Intricate gene regulatory networks direct the development of multi-cellular eukaryotes. Although transcription regulation is the best understood mechanism by which these networks are controlled, it has recently become clear that alternative splicing is an equally important regulatory mechanism. Despite the fact that the majority of eukaryotic genes encode pre-mRNAs that are alternatively spliced (at least 65% of human genes), very few splicing regulatory factors have been identified, and the target genes that are controlled by specific splicing regulators are largely unknown. Alternative splicing is primarily thought to be regulated by auxiliary splicing factors - proteins that are not core components of the spliceosome. Auxiliary splicing factors function by binding to the pre-mRNA where they modulate the association of the spliceosome with the regulated splice sites. Recent work from our group and others, however, suggests that components of the general splicing machinery may also play important roles in regulating alternative splicing. This may in part account for the apparent discrepancy between the numbers of known splicing regulators and regulatory targets. The goals of this research proposal are to identify proteins that regulate alternative splicing and their regulatory targets on a genome-wide level, and to determine the biochemical mechanisms by which these proteins regulate alternative splicing using Drosophila melaongaster as a model system. We will first perform microarray experiments to identify alternatively spliced exons that are regulated by the entire complement of RNA binding proteins encoded by the Drosophila genome and to build a model of the splicing regulatory networks in Drosophila. These experiments will be complemented by a combination of biochemical, genetic, genomic, and bioinformatics experiments designed to determine the mechanisms by which both auxiliary and general splicing factors function to control alternative splicing. Together these experiments will provide tremendous insight into the mechanisms of alternative splicing. Given that the proteins involved in regulating alternative splicing in Drosophila and humans are similar, that a number of human diseases are caused by defects in the normal patterns of alternative splicing, and that the human homologs of many of the proteins we will be studying have been implicated in human diseases, it is likely that the discoveries we make will be of direct relevance to human health. ? ? ?
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