Genetic interactions between the 5' and 3' splice site consensus sequences and U6 snRNA during the second catalytic step of pre-mRNA splicing.
The YAG/ consensus sequence at the 3' end of introns (the slash indicates the location of the 3' splice site) is essential for catalysis of the second step of pre-mRNA splicing. Little is known about the interactions formed by these three nucleotides in the spliceosome. Although previous observations have suggested that the G of the YAG/ interacts with the first nucleotide of the /GUA consensus sequence at the 5' end of the intron, additional interactions have not been identified. Here we report several striking genetic interactions between A+3 of the 5' /GUA with Y-3 of the 3' YAG/ and G50 of the highly conserved ACAGAG motif in U6 snRNA. Two mutations in U6 G50 of the ACAGAG can weakly suppress two mutations in A+3 of the 5' /GUA. This suppression is significantly enhanced upon the inclusion of a specific mutation Y-3 in the 3' YAG/. RNA analysis confirmed that the severe splicing defect observed in A+3 and Y-3 double mutants can be rescued to near wild-type levels by the mutations in U6 G50. The contributions of each mutation to the genetic interaction and the strong position specificity of suppression, combined with previous findings, support a model in which the 5' /GUA and the GAG of U6 function in binding the 3' YAG/ during the second catalytic step.
Collins
C A
CA
Department of Biochemistry and Biophysics, University of California, San Francisco 94143, USA.
Guthrie
C
C
D001483:Base Sequence; D016384:Consensus Sequence; D007438:Introns / Q000235:genetics; D009154:Mutation; D012322:RNA Precursors / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics*; D013489:Suppression, Genetic
D012322:RNA Precursors; D022821:RNA Splice Sites; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA