Exon sequences at the splice junctions affect splicing fidelity and alternative splicing

Identification of splice sites is essential for the expression of most eukaryotic genes, allowing accurate splicing of pre-mRNAs. The splice sites are recognized by the splicing machinery based on sequences within the pre-mRNA. Here, we show that the exon sequences at the splice junctions play a significant, previously unrecognized role in the selection of 3' splice sites during the second step of splicing. The influence of the exon sequences was enhanced by the Prp18 mutant Prp18DeltaCR, and the strength of an exon sequence in Prp18DeltaCR splicing predicted its effect in wild-type splicing. Analysis of the kinetics of splicing in vitro demonstrated that 3' splice sites were chosen competitively during the second step, likely at the same time as exon ligation. In wild-type yeast, splice site selection for two genes studied was altered by point mutations in their exon bases, affecting splicing fidelity and alternative splicing. Finally, we note that the degeneracy of the genetic code allows competing 3' splice sites to be eliminated from coding regions, and we suggest that the evolution of the splicing signals and the genetic code are connected.