Alternative pre-mRNA splicing in Drosophila. D. Rio - PI Alternative pre-mRNA splicing is an important mechanism for regulating gene expression in metazoans. Indeed, RNA processing is a conduit through which genomic information is transferred to proteomic information. Because most eukaryotic genes are split and have the potential for alternative splicing, a dramatic increase in proteomic diversity among cells, tissues and organisms is a direct consequence of alternative splicing. In fact, it is now recognized that 30-40% of the known human and mouse disease gene mutations affect the splicing process. Thus, understanding how introns are recognized and how patterns of alternative splicing are set up may allow therapeutic intervention. Splicing silencers are a major type of RNA control element generating tissue- or cell type-specific splicing patterns in metazoans. Our previous work has focused on characterization of the tissue-specific Drosophila P element splicing silencer element. Recent work from our group has focused on tissue-specific regulation of alternative splicing and how the action of hnRNP proteins, PSI and the hnRNP A/B family proteins, hrp48, 40, 38 and 36 results in splicing silencer function. We wish to extend these studies to investigate the function of a Drosophila paralog of U2AF, called LS2, that functions as a splicing repressor. Building on previous R21 funding, we will continue investigations of the microRNA pathway protein, Ago-2, in alternative splicing pathways. Finally, using a variety of approaches, we want to identify and functionally characterize new cellular splicing silencers that are controlled by multiple splicing factors. One advantage this proposal has is the small genome size and well-annotated Drosophila genome that allows a comprehensive analysis of both alternative splicing patterns and the genome-wide distribution of RNA binding proteins. Because most eukaryotic genes are split and have the potential for alternative splicing, increased proteomic diversity among cells, tissues and organisms is a direct consequence of alternative splicing. Because the protein factors involved in controlling patterns of alternative splicing have homologs in humans and like humans Drosophila has extensive alternative splicing, results obtained in this system will have direct relevance and application to human health.