Alternative pre-mRNA splicing plays a central role in generating the complex proteome of metazoans. About 60% of human genes are alternatively spliced and a compromise in this process is known to cause multiple human diseases. An understanding of the mecha nisms that regulate alternative splicing will be essential for a functional interpretation of the genome. We propose to study the co-transcriptional recruitment of splicing regulatory proteins-specifically SR proteins- onto the pre-mRNA. Such an analysis w ould be valuable since the coupling of splicing to transcription has been shown to contribute to alternative splicing regulation. My approach will involve analysis of RNA while it is still attached to the chromosomal DNA, during the act of transcription by RNA polymeraseII. Recruitment of the basic splicing machinery to target genes by this novel method has been shown in the host lab. I propose to examine the detectability and distribution of SR proteins on a candidate gene that undergoes alternative splici ng to encode either calcitonin or the neuropeptide CGRP. Through chromosomal immunoprecipitation (CHIP) I will determine the sites along the gene at which the SR proteins are recruited. Since the splicing of this gene can be manipulated in cell culture, I will be able to determine the consequential changes in SR protein recruitment. This will be followed by analysis of changes in recruitment pattern when critical sequences in the gene are altered. In a complementary approach we will alter the levels of the SR protein and analyse its affect on alternative splicing regulation. Lastly, I will extend the analysis to a global scale by carrying out CHIP followed by hybridization to DNA microarrays, designed to detect genomic intron and exon regions. This study wil l identify the yet unknown endogenouos gene targets for the SR proteins. Thus, I will be able to build a comprehensive picture of splicing factor recruitment and its regulation of alternative splicing.
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