Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3 splice sites. A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5 splice sites within an alternative exon. In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites. Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5 splice site choice when placed between competing 5 splice sites in an internal exon. The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5 splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5 splice site. Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5 splice sites were utilized. Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR) n repeat interdigitated with two enhancer-specific sequences. The entire enhancer was necessary for maximal splice site selectivity; however, a 5-to 7-nucleotide region from the 3 end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5 splice site selectivity was different than that shown to be maximally important for activation of a 3 splice site, suggesting that enhancer environment can have a major impact on activity. These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites. Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in enhancer sequence can have profound effects on the splicing pathway.Sequences within exons in addition to splice sites have emerged in the last several years as powerful determinants of splicing efficiency (2, 13, 24). Of these sequences, the purinerich exon enhancers containing the generic core sequence (GAR) n (R ϭ G or A) (42) have received the most attention (4,6,9,17,20,23,25,32,(41)(42)(43). Even short purine-rich enhancers can have major effects on the efficiency of exon inclusion. Most characterized purine-rich enhancers reside in alternative exons and have been shown to be essential sequence elements for exon inclusion, usually via the activation of weak 3Ј splice sites (2,14,26). Exon enhancers are often interchangeable in their ability to activate weak 3Ј splice sites, not only between genes (8,17,20,36,(41)(42)(43), but also between species (13), suggesting that either most enhancers bind the same factors or the bound factors have interchangeable activities.Purine-rich enhancers bind to members of the arginineserine-rich class of splicing factors (29, 44), the S/R proteins (re...
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