The large subunit of the mammalian U2AF heterodimer
(U2AF65) is essential for splicing in vitro.
To expand our understanding of how this protein functions
in vivo, we have created a null allele of the gene encoding
the Schizosaccharomyces pombe ortholog, U2AF59,
and employed it in a variety of genetic complementation
assays. First, analysis of an extensive series of double
amino acid substitutions indicates that this splicing factor
is surprisingly refractory to mutations. Second, despite
extensive structural conservation, we find that metazoan
large subunit orthologs cannot substitute in vivo for fission
yeast U2AF59. Third, because the activity of
U2AF65 in vitro involves binding to the 3′
polypyrimidine tract, we examined the splicing of introns
containing or lacking this feature in a U2AF59
mutant described here as well as a previously isolated
temperature-sensitive mutant (Potashkin et al., 1993, Science
262:573–575). Our data indicate that all four
introns tested, including two that lack extensive runs
of pyrimidines between the branchpoint and 3′ splice
site, show splicing defects upon shifting to the nonpermissive
condition. In all cases, splicing is blocked prior to the
first transesterification reaction in the mutants, consistent
with the role inferred for human U2AF65 based
on in vitro experiments.