http://www.silencejournal.com The latest research articles published by Silence 2013-05-20T00:00:00Z Background: Post-transcriptional regulation of gene expression by small RNAs and RNA binding proteins is of fundamental importance in development of complex organisms, and dysregulation of regulatory RNAs can influence onset, progression and potential treatment of many diseases. Post-transcriptional regulation by small RNAs is mediated through partial complementary binding to messenger RNAs leaving nucleotide signatures or motifs throughout the entire transcriptome. Computational methods for discovery and analysis of sequence motifs in high-throughput mRNA expression profiling experiments are becoming increasingly important tools for the identification of post-transcriptional regulatory motifs and the inference of the regulators and their targets. Results: cWords is a method designed for regulatory motif discovery in differential case--control mRNA expression datasets. We have improved the algorithms and statistical methods of cWords, resulting in at least a factor 100 speed gain over the previous implementation. On a benchmark dataset of 19 microRNA (miRNA) perturbation experiments cWords showed equal or better performance than two comparable methods, miReduce and Sylamer. We have developed rigorous motif clustering and visualization that accompany the cWords analysis for more intuitive and effective data interpretation. To demonstrate the versatility of cWords we show that it can also be used for identification of potential siRNA off-target binding. Moreover, cWords analysis of an experiment profiling mRNAs bound by Argonaute ribonucleoprotein particles discovered endogenous miRNA binding motifs. Conclusions: cWords is an unbiased, flexible and easy-to-use tool designed for regulatory motif discovery in differential case--control mRNA expression datasets. cWords is based on rigorous statistical methods that demonstrate comparable or better performance than other existing methods. Rich visualization of results promotes intuitive and efficient interpretation of data. cWords is available as a stand-alone Open Source program at Github https://github.com/simras/cWords and as a web-service at: http://servers.binf.ku.dk/cwords/. http://www.silencejournal.com/content/4/1/2 Simon Rasmussen Anders Jacobsen Anders Krogh Silence 2013, null:2 2013-05-20T00:00:00Z doi:10.1186/1758-907X-4-2 /content/figures/1758-907X-4-2-toc.gif Silence 1758-907X ${item.volume} 2 2013-05-20T00:00:00Z PDF Background: DNA methylation ensures genome integrity and regulates gene expression in diverse eukaryotes. In Arabidopsis, methylation occurs in three sequence contexts: CG, CHG and CHH. The initial establishment of DNA methylation at all three sequence contexts occurs through a process known as RNA-directed DNA methylation (RdDM), in which small RNAs bound by Argonaute4 (AGO4) guide DNA methylation at homologous loci through the de novo methyltransferase DRM2. Once established, DNA methylation at each of the three sequence contexts is maintained through different mechanisms. Although some players involved in RdDM and maintenance methylation have been identified, the underlying molecular mechanisms are not fully understood. To aid the comprehensive identification of players in DNA methylation, we generated a transgenic reporter system that permits genetic and chemical genetic screens in Arabidopsis. Results: A dual 35S promoter (d35S) driven luciferase (LUC) reporter was introduced into Arabidopsis and LUCL, a line with a low basal level of luciferase activity, was obtained. LUCL was found to be a multi-copy, single-insertion transgene that contains methylated cytosines in CG, CHG and CHH contexts, with the highest methylation in the CG context. Methylation was present throughout the promoter and LUC coding region. Treatment with an inhibitor of cytosine methylation de-repressed luciferase activity. A mutation in MET1, which encodes the CG maintenance methyltransferase, drastically reduced CG methylation and de-repressed LUC expression. Mutations in AGO4 and DRM2 also de-repressed LUC expression, albeit to a smaller extent than loss of MET1. Using LUCL as a reporter line, we performed a chemical screen for compounds that de-repress LUC expression, and identified a chemical, methotrexate, known to be involved in biogenesis of the methyl donor. Conclusion: We developed a luciferase-based reporter system, LUCL, which reports both RdDM and CG maintenance methylation in Arabidopsis. The low basal level of LUCL expression provides an easy readout in genetic and chemical genetic screens that will dissect the mechanisms of RdDM and methylation maintenance. http://www.silencejournal.com/content/4/1/1 Thanh Theresa Dinh Michael O¿Leary So Youn Won Shengben Li Lorena Arroyo Xigang Liu Andrew Defries Binglian Zheng Sean Cutler Xuemei Chen Silence 2013, null:1 2013-04-05T00:00:00Z doi:10.1186/1758-907X-4-1 /content/figures/1758-907X-4-1-toc.gif Silence 1758-907X ${item.volume} 1 2013-04-05T00:00:00Z XML Background: High throughput DNA sequencing technology has enabled quantification of all the RNAs in a cell or tissue, a method widely known as RNA sequencing (RNA-Seq). However, non-coding RNAs such as rRNA are highly abundant and can consume >70% of sequencing reads. A common approach is to extract only polyadenylated mRNA; however, such approaches are blind to RNAs with short or no poly(A) tails, leading to an incomplete view of the transcriptome. Another challenge of preparing RNA-Seq libraries is to preserve the strand information of the RNAs.DesignHere, we describe a procedure for preparing RNA-Seq libraries from 1 to 4 μg total RNA without poly(A) selection. Our method combines the deoxyuridine triphosphate (dUTP)/uracil-DNA glycosylase (UDG) strategy to achieve strand specificity with AMPure XP magnetic beads to perform size selection. Together, these steps eliminate gel purification, allowing a library to be made in less than two days. We barcode each library during the final PCR amplification step, allowing several samples to be sequenced in a single lane without sacrificing read length. Libraries prepared using this protocol are compatible with Illumina GAII, GAIIx and HiSeq 2000 platforms.DiscussionThe RNA-Seq protocol described here yields strand-specific transcriptome libraries without poly(A) selection, which provide approximately 90% mappable sequences. Typically, more than 85% of mapped reads correspond to protein-coding genes and only 6% derive from non-coding RNAs. The protocol has been used to measure RNA transcript identity and abundance in tissues from flies, mice, rats, chickens, and frogs, demonstrating its general applicability. http://www.silencejournal.com/content/3/1/9 Zhao Zhang William Theurkauf Zhiping Weng Phillip Zamore Silence 2012, null:9 2012-12-28T00:00:00Z doi:10.1186/1758-907X-3-9 /content/figures/1758-907X-3-9-toc.gif Silence 1758-907X ${item.volume} 9 2012-12-28T00:00:00Z XML Background: The processing of a microRNA results in an intermediate duplex of two potential mature products that derive from the two arms (5′ and 3′) of the precursor hairpin. It is often suggested that one of the sequences is degraded and the other is incorporated into the RNA-induced silencing complex. However, both precursor arms may give rise to functional levels of mature microRNA and the dominant product may change from species to species, from tissue to tissue, or between developmental stages. Therefore, both arms of the precursor have the potential to produce functional mature microRNAs. Results: We have investigated the relationship between predicted mRNA targets of mature sequences derived from the 5′ and 3′ arms of the same pre-microRNAs. Using six state-of-the-art target prediction algorithms, we find that 5′/3′ microRNA pairs target different sites in 3′ untranslated regions of mRNAs. We also find that these pairs do not generally target overlapping sets of genes, or functionally related genes. Conclusions: We show that alternative mature products produced from the same precursor microRNAs have different targeting properties and therefore different biological functions. These data strongly suggest that developmental or evolutionary changes in arm choice will have significant functional consequences. http://www.silencejournal.com/content/3/1/8 Antonio Marco Jamie MacPherson Matthew Ronshaugen Sam Griffiths-Jones Silence 2012, null:8 2012-09-27T00:00:00Z doi:10.1186/1758-907X-3-8 /content/figures/1758-907X-3-8-toc.gif Silence 1758-907X ${item.volume} 8 2012-09-27T00:00:00Z XML This month, Silence launches a new series on methods and protocols to study silencing pathways and analyze nucleic acids and proteins. http://www.silencejournal.com/content/3/1/7 Phillip Zamore Silence 2012, null:7 2012-06-07T00:00:00Z doi:10.1186/1758-907X-3-7 /content/figures/1758-907X-3-7-toc.gif Silence 1758-907X ${item.volume} 7 2012-06-07T00:00:00Z XML Background: Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis. Results: We introduced a dual 35 S promoter (d35S) driven luciferase reporter, LUCH, into Arabidopsis and isolated a line with a moderate level of luciferase activity. LUCH produced transgene-specific 24 nucleotide siRNAs and its d35S contained methylated cytosine in CG, CHG and CHH contexts. Treatment of the transgenic line with an inhibitor of cytosine methylation de-repressed luciferase activity. Mutations in several components of the RdDM pathway but not the maintenance methylation genes resulted in reduced d35S methylation, especially CHH methylation, and de-repression of luciferase activity. A mutation in MOM1, which is known to cooperate with RdDM to silence transposons, reduced d35S DNA methylation and de-repressed LUCH expression. A mutation in ROS1, a cytosine demethylation enzyme, increased d35S methylation and reduced LUCH expression. Conclusion: We developed a luciferase-based reporter, LUCH, which reports both DNA methylation directed by small RNAs and active demethylation by ROS1 in Arabidopsis. The moderate basal level of LUCH expression allows for bi-directional genetic screens that dissect the mechanisms of DNA methylation as well as demethylation. http://www.silencejournal.com/content/3/1/6 So Youn Won Shengben Li Binglian Zheng Yuanyuan Zhao Dongming Li Xin Zhao Huilan Yi Lei Gao Thanh Dinh Xuemei Chen Silence 2012, null:6 2012-06-07T00:00:00Z doi:10.1186/1758-907X-3-6 Novel screen for transcriptional gene silencing A luciferase-based reporter system, LUCH, is used to screen for bidirectional mutants that enhance or suppress transcriptional gene silencing, elucidating the mechanisms of DNA methylation and demethylation. /content/figures/1758-907X-3-6-toc.gif Silence 1758-907X ${item.volume} 6 2012-06-07T00:00:00Z XML To reduce the losses caused by plant pathogens, plant biologists have adopted numerous methods to engineer resistant plants. Among them, RNA silencing-based resistance has been a powerful tool that has been used to engineer resistant crops during the last two decades. Based on this mechanism, diverse approaches were developed. In this review, we focus on the application of RNA silencing to produce plants that are resistant to plant viruses such as RNA and DNA viruses, viroids, insects, and the recent expansion to fungal pathogens. http://www.silencejournal.com/content/3/1/5 Cheng-Guo Duan Chun-Han Wang Hui-Shan Guo Silence 2012, null:5 2012-05-31T00:00:00Z doi:10.1186/1758-907X-3-5 RNA silencing: a powerful tool against plant disease Hui-Shan Guo and colleagues review the application of RNA silencing in engineering plants that are resistant to plant viruses, viroids, insects and, more recently, fungal pathogens. /content/figures/1758-907X-3-5-toc.gif Silence 1758-907X ${item.volume} 5 2012-05-31T00:00:00Z XML Background: The use of nucleic acid-modifying enzymes has driven the rapid advancement in molecular biology. Understanding their function is important for modifying or improving their activity. However, functional analysis usually relies upon low-throughput experiments. Here we present a method for functional analysis of nucleic acid-modifying enzymes using next generation sequencing.FindingsWe demonstrate that sequencing data of libraries generated by RNA ligases can reveal novel secondary structure preferences of these enzymes, which are used in small RNA cloning and library preparation for NGS. Using this knowledge we demonstrate that the cloning bias in small RNA libraries is RNA ligase-dependent. We developed a high definition (HD) protocol that reduces the RNA ligase-dependent cloning bias. The HD protocol doubled read coverage, is quantitative and found previously unidentified microRNAs. In addition, we show that microRNAs in miRBase are those preferred by the adapters of the main sequencing platform. Conclusions: Sequencing bias of small RNAs partially influenced which microRNAs have been studied in depth; therefore most previous small RNA profiling experiments should be re-evaluated. New microRNAs are likely to be found, which were selected against by existing adapters. Preference of currently used adapters towards known microRNAs suggests that the annotation of all existing small RNAs, including miRNAs, siRNAs and piRNAs, has been biased. http://www.biomedcentral.com//3/1/4 Karim Sorefan Helio Pais Adam Hall Ana Kozomara Sam Griffiths-Jones Vincent Moulton Tamas Dalmay Silence 2012, null:4 2012-05-30T00:00:00Z doi:10.1186/1758-907X-3-4 /content/figures/1758-907X-3-4-toc.gif Silence 1758-907X ${item.volume} 4 2012-05-30T00:00:00Z XML 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