A 32-NUCLEOTIDE EXON-SPLICING ENHANCER REGULATES USAGE OF COMPETING 5'-SPLICE SITES IN A DIFFERENTIAL INTERNAL EXON

Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this r eport, we examined alternative splicing of a 1-kb internal exon from t he human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon R NA via utilization of the exon-internal 5' splice site, inclusion of t he differential exon required a long purine-rich sequence located betw een the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicin g enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice si te, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the i nternal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' spli ce site. In the absence of the internal 5' splice site, the ESE activa ted a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regula te competition between two 5' splice sites-within a differential inter nal exon.