A SUBSET OF SR PROTEINS ACTIVATES SPLICING OF THE CARDIAC TROPONIN-T ALTERNATIVE EXON BY DIRECT INTERACTIONS WITH AN EXONIC ENHANCER

The cardiac troponin T pre-mRNA contains an exonic splicing enhancer t hat is required for inclusion of the alternative exon 5. Here we shaw that enhancer activity is exquisitely sensitive to changes in the sequ ence of a 9-nucleotide motif (GAGGAAGAA) even when its purine content is preserved. A series of mutations that increased or decreased the le vel of exon inclusion in vivo were used to correlate enhancer strength with RNA-protein interactions in vitro. Analyses involving UV cross-l inking and immunoprecipitation indicated that only four (SRp30a, SRp40 , SRp55, and SRp75) of six essential splicing factors known as SR prot eins bind to the active enhancer RNA. Moreover, purified SRp40 and SRp 55 activate splicing of exon 5 when added to a splicing-deficient S100 extract. Purified SRp30b did not stimulate splicing in S100 extracts, which is consistent with its failure to bind the enhancer RNA. In vit ro competition of SR protein splicing activity and UV cross-linking de monstrated that the sequence determinants for SR protein binding were precisely coincident with the sequence determinants of enhancer streng th. Thus, a subset of SR proteins interacts directly with the exonic e nhancer to promote inclusion of a poorly defined alternative exon. Ind ependent regulation of the levels of SR proteins may, therefore, contr ibute to the developmental regulation of exon inclusion.