Selection of pre-mRNA splice sites is a highly
accurate process involving many trans-acting factors. Recently,
we described a role for U6 snRNA position G52 in selection
of the first intron nucleotide (+1G). Because some U2 alleles
suppress U6-G52 mutations, we investigated whether the
corresponding U2 snRNA region also influenced 5′
splice site selection. Our results demonstrate that U2
snRNAs mutated at position U23, but not adjacent nucleotides,
specifically affect 5′ splice site cleavage. Furthermore,
all U2 position U23 mutations are synthetic lethal with
the thermosensitive U6-G52U allele. Interestingly, the
U2-U23C substitution has an unprecedented hyperaccurate
splicing phenotype in which cleavage of introns with a
+1G substitution is reduced, whereas the strain grows with
wild-type kinetics. U2 position U23 forms the first base
pair with U6 position A59 in U2/U6 helix Ib. Restoration
of the helical structure suppresses 5′ splice site
cleavage defects, showing an important role for the helix
Ib structure in 5′ splice site selection. U2/U6 helix
Ib and helix II have recently been described as being functionally
redundant. This report demonstrates a unique role for helix
Ib in 5′ splice site selection that is not shared
with helix II.