Exonic splicing enhancers contribute to the use of both 3 ' and 5 ' splicesite usage of rat beta-tropomyosin pre-mRNA

The rat beta-tropomyosin gene encodes two tissue-specific isoforms that con tain the internal, mutually exclusive exons 6 (nonmuscle/smooth muscle) and 7 (skeletal muscle). We previously demonstrated that the 3' splice site of exon 6 can be activated by introducing a 9-nt polyuridine tract at its 3' splice site, or by strengthening the 5' splice site to a U1 consensus bindi ng site, or by joining exon 6 to the downstream common exon 8. Examination of sequences within exons 6 and 8 revealed the presence of two purine-rich motifs in exon 6 and three purine-rich motifs in exon 8 that could potentia lly represent exonic splicing enhancers (ESEs). In this report we carried o ut substitution mutagenesis of these elements and show that some of them pl ay a critical role in the splice site usage of exon 6 in vitro and in vivo. Using UV crosslinking, we have identified SF2/ASF as one of the cellular f actors that binds to these motifs. Furthermore, we show that substrates tha t have mutated ESEs are blocked prior to A-complex formation, supporting a role for SF2/ASF binding to the ESEs during the commitment step in splicing . Using pre-mRNA substrates containing exons 5 through 8, we show that the ESEs within exon 6 also play a role in cooperation between the 3' and 5' sp lice sites flanking this exon. The splicing of exon 6 to 8 (i.e., 5' splice site usage of exon 6) was enhanced with pre-mRNAs containing either the po lyuridine tract in the 3' splice site or consensus sequence in the 5' splic e site around exon 6. We show that the ESEs in exon 6 are required for this effect. However, the ESEs are not required when both the polyuridine and c onsensus splice site sequences around exon 6 were present in the same pre-m RNA. These results support and extend the exon-definition hypothesis and de monstrate that sequences at the 3' splice site can facilitate use of a down stream 5' splice site. In addition, the data support the hypothesis that ES Es can compensate for weak splice sites, such as those found in alternative ly spliced exons, thereby providing a target for regulation.