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

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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.