Mutational analysis of 3 ' splice site selection during trans-splicing

trans-Splicing is essential for mRNA maturation in trypanosomatids. A conse rved AG dinucleotide serves as the 3' splice acceptor site, and analysis of native processing sites suggests that selection of this site is determined according to a 5'-3' scanning model. A series of stable gene replacement l ines were generated that carried point mutations at or near the 3' splice s ite within the intergenic region separating CUB2.65, the calmodulin-ubiquit in associated gene, and FUS1, the ubiquitin fusion gene of Trypanosoma cruz i. In one stable line, the elimination of the native 3' splice acceptor sit e led to the accumulation of Y-branched splicing intermediates, which serve d as templates for mapping the first trans-splicing branch points in T. cru zi, In other lines, point mutations shifted the position of the first conse nsus AG dinucleotide either upstream or downstream of the wild-type 3' spli ce acceptor site in this intergenic region, Consistent with the scanning mo del, the first AG dinucleotide downstream of the branch points was used as the predominant 3' splice acceptor site. In: all of the stable lines, the p oint mutations affected splicing efficiency in this region.