Regulation of human TMnm alternative pre-mRNA splicing
thesisposted on 15.12.2014, 10:31 by Neil. Thornton
Tissue-specific alternative splicing of pre-messenger mRNA is a common strategy for regulating mammalian gene expression. This mechanism is exemplified by several of the tropomyosin genes. One of these, the human tropomyosinnm (hTMnm) gene, contains two alternative centres, exon NM and exon SK. These exons are expressed tissue specifically and mutually exclusively; exon NM is expressed in non muscle cells and exon SK is expressed in skeletal muscle cells. Exon SK is specifically repressed in non muscle cells. This repression is dependent on sequence at the 5'-end of the exon that closely resembles a 5' splice site sequence. It is demonstrated here that U1 snRNP binds to this inhibitory sequence and mediates repression of exon SK in non muscle cells. In muscle cells, expression of exon SK is shown to require sequence within exon SK adjacent to the inhibitory sequence. Experiments in vivo showed that the activity of this enhancer sequence is dependent on a constitutively expressed factor(s). The sequence responded to SR proteins in vitro, thus highlighting a potential role for these proteins in regulation of hTMnm expression. Expression of mutants in muscle cells demonstrated that the inactivity of exon NM in these cells is dependent on the activity of the SK exon and on a reduction in the ability of the NM exon to be recognised by the splicing apparatus. It is proposed that interaction of U1 snRNP with the 5'-end of exon SK blocks recognition of the adjacent enhancer sequence in non muscle cells. In muscle cells, repression of exon SK is lifted and the activity of the enhancer sequence, together with a reduction in the activity of exon NM, allows exon SK to outcompete exon NM for recognition by the splicing apparatus.