The regulation of splicing of the human alpha sigma-tropomyosin pre-mRNA.

The human alpha-s-tropomyosin pre-mRNA, encoded by the hTMnm gene, is alternatively spliced. In skeletal muscle cells, amino acids 189-213 are encoded by a skeletal-muscle specific exon, called SK. In all other cell types, amino acids 189-213 are encoded by an alternative exon called NM. The pattern of splicing of SK and NM is mutually exclusive. Previous work had shown that in non-muscle cells, the selection of NM was determined by the intrinsic inactivity of the SK exon. SK inactivity was believed to be due to non-muscle-specific repression of the SK exon. The principal cis-acting sequence mediating this repression was believed to lie within the first fifteen nucleotides of the SK exon [Graham et al., 1992]. The aim of this thesis was to identify and characterise trans-acting factors which bound to the first fifteen nucleotides of the SK exon. It was hoped that this approach would identify the putative trans-acting repressor of the SK exon. Two factors which bound with apparent specificity were identified. The first of these was a 56 kDa protein, believed to be a member of the Y-box family of transcription factors. Irreproducibility of certain experiments, coupled with failure to successfully perform necessary confirmatory experiments mean that the status of p56 as a putative SK repressor remains uncertain. Data is presented to show that U1 snRNP also binds within the first fifteen nucleotides of SK, at a site which partially overlaps the 3' splice site. A hypothesis is proposed in which U1 snRNP is directly involved in the repression of SK in non-muscle cells. This finding is discussed in the context of related systems of alternative splicing.