MUSCLE-SPECIFIC SPLICING ENHANCERS REGULATE INCLUSION OF THE CARDIAC TROPONIN-T ALTERNATIVE EXON IN EMBRYONIC SKELETAL-MUSCLE

The alternative exon 5 of the striated muscle-specific cardiac troponi n T (cTNT) gene is included in mRNA from embryonic skeletal and cardia c muscle and excluded in mRNA from the adult, The embryonic splicing p attern is reproduced in primary skeletal muscle cultures for both the endogenous gene and transiently transfected minigenes, whereas in nonm uscle cell lines, minigenes express a default exon skipping pattern, U sing this experimental system, we previously showed that a purine-rich splicing enhancer in the alternative exon functions as a constitutive splicing element but not as a target for factors regulating cell-spec ific splicing, In this study, we identify four intron elements, one lo cated upstream and three located downstream of the alternative exon, w hich act in a positive manner to mediate the embryonic splicing patter n of exon inclusion. Synergistic interactions between at least three o f the four elements are necessary and sufficient to regulate splicing of a heterologous alternative exon and heterologous splice sites, Muta tions in these elements prevent activation of exon inclusion in muscle cells but do not affect the default level of exon inclusion in nonmus cle cells. Therefore, these elements function as muscle specific splic ing enhancers (MSEs) and are the first muscle-specific positive-acting splicing elements to be described. One MSE located downstream from th e alternative exon is conserved in the rat and chicken cTNT genes, A r elated sequence is found in a third muscle-specific gene, that encodin g skeletal troponin T, downstream from an alternative exon with a deve lopmental pattern of alternative splicing similar to that of rat and c hicken cTNT. Therefore, the MSEs identified in the cTNT gene may play a role in developmentally regulated alternative splicing in a number o f different genes.