Smooth muscle-specific switching of alpha-tropomyosin mutually exclusive exon selection by specific inhibition of the strong default exon.

Exons 2 and 3 of alpha-tropomyosin are spliced in a strict mutually exclusive manner. Exon 3 is a default choice, being selected in almost all cell types where the gene is expressed. The default selection arises from a competition between the two exons, in which the stronger branch point/pyrimidine tract elements of exon 3 win. Exon 2 is selected predominantly or exclusively only in smooth muscle cells. We show here that the basis for the smooth muscle-specific switching of exon selection is inhibition of exon 3. Exon 3 is still skipped with smooth muscle specificity, even in the absence of exon 2. We have defined two conserved sequence elements, one in each of the introns flanking exon 3, that are essential for this regulation. Mutation of either element severely impairs regulated suppression of exon 3. No other exon or intron sequences appear to be necessary for regulation. We have also demonstrated skipping of exon 3 that is dependent upon both regulatory elements in an in vitro splicing assay. We further show that both splice sites of exon 3 must be inhibited in a concerted fashion to switch to selection of exon 2. This may relate to the requirement for negative elements on both sides of the exon.