Silence最新文献

{"title":"Effect of small interfering RNA 3'-end overhangs on chemosensitivity to thymidylate synthase inhibitors.","authors":"John C Schmitz,&nbsp;Edward Chu","doi":"10.1186/1758-907X-2-1","DOIUrl":"https://doi.org/10.1186/1758-907X-2-1","url":null,"abstract":"<p><strong>Background: </strong>Small interfering RNAs (siRNAs) are double-stranded RNAs that effectively inhibit expression of its complimentary target mRNA. Standard siRNAs contain two nucleotide overhangs on their 3' end. While these overhangs are usually comprised of deoxythymidines (dT), it has been shown that any nucleotide can be used on the 3' end without affecting RNAi silencing.</p><p><strong>Results: </strong>It was recently shown that extension of the 3' end to five or eight dT molecules allows siRNAs to be effectively complexed with linear polyethylenimine (PEI), leading to enhanced cellular uptake and intracellular release. Here, we provide further evidence that only extended or 'sticky' siRNAs complexed with PEI result in significant target knockdown. However, when investigating the potential effects of these extended siRNAs on growth of human colon cancer RKO cells, we observed a dose-dependent reversal of cytotoxicity of a thymidylate synthase-targeted siRNA. In contrast, siRNAs with uridine overhangs maintained their growth inhibitory effects. We further demonstrated that dT-containing siRNAs prevented the cytotoxic effects of thymidylate synthase (TS) inhibitor compounds, such as ZD1694 and 5'-fluoro-deoxyuridine, while having no deleterious effect on cisplatin toxicity. We show that this rescue effect results from the rapid degradation of the siRNA.</p><p><strong>Conclusions: </strong>Given that TS is an important enzyme for cell growth and proliferation and that its expression is controlled by multiple pathways, the rescue of its growth inhibitory effects may have unintended consequences. As siRNAs are being developed as therapeutic molecules, it will be important to avoid such off-target effects due to dT release. Hence, siRNAs should contain only uridine residues in their 3'-end overhangs.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"2 1","pages":"1"},"PeriodicalIF":0.0,"publicationDate":"2011-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-2-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29609744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MicroR159 regulation of most conserved targets in Arabidopsis has negligible phenotypic effects.","authors":"Robert S Allen,&nbsp;Junyan Li,&nbsp;Maria M Alonso-Peral,&nbsp;Rosemary G White,&nbsp;Frank Gubler,&nbsp;Anthony A Millar","doi":"10.1186/1758-907X-1-18","DOIUrl":"https://doi.org/10.1186/1758-907X-1-18","url":null,"abstract":"<p><strong>Background: </strong>A current challenge of microRNA (miRNA) research is the identification of biologically relevant miRNA:target gene relationships. In plants, high miRNA:target gene complementarity has enabled accurate target predictions, and slicing of target mRNAs has facilitated target validation through rapid amplification of 5' cDNA ends (5'-RACE) analysis. Together, these approaches have identified more than 20 targets potentially regulated by the deeply conserved miR159 family in Arabidopsis, including eight MYB genes with highly conserved miR159 target sites. However, genetic analysis has revealed the functional specificity of the major family members, miR159a and miR159b is limited to only two targets, MYB33 and MYB65. Here, we examine the functional role of miR159 regulation for the other potential MYB target genes.</p><p><strong>Results: </strong>For these target genes, functional analysis failed to identify miR159 regulation that resulted in any major phenotypic impact, either at the morphological or molecular level. This appears to be mainly due to the quiescent nature of the remaining family member, MIR159c. Although its expression overlaps in a temporal and spatial cell-specific manner with a subset of these targets in anthers, the abundance of miR159c is extremely low and concomitantly a mir159c mutant displays no anther defects. Examination of potential miR159c targets with conserved miR159 binding sites found neither their spatial or temporal expression domains appeared miR159 regulated, despite the detection of miR159-guided cleavage products by 5'-RACE. Moreover, expression of a miR159-resistant target (mMYB101) resulted predominantly in plants that are indistinguishable from wild type. Plants that displayed altered morphological phenotypes were found to be ectopically expressing the mMYB101 transgene, and hence were misrepresentative of the in vivo functional role of miR159.</p><p><strong>Conclusions: </strong>This study presents a novel explanation for a paradox common to plant and animal miRNA systems, where among many potential miRNA-target relationships usually only a few appear physiologically relevant. The identification of a quiescent miR159c:target gene regulatory module in anthers provides a likely rationale for the presence of conserved miR159 binding sites in many targets for which miR159 regulation has no obvious functional role. Remnants from the demise of such modules may lead to an overestimation of miRNA regulatory complexity when investigated using bioinformatic, 5'-RACE or transgenic approaches.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"1 1","pages":"18"},"PeriodicalIF":0.0,"publicationDate":"2010-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-1-18","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29427976","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A structural-based statistical approach suggests a cooperative activity of PUM1 and miR-410 in human 3'-untranslated regions.","authors":"Limor Leibovich,&nbsp;Yael Mandel-Gutfreund,&nbsp;Zohar Yakhini","doi":"10.1186/1758-907X-1-17","DOIUrl":"https://doi.org/10.1186/1758-907X-1-17","url":null,"abstract":"<p><strong>Background: </strong>Micro (mi)RNAs comprise a large family of small non-coding RNAs that are thought to regulate a large fraction of protein-coding genes. Generally, miRNAs downregulate messenger (m)RNA expression by binding to the 3' untranslated regions (UTRs) of the RNA molecules. An important factor for binding specificity is the matching in the seed region. In addition, target site accessibility is thought to be crucial for efficient repression of miRNA targets. Several recent studies indicated that miRNA repression can be facilitated by RNA-binding proteins. In this study, we examine the conjecture that RNA-binding proteins are involved in ushering miRNAs to bind targets that are initially less accessible.</p><p><strong>Results: </strong>We analyzed human 3'-UTR sequences containing potential binding sites of 153 conserved miRNA families, and ranked sequences around the sites according to their miRNA accessibility. By applying a rank-based motif search tool to these miRNA targets, we found motifs that are enriched among less accessible targets. As expected from our ranking method, most of the significant motifs were GC-rich. However, one AU-rich motif was found to be enriched among miR-410 less accessible targets. This motif resembles the Pumilio homolog 1 (PUM1) consensus binding site. We observed a stronger enrichment of the PUM1 motif in conserved targets than in non-conserved targets; moreover, the enrichment of this motif was found to be conserved in a subset of placental mammals. Further, we analyzed publicly available gene expression data, and found that the mutual expression of PUM1 and miR-410 has a greater negative influence on the expression of low accessibility targets than on other targets, an effect that was stronger than when considering both miR-410 and PUM1 separately.</p><p><strong>Conclusions: </strong>Taken together, our findings suggest a cooperative relationship between miR-410 and PUM1 in regulating human highly structured 3'-UTRs. This kind of cooperation can allow a second level of regulation of such targets. Considering cases in which miRNAs bind low accessibility targets may help to improve current miRNA prediction tools and to obtain a better understanding of the mechanisms underlying miRNA regulation activity.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"1 1","pages":"17"},"PeriodicalIF":0.0,"publicationDate":"2010-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-1-17","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29294638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR).","authors":"Yosef Landesman,&nbsp;Nenad Svrzikapa,&nbsp;Armand Cognetta,&nbsp;Xuemei Zhang,&nbsp;Brian R Bettencourt,&nbsp;Satya Kuchimanchi,&nbsp;Keri Dufault,&nbsp;Sarfraz Shaikh,&nbsp;Maple Gioia,&nbsp;Akin Akinc,&nbsp;Renta Hutabarat,&nbsp;Rachel Meyers","doi":"10.1186/1758-907X-1-16","DOIUrl":"https://doi.org/10.1186/1758-907X-1-16","url":null,"abstract":"<p><strong>Background: </strong>While increasing numbers of small interfering RNA (siRNA) therapeutics enter into clinical trials, the quantification of siRNA from clinical samples for pharmacokinetic studies remains a challenge. This challenge is even more acute for the quantification of chemically modified and formulated siRNAs such as those typically required for systemic delivery.</p><p><strong>Results: </strong>Here, we describe a novel method, heating-in-Triton quantitative reverse transcription PCR (HIT qRT-PCR) that improves upon the stem-loop RT-PCR technique for the detection of formulated and chemically modified siRNAs from plasma and tissue. The broad dynamic range of this assay spans five orders of magnitude and can detect as little as 70 pg duplex in 1 g of liver or in 1 ml of plasma. We have used this assay to quantify intravenously administrated siRNA in rodents and have reliably correlated target reduction with tissue drug concentrations. We were able to detect siRNA in rat liver for at least 10 days post injection and determined that for a modified factor VII (FVII) siRNA, on average, approximately 500 siRNA molecules per cell are required to achieve a 50% target reduction.</p><p><strong>Conclusions: </strong>HIT qRT-PCR is a novel approach that simplifies the in vivo quantification of siRNA and provides a highly sensitive and reproducible tool to measure the silencing efficiency of chemically modified and formulated siRNAs.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"1 1","pages":"16"},"PeriodicalIF":0.0,"publicationDate":"2010-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-1-16","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29209088","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An endogenous F-box protein regulates ARGONAUTE1 in Arabidopsis thaliana.","authors":"K Earley, Mr Smith, R Weber, Bd Gregory, Rs Poethig","doi":"10.1186/1758-907X-1-15","DOIUrl":"10.1186/1758-907X-1-15","url":null,"abstract":"<p><p> ARGONAUTE1 (AGO1) mediates microRNA- and small interfering RNA-directed posttranscriptional gene silencing in Arabidopsis thaliana. Mutant alleles of SQUINT (SQN) slightly reduce AGO1 activity and have weak effects on shoot morphology. A screen for mutations that suppress the sqn phenotype produced loss-of-function mutations in the F-box gene FBW2. Mutations in FBW2 not only suppress sqn but also suppress many of the developmental phenotypes of weak, but not null, alleles of AGO1 by increasing AGO1 protein levels. Conversely, over-expression of FBW2 decreases the abundance of the AGO1 protein but not AGO1 messenger RNA, further indicating that FBW2 regulates AGO1 protein levels. fbw2 mutants have no obvious morphological phenotype, but display a reduced sensitivity to abscisic acid (ABA) that can be attributed to increased AGO1 activity. Our results indicate that FBW2 is a novel negative regulator of AGO1 and suggest that it plays a role in ABA signalling and/or response.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"1 1","pages":"15"},"PeriodicalIF":0.0,"publicationDate":"2010-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2914764/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29117695","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A status report on RNAi therapeutics.","authors":"Akshay K Vaishnaw,&nbsp;Jared Gollob,&nbsp;Christina Gamba-Vitalo,&nbsp;Renta Hutabarat,&nbsp;Dinah Sah,&nbsp;Rachel Meyers,&nbsp;Tony de Fougerolles,&nbsp;John Maraganore","doi":"10.1186/1758-907X-1-14","DOIUrl":"https://doi.org/10.1186/1758-907X-1-14","url":null,"abstract":"<p><p> Fire and Mello initiated the current explosion of interest in RNA interference (RNAi) biology with their seminal work in Caenorhabditis elegans. These observations were closely followed by the demonstration of RNAi in Drosophila melanogaster. However, the full potential of these new discoveries only became clear when Tuschl and colleagues showed that 21-22 bp RNA duplexes with 3\" overhangs, termed small interfering (si)RNAs, could reliably execute RNAi in a range of mammalian cells. Soon afterwards, it became clear that many different human cell types had endogenous machinery, the RNA-induced silencing complex (RISC), which could be harnessed to silence any gene in the genome. Beyond the availability of a novel way to dissect biology, an important target validation tool was now available. More importantly, two key properties of the RNAi pathway - sequence-mediated specificity and potency - suggested that RNAi might be the most important pharmacological advance since the advent of protein therapeutics. The implications were profound. One could now envisage selecting disease-associated targets at will and expect to suppress proteins that had remained intractable to inhibition by conventional methods, such as small molecules. This review attempts to summarize the current understanding on siRNA lead discovery, the delivery of RNAi therapeutics, typical in vivo pharmacological profiles, preclinical safety evaluation and an overview of the 14 programs that have already entered clinical practice.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"1 1","pages":"14"},"PeriodicalIF":0.0,"publicationDate":"2010-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-1-14","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29109577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"RNA silencing in plants: Flash report!","authors":"Rebecca A Mosher,&nbsp;Mathew G Lewsey,&nbsp;Padubidri V Shivaprasad","doi":"10.1186/1758-907X-1-13","DOIUrl":"https://doi.org/10.1186/1758-907X-1-13","url":null,"abstract":"<p><p> Earlier this year plant scientists met in Santa Fe, New Mexico at the Keystone Symposium \"RNA Silencing Mechanisms in Plants\". Sessions included small RNA biogenesis and signalling, development and stress responses, small RNA-directed DNA methylation, and interaction with pathogens. This report highlights some of the prominent and recurring themes at the meeting and emerging arenas of future research.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"1 1","pages":"13"},"PeriodicalIF":0.0,"publicationDate":"2010-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-1-13","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29091592","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Naturally occurring variations in sequence length creates microRNA isoforms that differ in argonaute effector complex specificity.","authors":"H Alexander Ebhardt,&nbsp;Amber Fedynak,&nbsp;Richard P Fahlman","doi":"10.1186/1758-907X-1-12","DOIUrl":"https://doi.org/10.1186/1758-907X-1-12","url":null,"abstract":"<p><strong>Background: </strong>Micro(mi)RNAs are short RNA sequences, ranging from 16 to 35 nucleotides (miRBase; http://www.mirbase.org). The majority of the identified sequences are 21 or 22 nucleotides in length. Despite the range of sequence lengths for different miRNAs, individual miRNAs were thought to have a specific sequence of a particular length. A recent report describing a longer variant of a previously identified miRNA in Arabidopsis thaliana prompted this investigation for variations in the length of other miRNAs.</p><p><strong>Results: </strong>In this paper, we demonstrate that a fifth of annotated A. thaliana miRNAs recorded in miRBase V.14 have stable miRNA isoforms that are one or two nucleotides longer than their respective recorded miRNA. Further, we demonstrate that miRNA isoforms are co-expressed and often show differential argonaute complex association. We postulate that these extensions are caused by differential cleavage of the parent precursor miRNA.</p><p><strong>Conclusions: </strong>Our systematic analysis of A. thaliana miRNAs reveals that miRNA length isoforms are relatively common. This finding not only has implications for miRBase and miRNA annotation, but also extends to miRNA validation experiments and miRNA localization studies. Further, we predict that miRNA isoforms are present in other plant species also.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"1 1","pages":"12"},"PeriodicalIF":0.0,"publicationDate":"2010-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-1-12","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29042679","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"How do miRNAs mediate translational repression?","authors":"Shuo Gu,&nbsp;Mark A Kay","doi":"10.1186/1758-907X-1-11","DOIUrl":"https://doi.org/10.1186/1758-907X-1-11","url":null,"abstract":"<p><p> Micro(mi)RNAs regulate gene expression by what are believed to be related but separate mechanistic processes. The relative contribution that each process plays, their mechanistic overlap, and the degree by which they regulate complex genetic networks is still being unraveled. One process by which miRNAs inhibit gene expression occurs through translational repression. In recent years, there has been a plethora of studies published, which have resulted in various molecular models of how miRNAs impair translation. At first evaluation, it appears that these models are quite different and incompatible with one another. In this paper, we focus on possible explanations for the various interpretations of these data sets, and provide a model that we believe is consistent with many of the observations published to date.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"1 1","pages":"11"},"PeriodicalIF":0.0,"publicationDate":"2010-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-1-11","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28980457","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Specificity and functionality of microRNA inhibitors.","authors":"Barbara Robertson,&nbsp;Andrew B Dalby,&nbsp;Jon Karpilow,&nbsp;Anastasia Khvorova,&nbsp;Devin Leake,&nbsp;Annaleen Vermeulen","doi":"10.1186/1758-907X-1-10","DOIUrl":"https://doi.org/10.1186/1758-907X-1-10","url":null,"abstract":"<p><strong>Background: </strong>Micro(mi)RNAs regulate gene expression through translational attenuation and messenger (m)RNA degradation, and are associated with differentiation, homeostasis and disease. Natural miRNA target recognition is determined primarily by perfect complementarity in a seed region (nucleotide positions 2 to 7) with additional interactions contributing in a sequence- and target-specific manner. Synthetic miRNA target analogs, which are fully complementary, chemically modified oligonucleotides, have been used successfully to inhibit miRNA function.</p><p><strong>Results: </strong>In this paper, we present a first systematic study to evaluate the effect of mismatches in the target site on synthetic inhibitor activity. Panels of miRNA inhibitors containing two-nucleotide mismatches across the target site were tested against three miRNAs (miR-21, miR-22 and miR-122). The results showed that the function of inhibitors vary as mismatch positions in the inhibitors change.</p><p><strong>Conclusions: </strong>The data indicate that features important for natural miRNA target recognition (such as seed region complementarity) are also important for inhibitor functionality. In addition, base pairing at a second, more 3' region appears to be equally important in determining the efficacy of synthetic inhibitors. Considering the importance of these inhibitor regions and the expression of closely related miRNA sequences will enable researchers to interpret results more accurately in future experiments.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"1 1","pages":"10"},"PeriodicalIF":0.0,"publicationDate":"2010-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-1-10","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28894783","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}