Mutational analysis of 3' splice site selection during trans-splicing.
trans-Splicing is essential for mRNA maturation in trypanosomatids. A conserved 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 lines were generated that carried point mutations at or near the 3' splice site within the intergenic region separating CUB2.65, the calmodulin-ubiquitin associated gene, and FUS1, the ubiquitin fusion gene of Trypanosoma cruzi. In one stable line, the elimination of the native 3' splice acceptor site led to the accumulation of Y-branched splicing intermediates, which served as templates for mapping the first trans-splicing branch points in T. cruzi. In other lines, point mutations shifted the position of the first consensus AG dinucleotide either upstream or downstream of the wild-type 3' splice acceptor site in this intergenic region. Consistent with the scanning model, 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 point mutations affected splicing efficiency in this region.
Department of Microbiology and Immunology, University of Tennessee, Memphis, Tennessee 38163, USA.
H S
HS
Hummel
R D
RD
Gillespie
J
J
Swindle
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D018274:Electroporation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013997:Time Factors; D014170:Transformation, Genetic; D014349:Trypanosoma cruzi / Q000235:genetics
D012333:RNA, Messenger; D015719:Single-Strand Specific DNA and RNA Endonucleases