Alternative splicing increases the coding capacity of genes
through the production 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 exon. 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 studies have shown that ESEs can stimulate
splicing of individual U12-dependent introns, there is no direct
evidence that the U12-dependent splicing system can respond
to ESEs in vivo. To investigate the ability of U12-dependent
introns to use alternative splice sites and to respond to ESEs
in an in vivo context, we have constructed two sets of artificial
minigenes with alternative splicing pathways and evaluated the
effects of ESEs on their alternative splicing patterns. In
minigenes with alternative U12-dependent 3′ splice sites,
a purine-rich ESE promotes splicing to the immediately upstream
3′ splice site. As a control, a mutant ESE has no stimulatory
effect. In minigene constructs with two adjacent U12-dependent
introns, the predominant in vivo splicing pattern results in
the skipping of the internal exon. Insertion of a purine-rich
ESE into the internal exon promotes the inclusion of the internal
exon. These results show that U12-dependent introns can participate
in alternative splicing pathways and that U12-dependent splice
sites can respond to enhancer elements in vivo.