USAGE OF CRYPTIC SPLICE SITES IN CITRULLINEMIA FIBROBLASTS SUGGESTS ROLE OF POLYADENYLATION IN SPLICE-SITE SELECTION DURING TERMINAL EXON DEFINITION

Citrullinemia is a human genetic disease caused by a deficient arginin osuccinate 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., SA(1275), SA(1636), and SA(1663), 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., SA(1663) > SA(1636) > SA(1275) However , when these cryptic sites were relocated to the internal exon, SA(163 6) was used the most. The splice-site strength of SA(1636) was at leas t 10-fold higher than that of SA(1663) in this situation. The results suggest that the preferential usage of SA(1663) residing on the termin al 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 sel ection of splice site can be revealed only when the sites being select ed do not differ too much in their strength. By using a naturally occu rring human mutant gene as a model, this study reveals that polyadenyl ation may play an important role in the selection of splice site durin g terminal exon definition.