ROLE OF AN INHIBITORY PYRIMIDINE ELEMENT AND POLYPYRIMIDINE TRACT BINDING-PROTEIN IN REPRESSION OF A REGULATED ALPHA-TROPOMYOSIN EXON

Splicing of exons 2 and 3 of alpha-tropomyosin (TM) involves mutually exclusive selection of either exon 3, which occurs in most cells, or o f exon 2 in smooth muscle (SM) cells. The SM-specific selection of exo n 2 results from the inhibition of exon 3. At least two essential cis- acting elements are required for exon 3 inhibition, the upstream and d ownstream regulatory elements (URE and DRE). These elements are essent ial for repression of TM exon 3 in SM cells, and also mediate a low le vel of repression of exon 3 in an in vitro 5' splice site competition assay in HeLa extracts. Here, we show that the DRE consists of at leas t two discrete components, a short region containing a number of UGC m otifs, and aln essential pyrimidine-rich tract (DY). We show that the specific sequence of the DY element is important and that DY is able t o bind to factors in HeLa nuclear extracts that mediate a low backgrou nd level of exon 3 skipping. Deletion of a sequence within DY identifi ed as an optimal binding site for PTB impairs (1) regulation of splici ng in vivo, (2) skipping of exon 3 in an in vitro 5' splice site compe tition, (3) the ability of DY competitors to affect the 5' splice site competition in vitro, and (4) binding of PTB to DY. Addition of recom binant PTB to in vitro splicing reactions is able to partially reverse the effects of the DY competitor RNA. The data are consistent with a model for regulation of TM splicing that involves the participation of both tissue-specific and general inhibitory factors and in which PTB plays a role in repressing both splice sites of exon 3.