phenomenon that has been reported in different organisms. These data suggest a significant role of TE-associated small RNAs in the evolution of regulatory networks in the germline.

  7. Disease onset and aging in the world of circular RNAs

    Science.gov (United States)

    Maiese, Kenneth

    2016-01-01

    Circular ribonucleic acids (circRNAs) are non-coding RNAs of approximately 100 nucleotides in length with thousands of members in mammalian cells. The presence of circRNAs is believed to be even greater than that of messenger RNAs. Identification of circRNAs occurred approximately 37 years ago with the subsequent demonstration that covalent bonds are necessary for the unique circular structure of these ribonucleic acids. However, present understanding of the complex biological role of circRNAs remains limited and requires further elucidation. CircRNAs may impact aging, multiple disorders, function as biomarkers, and are able to regulate gene expression by acting as effective microRNA (miRNA) sponges. New work suggests that circRNAs are vital for the modulation of cellular senescence and programmed cell death pathways such as apoptosis. These non-coding RNAs can control cell cycle progression, cellular proliferation, and cellular survival impacting disorders linked to aging, cardiovascular disease, and atherosclerosis through pathways that involve cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase inhibitor 1 (p21), and mammalian forkhead transcription factors. In addition, circRNAs can oversee cellular metabolism and disorders such as diabetes mellitus through the regulation of insulin signaling as well as limit tumor progression through Wnt signaling and β-catenin pathways. Further understanding of the biology of circRNAs offers great promise for the targeting of novel strategies against a wide spectrum of disease entities. PMID:27642518

  8. Disease onset and aging in the world of circular RNAs.

    Science.gov (United States)

    Maiese, Kenneth

    Circular ribonucleic acids (circRNAs) are non-coding RNAs of approximately 100 nucleotides in length with thousands of members in mammalian cells. The presence of circRNAs is believed to be even greater than that of messenger RNAs. Identification of circRNAs occurred approximately 37 years ago with the subsequent demonstration that covalent bonds are necessary for the unique circular structure of these ribonucleic acids. However, present understanding of the complex biological role of circRNAs remains limited and requires further elucidation. CircRNAs may impact aging, multiple disorders, function as biomarkers, and are able to regulate gene expression by acting as effective microRNA (miRNA) sponges. New work suggests that circRNAs are vital for the modulation of cellular senescence and programmed cell death pathways such as apoptosis. These non-coding RNAs can control cell cycle progression, cellular proliferation, and cellular survival impacting disorders linked to aging, cardiovascular disease, and atherosclerosis through pathways that involve cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase inhibitor 1 (p21), and mammalian forkhead transcription factors. In addition, circRNAs can oversee cellular metabolism and disorders such as diabetes mellitus through the regulation of insulin signaling as well as limit tumor progression through Wnt signaling and β-catenin pathways. Further understanding of the biology of circRNAs offers great promise for the targeting of novel strategies against a wide spectrum of disease entities.

  9. Identifying microRNAs and transcript targets in Jatropha seeds.

    Directory of Open Access Journals (Sweden)

    Vanessa Galli

    Full Text Available MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs.

  10. The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Doller, Anke; Badawi, Amel [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Schmid, Tobias; Brauß, Thilo [Institut für Biochemie I (Pathobiochemie), Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Pleli, Thomas [Medizinische Klinik 1, Schwerpunkt Gastroenterologie und Hepatologie, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Meyer zu Heringdorf, Dagmar [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Piiper, Albrecht [Medizinische Klinik 1, Schwerpunkt Gastroenterologie und Hepatologie, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Pfeilschifter, Josef [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Eberhardt, Wolfgang, E-mail: w.eberhardt@em.uni-frankfurt.de [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany)

    2015-01-01

    The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D{sub 1} encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E{sub 2} synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on

  11. MicroRNAs as regulatory elements in psoriasis

    Directory of Open Access Journals (Sweden)

    Liu Yuan

    2016-01-01

    Full Text Available Psoriasis is a chronic, autoimmune, and complex genetic disorder that affects 23% of the European population. The symptoms of Psoriatic skin are inflammation, raised and scaly lesions. microRNA, which is short, nonprotein-coding, regulatory RNAs, plays critical roles in psoriasis. microRNA participates in nearly all biological processes, such as cell differentiation, development and metabolism. Recent researches reveal that multitudinous novel microRNAs have been identified in skin. Some of these substantial novel microRNAs play as a class of posttranscriptional gene regulator in skin disease, such as psoriasis. In order to insight into microRNAs biological functions and verify microRNAs biomarker, we review diverse references about characterization, profiling and subtype of microRNAs. Here we will share our opinions about how and which microRNAs are as regulatory in psoriasis.

  12. Long noncoding RNAs during normal and malignant hematopoiesis.

    Science.gov (United States)

    Alvarez-Dominguez, Juan R; Hu, Wenqian; Gromatzky, Austin A; Lodish, Harvey F

    2014-01-01

    Long noncoding RNAs (lncRNAs) are increasingly recognized to contribute to cellular development via diverse mechanisms during both health and disease. Here, we highlight recent progress on the study of lncRNAs that function in the development of blood cells. We emphasize lncRNAs that regulate blood cell fates through epigenetic control of gene expression, an emerging theme among functional lncRNAs. Many of these noncoding genes and their targets become dysregulated during malignant hematopoiesis, directly implicating lncRNAs in blood cancers such as leukemia. In a few cases, dysregulation of an lncRNA alone leads to malignant hematopoiesis in a mouse model. Thus, lncRNAs may be not only useful as markers for the diagnosis and prognosis of cancers of the blood, but also as potential targets for novel therapies.

  13. The therapeutic potential of MicroRNAs in cancer

    DEFF Research Database (Denmark)

    Thorsen, Susanne; Obad, S.; Jensen, N.F.

    2012-01-01

    MicroRNAs (miRNAs) have been uncovered as important posttranscriptional regulators of nearly every biological process in the cell. Furthermore, mounting evidence implies that miRNAs play key roles in the pathogenesis of cancer and that many miRNAs can function either as oncogenes or tumor...... suppressors. Thus, miRNAs have rapidly emerged as promising targets for the development of novel anticancer therapeutics. The development of miRNA-based cancer therapeutics relies on restoring the activity of tumor suppressor miRNAs using double-stranded miRNA mimics or inhibition of oncogenic miRNAs using...... single-stranded antisense oligonucleotides, termed antimiRs. In the present review, we focus on recent advancements in the discovery and development of miRNA-based cancer therapeutics using these 2 approaches. In addition, we summarize selected studies, in which modulation of miRNA activity...

  14. Discoveries and functions of virus-encoded MicroRNAs

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, ChinaVirus-encoded microRNAs (miRNAs) are a new kind of miRNAs that regulate the expression of target gene in host cells or viruses through inducing cleavage of mRNA, repressing translation, etc., and change the processes of host cells or replicate viruses to escape or resist immune surveillance of host and protect viruses themselves. It has become a hot topic to discover viral genes encoding miRNAs and their target genes, and to identify their functions. This review provides background information on the history of virally encoded miRNAs including their genomic distribution, functions and mechanisms.In addition, we discuss the similarities and differences between virus- and host-encoded miRNAs, the future directions of researches in viral miRNAs and their applications in diseases control and therapy.

  15. Progress, challenges and new concepts in microRNAs

    Institute of Scientific and Technical Information of China (English)

    ZHANG YouYi

    2011-01-01

    MicroRNAs are a group of small noncoding RNAs.They have rapidly gained attention in the field as novel regulators of cellular morphology and function.Currently hundreds of microRNAs have been described in human genome.MicroRNAs play important roles in posttranscriptional regulation of gene expression by inhibiting protein translation and/or promoting mRNA degradation.MicroRNAs were initially thought to be subtle regulators of gene expression,but increasing evidence demonstrates that the regulatory functions of microRNAs are crucial for the cell.MicroRNAs have been found to be involved in the development,tissue homeostasis as well as diseases.

  16. The role of RNases in the regulation of small RNAs.

    Science.gov (United States)

    Saramago, Margarida; Bárria, Cátia; Dos Santos, Ricardo F; Silva, Inês J; Pobre, Vânia; Domingues, Susana; Andrade, José M; Viegas, Sandra C; Arraiano, Cecília M

    2014-04-01

    Ribonucleases (RNases) are key factors in the control of biological processes, since they modulate the processing, degradation and quality control of RNAs. This review gives many illustrative examples of the role of RNases in the regulation of small RNAs (sRNAs). RNase E and PNPase have been shown to degrade the free pool of sRNAs. RNase E can also be recruited to cleave mRNAs when they are interacting with sRNAs. RNase III cleaves double-stranded structures, and can cut both the sRNA and its RNA target when they are hybridized. Overall, ribonucleases act as conductors in the control of sRNAs. Therefore, it is very important to further understand their role in the post-transcriptional control of gene expression.

  17. DNA methyltransferase controls stem cell aging by regulating BMI1 and EZH2 through microRNAs.

    Directory of Open Access Journals (Sweden)

    Ah-Young So

    Full Text Available Epigenetic regulation of gene expression is well known mechanism that regulates cellular senescence of cancer cells. Here we show that inhibition of DNA methyltransferases (DNMTs with 5-azacytidine (5-AzaC or with specific small interfering RNA (siRNA against DNMT1 and 3b induced the cellular senescence of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs and increased p16(INK4A and p21(CIP1/WAF1 expression. DNMT inhibition changed histone marks into the active forms and decreased the methylation of CpG islands in the p16(INK4A and p21(CIP1/WAF1 promoter regions. Enrichment of EZH2, the key factor that methylates histone H3 lysine 9 and 27 residues, was decreased on the p16(INK4A and p21(CIP1/WAF1 promoter regions. We found that DNMT inhibition decreased expression levels of Polycomb-group (PcG proteins and increased expression of microRNAs (miRNAs, which target PcG proteins. Decreased CpG island methylation and increased levels of active histone marks at genomic regions encoding miRNAs were observed after 5-AzaC treatment. Taken together, DNMTs have a critical role in regulating the cellular senescence of hUCB-MSCs through controlling not only the DNA methylation status but also active/inactive histone marks at genomic regions of PcG-targeting miRNAs and p16(INK4A and p21(CIP1/WAF1 promoter regions.

  18. Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs.

    Science.gov (United States)

    Babiarz, Joshua E; Ruby, J Graham; Wang, Yangming; Bartel, David P; Blelloch, Robert

    2008-10-15

    Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by a complex of TRBP and Dicer. dgcr8Delta/Delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1Delta/Delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild-type, dgcr8Delta/Delta, and dicer1Delta/Delta mESCs. Several of the resulting DGCR8-independent, Dicer-dependent RNAs were noncanonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of shRNAs. Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small RNA-generating pathways and show that mammalian siRNAs exist in cell types other than oocytes.

  19. Long Noncoding RNAs, Chromatin, and Development

    Directory of Open Access Journals (Sweden)

    Daniel P. Caley

    2010-01-01

    Full Text Available The way in which the genome of a multicellular organism can orchestrate the differentiation of trillions of cells and many organs, all from a single fertilized egg, is the subject of intense study. Different cell types can be defined by the networks of genes they express. This differential expression is regulated at the epigenetic level by chromatin modifications, such as DNA and histone methylation, which interact with structural and enzymatic proteins, resulting in the activation or silencing of any given gene. While detailed mechanisms are emerging on the role of different chromatin modifications and how these functions are effected at the molecular level, it is still unclear how their deposition across the epigenomic landscape is regulated in different cells. A raft of recent evidence is accumulating that implicates long noncoding RNAs (lncRNAs in these processes. Most genomes studied to date undergo widespread transcription, the majority of which is not translated into proteins. In this review, we will describe recent work suggesting that lncRNAs are more than transcriptional "noise", but instead play a functional role by acting as tethers and guides to bind proteins responsible for modifying chromatin and mediating their deposition at specific genomic locations. We suggest that lncRNAs are at the heart of developmental regulation, determining the epigenetic status and transcriptional network in any given cell type, and that they provide a means to integrate external differentiation cues with dynamic nuclear responses through the regulation of a metastable epigenome. Better characterization of the lncRNA-protein "interactome" may eventually lead to a new molecular toolkit, allowing researchers and clinicians to modulate the genome at the epigenetic level to treat conditions such as cancer.

  20. Small Regulatory RNAs of Rickettsia conorii

    Science.gov (United States)

    Narra, Hema P.; Schroeder, Casey L. C.; Sahni, Abha; Rojas, Mark; Khanipov, Kamil; Fofanov, Yuriy; Sahni, Sanjeev K.

    2016-01-01

    Small regulatory RNAs comprise critically important modulators of gene expression in bacteria, yet very little is known about their prevalence and functions in Rickettsia species. R. conorii, the causative agent of Mediterranean spotted fever, is a tick-borne pathogen that primarily infects microvascular endothelium in humans. We have determined the transcriptional landscape of R. conorii during infection of Human Microvascular Endothelial Cells (HMECs) by strand-specific RNA sequencing to identify 4 riboswitches, 13 trans-acting (intergenic), and 22 cis-acting (antisense) small RNAs (termed ‘Rc_sR’s). Independent expression of four novel trans-acting sRNAs (Rc_sR31, Rc_sR33, Rc_sR35, and Rc_sR42) and known bacterial sRNAs (6S, RNaseP_bact_a, ffs, and α-tmRNA) was next confirmed by Northern hybridization. Comparative analysis during infection of HMECs vis-à-vis tick AAE2 cells revealed significantly higher expression of Rc_sR35 and Rc_sR42 in HMECs, whereas Rc_sR31 and Rc_sR33 were expressed at similar levels in both cell types. We further predicted a total of 502 genes involved in all important biological processes as potential targets of Rc_sRs and validated the interaction of Rc_sR42 with cydA (cytochrome d ubiquinol oxidase subunit I). Our findings constitute the first evidence of the existence of post-transcriptional riboregulatory mechanisms in R. conorii and interactions between a novel Rc_sR and its target mRNA. PMID:27834404

  1. Which Depressive Symptoms and Medication Side Effects Are Perceived by Patients as Interfering Most with Occupational Functioning?

    Directory of Open Access Journals (Sweden)

    Raymond W. Lam

    2012-01-01

    Full Text Available Background. Major depressive disorder (MDD is associated with significant impairment in occupational functioning. This study sought to determine which depressive symptoms and medication side effects were perceived by patients with MDD to have the greatest interference on work functioning. Methods. 164 consecutive patients with MDD by DSM-IV criteria completed a standard assessment that included a self-rated questionnaire about the degree to which symptoms and side effects interfered with work functioning. Results. The symptoms perceived by patients as interfering most with work functioning were fatigue and low energy, insomnia, concentration and memory problems, anxiety, and irritability. The medication side effects rated as interfering most with work functioning were daytime sedation, insomnia, headache, and agitation/anxiety. There were no differences between men and women in symptoms or side effects that were perceived as interfering with work functioning. Limitations. This was a cross-sectional study; only subjective assessments of work functioning were obtained; the fact that patients were using varied medications acts as a potential confound. Conclusions. Specific depressive symptoms and medication side effects were perceived by patients as interfering more with occupational functioning than others. These factors should be considered in treatment selection (e.g., in the choice of antidepressant in working patients with MDD.

  2. Specificity of insulin signalling in human skeletal muscle as revealed by small interfering RNA.

    Science.gov (United States)

    Krook, A; Zierath, J R

    2009-07-01

    Insulin action on metabolically active tissues is a complex process involving positive and negative feedback regulation to control whole body glucose homeostasis. At the cellular level, glucose and lipid metabolism, as well as protein synthesis, are controlled through canonical insulin signalling cascades. The discovery of small interfering RNA (siRNA) allows for the molecular dissection of critical components of the regulation of metabolic and gene regulatory events in insulin-sensitive tissues. The application of siRNA to tissues of human origin allows for the molecular dissection of the mechanism(s) regulating glucose and lipid metabolism. Penetration of the pathways controlling insulin action in human tissue may aid in discovery efforts to develop diabetes prevention and treatment strategies. This review will focus on the use of siRNA to validate critical regulators controlling insulin action in human skeletal muscle, a key organ important for the control of whole body insulin-mediated glucose uptake and metabolism.

  3. The Deterministic Sum Capacity of a Multiple Access Channel Interfering with a Point to Point Link

    CERN Document Server

    Bühler, Jörg

    2011-01-01

    In this paper, we use the linear deterministic approximation model to study a two user multiple access channel mutually interfering with a point to point link, which represents a basic setup of a cellular system. We derive outer bounds on the achievable sum rate and construct coding schemes achieving the outer bounds. For a large parameter range, the sum capacity is identical to the sum capacity of the interference channel obtained by silencing the weaker user in the multiple access channel. For other interference configurations, the sum rate can be increased using interference alignment, which exploits the channel gain difference of the users in the multiple access channel. From these results, lower bounds on the generalized degrees of freedom for the Gaussian counterpart are derived.

  4. The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles.

    Directory of Open Access Journals (Sweden)

    Christopher M Ziegler

    2016-03-01

    Full Text Available Arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. Persistently-infected rodents harbor high levels of defective interfering (DI particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. Little is known about what drives the production of DI particles. We show that neither the PPXY late domain encoded within the lymphocytic choriomeningitis virus (LCMV matrix protein nor a functional endosomal sorting complex transport (ESCRT pathway is absolutely required for the generation of standard infectious virus particles. In contrast, DI particle release critically requires the PPXY late domain and is ESCRT-dependent. Additionally, the terminal tyrosine in the PPXY motif is reversibly phosphorylated and our findings indicate that this posttranslational modification may regulate DI particle formation. Thus we have uncovered a new role for the PPXY late domain and a possible mechanism for its regulation.

  5. Small interfering RNA delivery by polyethylenimine-functionalised porous silicon nanoparticles.

    Science.gov (United States)

    Hasanzadeh Kafshgari, M; Alnakhli, M; Delalat, B; Apostolou, S; Harding, F J; Mäkilä, E; Salonen, J J; Kuss, B J; Voelcker, N H

    2015-12-01

    In this study, thermally hydrocarbonised porous silicon nanoparticles (THCpSiNPs) capped with polyethylenimine (PEI) were fabricated, and their potential for small interfering RNA (siRNA) delivery was investigated in an in vitro glioblastoma model. PEI coating following siRNA loading enhanced the sustained release of siRNA, and suppressed burst release effects. The positively-charged surface improved the internalisation of the nanoparticles across the cell membrane. THCpSiNP-mediated siRNA delivery reduced mRNA expression of the MRP1 gene, linked to the resistence of glioblastoma to chemotherapy, by 63% and reduced MRP1-protein levels by 70%. MRP1 siRNA loaded nanoparticles did not induce cytotoxicity in glioblastoma cells, but markedly reduced cell proliferation. In summary, the results demonstrated that non-cytotoxic cationic THCpSiNPs are promising vehicles for therapeutic siRNA delivery.

  6. The inter-kingdom volatile signal indole promotes root development by interfering with auxin signalling.

    Science.gov (United States)

    Bailly, Aurélien; Groenhagen, Ulrike; Schulz, Stefan; Geisler, Markus; Eberl, Leo; Weisskopf, Laure

    2014-12-01

    Recently, emission of volatile organic compounds (VOCs) has emerged as a mode of communication between bacteria and plants. Although some bacterial VOCs that promote plant growth have been identified, their underlying mechanism of action is unknown. Here we demonstrate that indole, which was identified using a screen for Arabidopsis growth promotion by VOCs from soil-borne bacteria, is a potent plant-growth modulator. Its prominent role in increasing the plant secondary root network is mediated by interfering with the auxin-signalling machinery. Using auxin reporter lines and classic auxin physiological and transport assays we show that the indole signal invades the plant body, reaches zones of auxin activity and acts in a polar auxin transport-dependent bimodal mechanism to trigger differential cellular auxin responses. Our results suggest that indole, beyond its importance as a bacterial signal molecule, can serve as a remote messenger to manipulate plant growth and development.

  7. Variable selectivity of the Hitachi chemistry analyzer chloride ion-selective electrode toward interfering ions.

    Science.gov (United States)

    Wang, T; Diamandis, E P; Lane, A; Baines, A D

    1994-02-01

    Chloride measurements by ion-selective electrodes are vulnerable to interference by anions such as iodide, thiocyanate, nitrate, and bromide. We have found that the degree of interference of these anions on the Hitachi chemistry analyzer chloride electrode varies from electrode to electrode and this variation can even occur within the same lot of membrane. This variation is not dependent upon the length of time the cartridge has been in the analyzer because no correlation existed between the usage time and the electrode response to interfering ions. Neither is this variation due to the deterioration of the electrode because all electrodes tested had calibration slopes within the manufacturer's specification. Our study, however, showed that even after repeated exposure to a plasma sample containing 2 mM thiocyanate, the chloride electrode was still able to accurately measure the chloride in plasma without thiocyanate, thus confirming that a carryover effect does not exist from a previous thiocyanate-containing sample.

  8. Detailed balance study of time reversal invariance with interfering resonancesrefid="FN1">*

    Science.gov (United States)

    Mitchell, G. E.; Bilpuch, E. G.; Bybee, C. R.; Drake, J. M.; Shriner, J. F.

    1993-06-01

    Bunakov and Weidenmüller suggested that large enhancement of time reversal invariance violation may be observed near two interfering resonances via a test of detailed balance. In our (p, α) resonance data on 23Na, 27Al, 31P, 35Cl, and 39K, there are 33 pairs of adjacent resonances which have the same spin and parity. The difference in the differential cross sections for the (p, α 0) and (α, p 0) reactions was calculated for these resonance pairs using experimental values for the partial widths. The collision matrix elements were obtained for a Hamiltonian H = H0 + iH', following the approach of Moldauer. The differences show striking dependence on energy and angle and on the particular pair of resonances, with the relative sensitivity of the detailed balance test varying by many orders of magnitude. These preliminary results indicate that this class of experiments may be more sensitive than previous detailed balance tests.

  9. Interferência de plantas daninhas na cultura do quiabo Weed interference in okra crop

    Directory of Open Access Journals (Sweden)

    J.B. Santos

    2010-06-01

    Full Text Available Objetivou-se com este trabalho avaliar os períodos de interferência das plantas daninhas na cultura do quiabo (Abelmoschus esculentus na região do Médio Vale do Rio Doce, em Minas Gerais. O experimento foi conduzido em campo, entre maio e outubro de 2007. Utilizaram-se sementes do quiabo Santa Cruz-47, semeadas no espaçamento de 0,25 x 1 m. Foram estabelecidos diferentes períodos de controle das plantas daninhas na cultura, variando entre zero e 120 dias após a emergência (DAE. Foram avaliados 12 tratamentos, correspondendo a diferentes períodos de controle das plantas daninhas na cultura: capina após a emergência a partir dos 20, 40, 60, 80 e 100 dias; capina após a emergência até os 20, 40, 60, 80 e 100 dias; além de duas testemunhas com capina, ou não capinadas, ambas por 120 dias. Determinou-se o número de frutos por planta e o rendimento (produtividade, bem como os valores em dias para período anterior à interferência (PAI, período crítico de prevenção da interferência (PCPI e período total de prevenção da interferência (PTPI, considerando 5% de perdas. A partir das espécies encontradas na área experimental, avaliou-se também, em vasos, isoladamente ou em competição com o quiabeiro, a capacidade competitiva das principais plantas daninhas. Com base nos resultados, verificou-se que o PAI estimado foi de 25 DAE, indicando a época de início das capinas. Para o PCPI, o período observado foi de 75 dias, indicando PTPI de 100 DAE. Entre as plantas daninhas presentes, Eleusine indica apresentou maior capacidade competitiva sobre a cultura.An experiment was carried out under field conditions in Médio Vale do Rio Doce-MG, from May to October, 2007, to establish periods of weed interference in Abelmoschus esculentus crop. 'Santa Cruz-47' seeds were sown in a 0.25 x 1.0 m spacing, and weed control times varied from 0 to 120 days after emergence (DAE. Number of fruit per plant and yield as well as values in days

  10. Interferência de plantas daninhas na cultura do sorgo sacarino

    OpenAIRE

    Cézar Silva; Alexandre Ferreira da Silva; Welington Gonzaga do Vale; Leandro Galon; Fabiano André Petter; André May; Decio Karam

    2014-01-01

    Este trabalho teve como objetivo o estudo fitossociológico da comunidade infestante ao longo do ciclo de desenvolvimento do sorgo sacarino e a determinação do período crítico de prevenção à interferência das plantas daninhas na cultura. O delineamento experimental foi de blocos ao acaso com três repetições. Os tratamentos foram constituídos por períodos crescentes de controle ou convivência das plantas daninhas em diferentes estádios fenológicos da cultura: 0-3 (V3), 0-5 (V5), 0-7 (V7), 0-9 (...

  11. Nanograting formation on metals in air with interfering femtosecond laser pulses

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, Kenzo, E-mail: k-miyazaki@iae.kyoto-u.ac.jp, E-mail: kmiyazaki@wind.ocn.ne.jp [Laser Science Research Section, Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011 (Japan); Center for Collaborative Research and Community Cooperation, University of Miyazaki, Miyazaki 889-2192 (Japan); Miyaji, Godai, E-mail: gmiyaji@cc.tuat.ac.jp [Laser Science Research Section, Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011 (Japan); Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan); Inoue, Toshishige [Laser Science Research Section, Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2015-08-17

    It is demonstrated that a homogeneous nanograting having the groove period much smaller than the laser wavelength (∼800 nm) can be fabricated on metals in air through ablation induced by interfering femtosecond laser pulses (100 fs at a repetition rate of 10 Hz). Morphological changes on stainless steel and Ti surfaces, observed with an increase in superimposed shots of the laser pulses at a low fluence, have shown that the nanograting is developed through bonding structure change at the interference fringes, plasmonic near-field ablation to create parallel grooves on the fringe, and subsequent excitation of surface plasmon polaritons to regulate the groove intervals at 1/3 or 1/4 of the fringe period over the whole irradiated area. Calculation for a model target having a thin oxide layer on the metal substrate reproduces well the observed groove periods and explains the mechanism for the nanograting formation.

  12. Small interfering ribonucleic acid induces liquid-to-ripple phase transformation in a phospholipid membrane

    Science.gov (United States)

    Choubey, Amit; Nomura, Ken-ichi; Kalia, Rajiv K.; Nakano, Aiichiro; Vashishta, Priya

    2014-09-01

    Small interfering ribonucleic acid (siRNA) molecules play a pivotal role in silencing gene expression via the RNA interference mechanism. A key limitation to the widespread implementation of siRNA therapeutics is the difficulty of delivering siRNA-based drugs to cells. Here, we examine changes in the structure and dynamics of a dipalmitoylphosphatidylcholine bilayer in the presence of a siRNA molecule and mechanical barriers to siRNA transfection in the bilayer. Our all-atom molecular dynamics simulation shows that siRNA induces a liquid crystalline-to-ripple phase transformation in the bilayer. The ripple phase consists of a major region of non-interdigitated and a minor region of interdigitated lipid molecules with an intervening kink. In the ripple phase, hydrocarbon chains of lipid molecules have large compressive stresses, which present a considerable barrier to siRNA transfection.

  13. Interference Alignment Through User Cooperation for Two-cell MIMO Interfering Broadcast Channels

    CERN Document Server

    Shin, Wonjae; Lim, Jong-Bu; Shin, Changyong; Jang, Kyunghun

    2010-01-01

    This paper focuses on two-cell multiple-input multiple-output (MIMO) Gaussian interfering broadcast channels (MIMO-IFBC) with $K$ cooperating users on the cell-boundary of each BS. It corresponds to a downlink scenario for cellular networks with two base stations (BSs), and $K$ users equipped with Wi-Fi interfaces enabling to cooperate among users on a peer-to-peer basis. In this scenario, we propose a novel interference alignment (IA) technique exploiting user cooperation. Our proposed algorithm obtains the achievable degrees of freedom (DoF) of 2K when each BS and user have $M=K+1$ transmit antennas and $N=K$ receive antennas, respectively. Furthermore, the algorithm requires only a small amount of channel feedback information with the aid of the user cooperation channels. The simulations demonstrate that not only are the analytical results valid, but the achievable DoF of our proposed algorithm also outperforms those of conventional techniques.

  14. Cloned Defective Interfering Influenza RNA and a Possible Pan-Specific Treatment of Respiratory Virus Diseases

    Science.gov (United States)

    Dimmock, Nigel J.; Easton, Andrew J.

    2015-01-01

    Defective interfering (DI) genomes are characterised by their ability to interfere with the replication of the virus from which they were derived, and other genetically compatible viruses. DI genomes are synthesized by nearly all known viruses and represent a vast natural reservoir of antivirals that can potentially be exploited for use in the clinic. This review describes the application of DI virus to protect from virus-associated diseases in vivo using as an example a highly active cloned influenza A DI genome and virus that protects broadly in preclinical trials against different subtypes of influenza A and against non-influenza A respiratory viruses. This influenza A-derived DI genome protects by two totally different mechanisms: molecular interference with influenza A replication and by stimulating innate immunity that acts against non-influenza A viruses. The review considers what is needed to develop DI genomes to the point of entry into clinical trials. PMID:26184282

  15. Small interfering ribonucleic acid induces liquid-to-ripple phase transformation in a phospholipid membrane

    Energy Technology Data Exchange (ETDEWEB)

    Choubey, Amit; Nomura, Ken-ichi; Kalia, Rajiv K.; Nakano, Aiichiro; Vashishta, Priya [Collaboratory for Advanced Computing and Simulations, Department of Physics and Astronomy, Department