Most eukaryotic genes are interrupted by non-coding introns that must be removed from pre-mRNA transcripts for the production of functional proteins. The splicing of introns is remarkably efficient and rapid, an amazing feat considering that the signals that delineate introns from exons are not conserved in higher eukaryotes. A corollary of the importance of splicing in normal cell function is that approximately 15% of characterized genetic diseases involve mutations that cause defects in splicing. Further, 60% or more of human genes are subject to alternative splicing requiring that cells not only be able to accurately recognize the difference between introns and exons, but also between alternatively spliced exons. We have been studying the regulation of alternative splicing focusing on the molecular mechanisms by which specific splice sites are activated or repressed. In the first part of this proposal, experiments are designed to understand the regulation of the mutually exclusive exons 2 and 3 of the alpha- tropomyosin gene. Dissection of the cis-acting sequences needed to control splicing have identified elements flanking exon 3 that are needed to repress splicing in smooth muscle cells. PTB (hnRNP I) binds to one of these elements but the identity of factors that bind to a directly adjacent, conserved UGC repeat remains unknown. Biochemical purification is proposed to identify this factor and determine how it participates with PTB to repress splicing. In contrast to repression of exon 3, activation of exon 2 is needed in smooth muscle cells mediated by four purine-rich enhancer elements and one or more members of the SR protein family of splicing factors. In vivo and in vitro splicing assays will be used to identify which SR proteins are essential for exon 2 activation. In the second part of this proposal, regulation of splicing by SR proteins will be expanded to include characterization of a new SR-related protein (SRrp86) that can activate or repress specific SR family members. The specific targets of SRrp86 are unknown but preliminary data suggests that it functions by protein-protein interaction. Experiments are proposed to determine how such interaction alters SR protein activity and to identify specific SR protein targets. Overall, the experiments in this proposal seek to understand the mechanistic basis underlying the repression and activation of splicing by SR proteins and hnRNP proteins.
