Deciphering the RNA degradome: A new tool for small RNA target discovery

RNA沉默是一种复杂和高度保守的调控机制，目前已知参与多种过程，如发育、病原菌控制、基因组维持和对环境变化的响应。自最近发现以来，RNA沉默已经成为植物和动物分子生物学中一个快速发展的研究领域，具有重要的意义。这一领域的研究极大地得益于新型高通量测序技术(如454和Solexa/Illumina)的新发展，这些技术可以在一次运行中生成多个数据集，每个数据集都包含数百万个小RNA(SRNA)分子，这些分子是所有RNA沉默现象的关键参与者。目前sRNA研究中的一个关键挑战是了解最近测序并存放在公共数据库中的数百万sRNA的功能。迎接这一挑战的第一步是找出每个sRNA调控哪些基因(如果有的话)。已经设计了几种计算方法来预测植物和动物的sRNA靶标，但这些只是预测，没有实验验证就不能依赖。然而，在过去一年左右的时间里，一种新的高通量实验技术被描述用于对无上限mRNAs的5‘端进行测序，包括那些被sRNAs靶向并经历内切的转录本。在植物中，降解的mRNA片段提供了sRNAs与其互补的mRNA靶标之间相互作用的证据，从而导致mRNA的切割和降解。因此，以这种方式对生物体的‘’RNA降解组‘’进行测序的可能性将彻底改变植物中的靶标验证，因为它首次允许对被切割的mRNA进行基因组规模的测序。目前只有一种工具可用于退化穹顶分析，而且它有几个局限性，不适合对这类数据的大规模分析。在这项提案中，我们将开发和实施一种新的方法来高通量分析降解组数据，这将允许用户验证整个sRNAome中的靶标，并基于降解组证据产生sRNA/mRNA相互作用网络。该工具将在网上提供，并可通过UEA植物SRNA工具包下载，该工具包是一组用户友好的工具，允许通过高性能计算分析高通量植物SRNA数据集，而不需要专业知识或专门的生物信息学支持。

RNA silencing is a complex and highly conserved regulatory mechanism that is now known to be involved in diverse processes such as development, pathogen control, genome maintenance and response to environmental changes. Since its recent discovery, RNA silencing has become a fast moving area of research of great importance in both plant and animal molecular biology. Research in this field has greatly profited from new developments in novel high-throughput sequencing technologies (such as 454 and Solexa/Illumina) which, in a single run, can generate several datasets each containing millions of small RNA (sRNA) molecules, the key players in all RNA silencing phenomena. A key challenge in sRNA research at present is to understand the function of the millions of sRNAs that have been recently sequenced and deposited in public databases. A first step in meeting this challenge is to find which (if any) genes are being regulated by each sRNA. Several computational approaches have been devised for sRNA target prediction in plants and animals but these are only predictions and cannot be relied upon without experimental validation. However, in the last year or so a new high-throughput experimental technique has been described for sequencing of the 5'-ends of uncapped mRNAs including those transcripts that are targeted by sRNAs and subjected to endonucleolytic cleavage. In plants, degraded mRNA fragments provide evidence of the interaction between sRNAs and their complimentary mRNA targets that lead to cleavage and degradation of the mRNA. Thus the possibility of sequencing the ''RNA degradome' of an organism in this manner is set to revolutionise target validation in plants since it permits the genomic scale sequencing of cleaved mRNAs for the first time. Currently there is only one tool available for degradome analysis and it has several limitations which make it unsuitable for large-scale analysis of such data. In this proposal we will develop and implement a novel approach to high-throughput analysis of degradome data which will allow users to validate targets across the entire sRNAome and produce a network of sRNA/mRNA interactions based on degradome evidence. The tool will be made available online and for download through the UEA Plant sRNA Toolkit, a collection of user-friendly tools allowing the analysis of high-throughput plant sRNA datasets with high-performance computing without the need for expert knowledge or dedicated bioinformatics support.