Usage of cryptic splice sites in citrullinemia fibroblasts suggests role of polyadenylation in splice-site selection during terminal exon definition.

Abstract

Citrullinemia is a human genetic disease caused by a deficient argininosuccinate synthetase. In fibroblasts established from a citrullinemia patient with a mutation at the 3' splice site of the terminal intron of the gene, three cryptic 3' splice sites; i.e., SA1275, SA1636, and SA1663, residing on the terminal exon were activated. The usage of the cryptic sites showed a gradient, with the most downstream site having the highest usage; i.e., SA1663 > SA1636 > SA1275. However, when these cryptic sites were relocated to the internal exon, SA1636 was used the most. The splice-site strength of SA1636 was at least 10-fold higher than that of SA1663 in this situation. The results suggest that the preferential usage of SA1663 residing on the terminal exon may depend on its proximity to the poly(A) signal rather than on the strength of the splice site. Furthermore, when the strength of the downstream-most splice site increased, almost all the RNAs spliced to this site. However, in the presence of the wild-type splice site, all the RNAs were processed to the authentic site. Apparently, the selection of splice site can be revealed only when the sites being selected do not differ too much in their strength. By using a naturally occurring human mutant gene as a model, this study reveals that polyadenylation may play an important role in the selection of splice site during terminal exon definition.