http://www.silencejournal.com The latest research articles published by Silence 2012-02-10T00:00:00Z Background: MicroRNA (miRNA) target genes tend to have relatively long and conserved 3' untranslated regions (UTRs), but to what degree these characteristics contribute to miRNA targeting is poorly understood. Different high-throughput experiments have, for example, shown that miRNAs preferentially regulate genes with both short and long 3' UTRs and that target site conservation is both important and irrelevant for miRNA targeting. Results: We have analyzed several gene context-dependent features, including 3' UTR length, 3' UTR conservation, and messenger RNA (mRNA) expression levels, reported to have conflicting influence on miRNA regulation. By taking into account confounding factors such as technology-dependent experimental bias and competition between transfected and endogenous miRNAs, we show that two factors - target gene expression and competition - could explain most of the previously reported experimental differences. Moreover, we find that these and other target site-independent features explain about the same amount of variation in target gene expression as the target site-dependent features included in the TargetScan model. Conclusions: Our results show that it is important to consider confounding factors when interpreting miRNA high throughput experiments and urge special caution when using microarray data to compare average regulatory effects between groups of genes that have different average gene expression levels. http://www.silencejournal.com/content/3/1/3 Takaya Saito Pal Saetrom Silence 2012, null:3 2012-02-10T00:00:00Z doi:10.1186/1758-907X-3-3 /content/figures/1758-907X-3-3-toc.gif Silence 1758-907X ${item.volume} 3 2012-02-10T00:00:00Z XML An important challenge in biology has been to understand how cell-type-specific expression programs are orchestrated through regulated access to chromatin. Knowledge of the interaction between noncoding RNAs (ncRNAs) and chromatin regulators has the potential to help answer such questions, but how ncRNAs target chromatin regulators to specific sites in the genome is not well understood. Recently, Jeon and Lee proposed that DNA-binding proteins act as a bridge between ncRNAs and their target sites in chromatin. In this minireview, we examine their findings and place them in the wider context of how chromatin regulator-RNA complexes are targeted to specific sites in chromatin. http://www.silencejournal.com/content/3/1/2 Aditi Kanhere Richard Jenner Silence 2012, null:2 2012-01-31T00:00:00Z doi:10.1186/1758-907X-3-2 /content/figures/1758-907X-3-2-toc.gif Silence 1758-907X ${item.volume} 2 2012-01-31T00:00:00Z XML MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of gene expression in many developmental and cellular processes. Moreover, there is now ample evidence that perturbations in the levels of individual or entire families of miRNAs are strongly associated with the pathogenesis of a wide range of human diseases. Indeed, disease-associated miRNAs represent a new class of targets for the development of miRNA-based therapeutic modalities, which may yield patient benefits unobtainable by other therapeutic approaches. The recent explosion in miRNA research has accelerated the development of several computational and experimental approaches for probing miRNA functions in cell culture and in vivo. In this review, we focus on the use of antisense oligonucleotides (antimiRs) in miRNA inhibition for loss-of-function studies. We provide an overview of the currently employed antisense chemistries and their utility in designing antimiR oligonucleotides. Furthermore, we describe the most commonly used in vivo delivery strategies and discuss different approaches for assessment of miRNA inhibition and potential off-target effects. Finally, we summarize recent progress in antimiR mediated pharmacological inhibition of disease-associated miRNAs, which shows great promise in the development of novel miRNA-based therapeutics. http://www.silencejournal.com/content/3/1/1 Jan Stenvang Andreas Petri Morten Lindow Susanna Obad Sakari Kauppinen Silence 2012, null:1 2012-01-09T00:00:00Z doi:10.1186/1758-907X-3-1 /content/figures/1758-907X-3-1-toc.gif Silence 1758-907X ${item.volume} 1 2012-01-09T00:00:00Z XML For the past five years, evidence has accumulated that vector-mediated robust RNA interference (RNAi) expression can trigger severe side effects in small and large animals, from cytotoxicity and accelerated tumorigenesis to organ failure and death. The recurring notions in these studies that a critical parameter is the strength of RNAi expression and that Exportin-5 and the Argonaute proteins are rate-limiting mammalian RNAi, strongly imply dose-dependent saturation of the endogenous miRNA pathway as one of the underlying mechanisms. This minireview summarizes the relevant work and data leading to this intriguing model and highlights potential avenues by which to alleviate RNAi-induced toxicities in future clinical applications. http://www.silencejournal.com/content/2/1/8 Dirk Grimm Silence 2011, null:8 2011-10-26T00:00:00Z doi:10.1186/1758-907X-2-8 /content/figures/1758-907X-2-8-toc.gif Silence 1758-907X ${item.volume} 8 2011-10-26T00:00:00Z XML Background: MicroRNAs, post-transcriptional regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies that suppress microRNA functions, resulting in a sustainable infection. In this work we report that Helicobacter pylori, a human stomach-colonizing bacterium responsible for severe gastric inflammatory diseases and gastric cancers, downregulates an embryonic stem cell microRNA cluster in proliferating gastric epithelial cells to achieve cell cycle arrest. Results: Using a deep sequencing approach in the AGS cell line, a widely used cell culture model to recapitulate early events of H. pylori infection of gastric mucosa, we reveal that hsa-miR-372 is the most abundant microRNA expressed in this cell line, where, together with hsa-miR-373, it promotes cell proliferation by silencing large tumor suppressor homolog 2 (LATS2) gene expression. Shortly after H. pylori infection, miR-372 and miR-373 synthesis is highly inhibited, leading to the post-transcriptional release of LATS2 expression and thus, to a cell cycle arrest at the G1/S transition. This downregulation of a specific cell-cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells, a mechanism highly associated with the development of severe atrophic gastritis and intestinal-type gastric carcinoma. Conclusions: These data constitute a novel example of host-pathogen interplay involving microRNAs, and unveil the couple LATS2/miR-372 and miR-373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells, which may be relevant in inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections. http://www.silencejournal.com/content/2/1/7 Cedric Belair Jessica Baud Sandrine Chabas Cynthia Sharma Joerg Vogel Cathy Staedel Fabien Darfeuille Silence 2011, null:7 2011-10-25T00:00:00Z doi:10.1186/1758-907X-2-7 /content/figures/1758-907X-2-7-toc.gif Silence 1758-907X ${item.volume} 7 2011-10-25T00:00:00Z XML The study of miRNAs and other noncoding RNAs has revolutionised our understanding of gene expression regulation during cancer development and progression, creating one of the fastest-growing research fields in cancer with realistic therapeutic potential. The 2011 Non-coding RNAs and Cancer Symposium hosted by the University College London Cancer Institute focused on the function and regulation of noncoding RNAs during oncogenesis. http://www.silencejournal.com/content/2/1/6 Ohad Yogev Dimitris Lagos Silence 2011, null:6 2011-09-29T00:00:00Z doi:10.1186/1758-907X-2-6 /content/figures/1758-907X-2-6-toc.gif Silence 1758-907X ${item.volume} 6 2011-09-29T00:00:00Z XML Shortly after their discovery, repertoires of miRNA were identified, together with proteins involved in their biogenesis and action. It is now obvious that miRNA-mediated gene regulation itself is regulated at multiple levels. Identifying the regulatory mechanisms that underpin small RNA homeostasis by modulation of their biogenesis and action has become a key issue, which can be partly resolved by identifying mediators of Argonautes turnover. An emerging theme in the control of Argonaute stability and activity is through posttranslational modifications, which are the focus of this review. http://www.silencejournal.com/content/2/1/5 Michael Johnston Gyorgy Hutvagner Silence 2011, null:5 2011-09-26T00:00:00Z doi:10.1186/1758-907X-2-5 /content/figures/1758-907X-2-5-toc.gif Silence 1758-907X ${item.volume} 5 2011-09-26T00:00:00Z XML Background: MicroRNA (miRNA) are diverse in sequence and have a single known sequence bias: they tend to start with uridine (U). Results: Our analyses of fly, worm and mouse miRNA sequence data reveal that the 5′-U is recognized after miRNA production. Only one of the two strands can be assembled into Argonaute protein from a single miRNA/miRNA* molecule: in fly embryo lysate, a 5′-U promotes miRNA loading while decreasing the loading of the miRNA*. Conclusion: We suggest that recognition of the 5′-U enhances Argonaute loading by a mechanism distinct from its contribution to weakening base pairing at the 5′-end of the prospective miRNA and, as recently proposed in Arabidopsis and in humans, that it improves miRNA precision by excluding incorrectly processed molecules bearing other 5′-nt. http://www.silencejournal.com/content/2/1/4 Herve Seitz Jogender Tushir Phillip Zamore Silence 2011, null:4 2011-06-07T00:00:00Z doi:10.1186/1758-907X-2-4 /content/figures/1758-907X-2-4-toc.gif Silence 1758-907X ${item.volume} 4 2011-06-07T00:00:00Z XML Background: RNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms. We performed a small interfering (si)RNA screen for novel members of the transforming growth factor (TGF)-β pathway in a human keratinocyte cell line. The TGF-β pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-β responses have been strongly implicated in cancer. Results: We assayed how strongly single siRNAs targeting each of 6,000 genes affect the nuclear translocation of a green fluorescent protein (GFP)-SMAD2 reporter fusion protein. Surprisingly, we found no novel TGF-β pathway members, but we did find dominant off-target effects. All siRNA hits, whatever their intended direct target, reduced the mRNA levels of two known upstream pathway components, the TGF-β receptors 1 and 2 (TGFBR1 and TGFBR2), via micro (mi)RNA-like off-target effects. The scale of these off-target effects was remarkable, with at least 1% of the sequences in the unbiased siRNA library having measurable off-target effects on one of these two genes. It seems that relatively minor reductions of message levels via off-target effects can have dominant effects on an assay, if the pathway output is very dose-sensitive to levels of particular pathway components. In search of mechanistic details, we identified multiple miRNA-like sequence characteristics that correlated with the off-target effects. Based on these results, we identified miR-20a, miR-34a and miR-373 as miRNAs that inhibit TGFBR2 expression. Conclusions: Our findings point to potential improvements for miRNA/siRNA target prediction methods, and suggest that the type II TGF-β receptor is regulated by multiple miRNAs. We also conclude that the risk of obtaining misleading results in siRNA screens using large libraries with single-assay readout is substantial. Control and rescue experiments are essential in the interpretation of such screens, and improvements to the methods to reduce or predict RNAi off-target effects would be beneficial. http://www.silencejournal.com/content/2/1/3 Nikolaus Schultz Dina Marenstein Dino De Angelis Wei-Qing Wang Sven Nelander Anders Jacobsen Debora Marks Joan Massague Chris Sander Silence 2011, null:3 2011-03-14T00:00:00Z doi:10.1186/1758-907X-2-3 /content/figures/1758-907X-2-3-toc.gif Silence 1758-907X ${item.volume} 3 2011-03-14T00:00:00Z XML Prior to the advent of new, deep sequencing methods, small RNA (sRNA) discovery was dependent on Sanger sequencing, which was time-consuming and limited knowledge to only the most abundant sRNA. The innovation of large-scale, next-generation sequencing has exponentially increased knowledge of the biology, diversity and abundance of sRNA populations. In this review, we discuss issues involved in the design of sRNA sequencing experiments, including choosing a sequencing platform, inherent biases that affect sRNA measurements and replication. We outline the steps involved in preprocessing sRNA sequencing data and review both the principles behind and the current options for normalization. Finally, we discuss differential expression analysis in the absence and presence of biological replicates. While our focus is on sRNA sequencing experiments, many of the principles discussed are applicable to the sequencing of other RNA populations. http://www.silencejournal.com/content/2/1/2 Kevin McCormick Matthew Willmann Blake Meyers Silence 2011, null:2 2011-02-28T00:00:00Z doi:10.1186/1758-907X-2-2 /content/figures/1758-907X-2-2-toc.gif Silence 1758-907X ${item.volume} 2 2011-02-28T00:00:00Z XML