The sRNA Workbench

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 change. Since its recent discovery, RNA silencing has become a fast moving area of research of great importance in both plant and animal molecular biology. Its discovery has not only been a major breakthrough for our understanding of complex regulatory processes in living cells, but has also opened the door to the development of novel therapeutics for human diseases. The importance of these findings was highlighted by the award of the Nobel prize for medicine in 2006 for the discovery of RNA interference. Small RNA molecules (sRNAs) play a crucial role in the RNA silencing machinery in that they can interact with other nucleic acids in a sequence specific manner to mark them as targets for inactivation. To learn more about this mechanism and its implications, researchers employ cutting-edge sequencing technologies to obtain large datasets of sRNA sequences (typically between 1-10 million sequences about 20-30 nucleotides in length) from a number of organisms. With these technologies becoming more accessible and affordable it is now possible to obtain multiple large-scale data sets which allow the analysis of changes in the sRNA population. These changes can indicate the involvement of RNA silencing pathways in the biological process of interest, such as regulation of key developmental stages in an organism. However, there is a lack of software tools for the analysis of such experiments, which presents a major bottleneck to the exploitation of the technology, especially for biologists without direct bioinformatics support. In this project, we will build on our existing publicly available software tools for sRNA analysis, with the aim of providing an easy to use, downloadable workbench to provide a complete solution for the analysis of plant and animal sRNA datasets. This will provide biologists with a much needed resource to identify novel sRNAs and their function as part of wet-lab studies.

Novel sequencing technologies such as 454, Solexa/Illumina and ABI Solid have become an important tool for researchers in the field of RNA silencing, due to the fact that they can sequence millions of small (s)RNAs in a single experiment. This makes it possible to produce snapshots of the complex sRNA populations of living cells and hence gain insights into the production and function of this important class of regulatory non-coding RNA molecules. With sequencing technologies becoming ever more affordable, it is now possible to follow changes in the sRNA signature through time series or compare the effects of different treatments at a high resolution. At present, however, there are a lack of methods and software tools available to analyse such large-scale sRNA data. Building on cutting-edge sRNA tools that we have developed over recent years, we will develop an easy to use downloadable workbench to allow researchers to perform complex analyses using large-scale sRNA datasets. The core analytical tools forming the basis of this workbench will also be made available for download as command-line based tools so that expert bioinformatics users can easily integrate them into pipelines. This will also incorporate new algorithms that will be designed to perform analyses of multiple-sample sRNA datasets from both plants and animals and to generate networks of sRNA interactions. This work will be carried out by a PDRA managed by an interdisciplinary team with over four years experience in developing cutting-edge algorithms and tools for sRNA analysis, that have already led to some important new discoveries in sRNA research.

Since its establishment last year, the UEA small RNA Toolkit (UPsT) has been used extensively by sRNA researchers both in the UK and worldwide with an average of over 12,000 page views per month, 39% of which are from the UK. In addition, an average of 120-200 full analyses are performed per month by external users. It has an attractive website, is easy to use, and is the only complete solution for processing and analysing high-throughput plant 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, and publications are beginning to appear in which the tools are cited and used as a major component of the analysis. It is therefore clear that user demand for tools such as those to be developed in this project is strong and growing. 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. We will also promote the application of the tools developed in this project in the context of high-throughput annotation of genomes that are to be sequenced in the recently established BBSRC Genome Analysis Centre (TGAC) in Norwich. This will also allow us to investigate the potential of commercial exploitation of our new methodologies through SME's working in areas such as plant breeding. Previously the UPsT resource has been promoted through national and international conferences, publications, and linking from other relevant sites and resources. In addition, members of Moulton's group have demonstrated the application of the current tools at numerous workshops and conferences, including: MicroRNAs Europe. Cambridge, UK. 2008; SIROCCO Bioinformatics Day. Hinxton, UK. 2008; Genome Informatics, Hinxton, UK. 2009; Computational Challenges of the Next Generation of DNA Sequencing. Uppsala, Sweden. 2009; Computational Methods for RNA Analysis. Benasque, Spain. Moulton's group has also supported UPsT users in provision of tutorials on the tools at various workshops. We will continue to use a similar strategy to promote the tools developed in this project. As the workbench developed in this project will be easy to maintain and extend, it will be possible for future development of new features to be driven by short-term projects. In particular, PhD projects could lead to the development of new tools 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 in the increasingly important area of bioinformatics. We expect that the proposed tools will ultimately contribute towards improving quality of life through enabling molecular biologists to understand and exploit key molecular pathways in important crop plants such as tomato and grape, as well as to develop new RNA based therapeutics for diseases such as cancer.