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.