Although significant progress has been made towards computational annotation of exon-intron boundaries, the existence and the remarkable prevalence of alternative splicing has made these predictions complicated. The last few years have seen an impressive development of microarray-based profiling of alternative splicing, however, these tools depend on pre-existing knowledge of the existence of splice sites. Here we propose a novel exon discovery tool, which we have termed """""""" SMaRTTM ExonFinder"""""""". Spliceosome Mediated RNA Trans-splicing (SMaRTTM) is a novel platform technology that reprograms genes at the level of pre-mRNA splicing. SMaRTTM has therapeutic, diagnostic as well as genomic applications, and each application is dependent upon the nature of the sequences encoded by the pre-trans-splicing molecule (PTM). The objective of this proposal is to test """""""" SMaRTTM ExonFinder"""""""" approach as a tool to annotate previously unknown splice sites in the human genome. To achieve our objective we propose three aims: 1. to develop and test the effectiveness of SMaRTTM ExonFinder PTMs against known endogenous premRNA targets that will mark exon-intron boundaries with high selectivity; 2. to develop highly efficient ExonFinder PTMs that can be used to annotate splice sites of low abundance transcripts. To achieve this, we will use a SMaRTTM high throughput screen, recently developed and validated for the identification of PTMs with high trans-splicing efficiency. Finally, 3. we propose to use these optimized ExonFinder PTMs to efficiently detect the sub-set of splice sites active in different cells and tissues. This will serve as a proof-of-principle for the """""""" SMaRTTM ExonFinder"""""""" approach in the annotation of alternate splicing in normal and disease states. ? ?
