A 32-Nucleotide Exon-Splicing Enhancer Regulates Usage of Competing 5′ Splice Sites in a Differential Internal Exon

Abstract

Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5 splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5 splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5 splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5 splice site, 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 internal 5 splice site only in the presence of the competing 5 splice site and had no effect when placed downstream of the terminal 5 splice site. In the absence of the internal 5 splice site, the ESE activated 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 regulate competition between two 5 splice sites within a differential internal exon.