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

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.

Novel sequencing technologies, such as 454 and Solexa/Illumina, have become importants tool for researchers in the field of RNA silencing, due to the fact that they can sequence millions of sRNAs in a single experiment. Over recent years researchers have been using this technology to catalogue entire 'sRNAomes' from a variety of organisms but, at present, we only know the function of only relatively few sRNAs. In the past year or so a new high-throughput experimental technique has been described that allows researchers to sequence 5'-ends of uncapped mRNAs including all transcripts targeted by sRNAs and subjected to endonucleolytic cleavage. In plants, degraded mRNA fragments provide evidence of the interaction between sRNAs and their complementary 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. In this proposal we will develop a new bioinformatics method for plant degradome analysis which will allow the identification of all sRNAs that are able to target and cleave mRNAs. This method will take as input degradome and sRNA data generated using next generation sequencing technologies together with the relevant transcriptome. The tool will output all possible interactions between sRNAs and mRNAs in a format that can be viewed using a standard open source network visualisation tool. To facilitate accessibility of the tool to wet-lab biologists we will integrate it into the existing UEA Plant sRNA Tools website which will enable users to run jobs on a 100+ node compute cluster, thus removing any requirement for users to have access to high-performance computing facilities. In addition a standalone command-line version of the tool will be made freely available for use by researchers having appropriate resources.

Over the past year, The UEA small RNA Toolkit (UPsT) has been used extensively by plant small RNA researchers both in the UK and worldwide with an average of 30-50 analyses performed per week. It has an attractive website, is easy to use, and is the only complete solution for processing and analysing plant high-throughput sRNA data both in the UK and internationally. We regularly receive emails from users requesting extra tools and features such as those that we intend to implement in this project. Moreover, there are several UK groups working with next generation sequencing sRNA data, for example, in miRNA discovery. It is therefore clear that user demand for tools such as those to be developed in this project is strong. Moulton's group has a strong track record in publicly releasing and promoting both the algorithms and software that it develops. Indeed, all websites and software created as part of the project will be provided free to all under open source licenses. This will allow both academic and commercial users to directly benefit from the resources generated in this project. The Genome Analysis Centre (TGAC) will also promote the use of tools developed in this project in the context of high-throughput annotation of plant genomes that are being sequenced in this recently established genomics research institute. This will also allow us to investigate the potential of commercial exploitation of our new methodologies through links that TGAC is developing with SME's working in the area of plant breeding. Previously the UPsT resource has been promoted through national and international conferences, publications and through linking from other relevant sites and resources. In addition, members of the Moulton lab have demonstrated the application of the current tools at numerous workshops and conferences, and the helped users of UPsT in the provision of tutorials on the tools at various national and international workshops. We will continue to use a similar strategy to promote the tools developed in this project. The future maintenance of the UPsT tools requires minimal dedicated resources and it will be possible for future development of new features to be driven by short-term projects. For instance, a PhD project could lead to the development of new tool(s) required by the community. Such projects will facilitate training of PhD students in the process of developing and promoting cutting-edge research to a broad user base. The degradome assay is a powerful tool to experimentally identify targets of plant short RNAs at the genomic scale but, due to a lack of appropriate computational tools, at the moment plant scientists cannot fully analyse such data. The proposed software will enable plant scientists to fully utilise this powerful technique, which will generate a wealth of knowledge on the function of sRNAs. In this way, we expect that the proposed tools will ultimately contribute towards improving quality of life through enabling plant and molecular biologists to understand and exploit key molecular pathways in important crop plants such as tomato and grape.