Alternative splicing of U12-dependent introns in vivo responds to purine-rich enhancers

Alternative splicing increases the coding capacity of genes through the pro duction of multiple protein isoforms by the conditional use of splice sites and exons. Many alternative splice sites are regulated by the presence of purine-rich splicing enhancer elements (ESEs) located in the downstream exo n. Although the role of ESEs in alternative splicing of the major class U2- dependent introns is well established, no alternatively spliced minor class U12-dependent introns have so far been described. Although in vitro studie s have shown that ESEs can stimulate splicing of individual U12-dependent i ntrons, there is no direct evidence that the U12-dependent splicing system can respond to ESEs in vivo. To investigate the ability of U12-dependent in trons to use alternative splice sites and to respond to ESEs in an in vivo context, we have constructed two sets of artificial minigenes with alternat ive splicing pathways and evaluated the effects of ESEs on their alternativ e splicing patterns. In minigenes with alternative U12-dependent 3' splice sites, a purine-rich ESE promotes splicing to the immediately upstream 3' s plice site. As a control, a mutant ESE has no stimulatory effect. In minige ne constructs with two adjacent U12-dependent introns, the predominant in v ivo splicing pattern results in the skipping of the internal exon. Insertio n of a purine-rich ESE into the internal exon promotes the inclusion of the internal exon. These results show that U12-dependent introns can participa te in alternative splicing pathways and that U12-dependent splice sites can respond to enhancer elements in vivo.