Molecular genetic analysis of U2AF59 in Schizosaccharomyces pombe: differential sensitivity of introns to mutational inactivation.

The large subunit of the mammalian U2AF heterodimer (U2AF 65 ) 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, U2AF 59 , 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 U2AF 59 . Third, because the activity of U2AF 65 in vitro involves binding to the 39 polypyrimidine tract, we examined the splicing of introns containing or lacking this feature in a U2AF 59 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 39 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 U2AF 65 based on in vitro experiments.