Molecular genetic analysis of U2AF(59) 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 fun ctions in vivo, we have created a null allele of the gene encoding the Schi zosaccharomyces pombe ortholog, U2AF(59), and employed it in a variety of g enetic complementation assays. First, analysis of an extensive series of do uble amino acid substitutions indicates that this splicing factor is surpri singly refractory to mutations. Second, despite extensive structural conser vation, 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 3' polypyrimidine tract, we examined the spl icing of introns containing or lacking this feature in a U2AF(59) mutant de scribed here as well as a previously isolated temperature-sensitive mutant (Potashkin et al., 1993, Science 262:573-575). Our data indicate that all f our introns tested, including two that lack extensive runs of pyrimidines b etween the branchpoint and 3' splice site, show splicing defects upon shift ing 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.