MUTATIONS WITHIN THE YEAST U4 U6 SNRNP PROTEIN PRP4 AFFECT A LATE-STAGE OF SPLICEOSOME ASSEMBLY/

We showed previously that the yeast Prp4 protein is a spliceosomal fac tor that is tightly associated with the U4, U5, and U6 small nuclear R NAs. Moreover, Prp4 appears to associate very transiently with the spl iceosome before the U4 snRNA dissociates from the spliceosome. Prp4 be longs to the G beta-like protein family, which suggests that the Prp4 G beta motifs could mediate interactions with other components of the spliceosome. To investigate the function of the G beta motifs, we intr oduced mutations within the second WD-repeat of Prp4. Among the 35 new alleles found, 24 were pseudo wild-type mutants, 8 failed to grow at any temperature, and 3 were conditional sensitive mutants. The biochem ical defects of the three thermosensitive prp4 mutants have been exami ned by immunoprecipitation, native gel electrophoresis, and glycerol g radient centrifugation. First, we show that snRNP formation is not imp aired in these mutants and that Prp4 is present in the U4/U6 and U4/U6 -U5 snRNP particles. We also demonstrate that spliceosome assembly is largely unaffected despite the fact that the first step of splicing do es not occur. However, both Prp4 and U4 snRNA remain tightly associate d with the spliceosome and this blocks the transition toward an active form of the spliceosome. Our results suggest a possible role of Prp4 in mediating important conformational rearrangements of proteins withi n the spliceosome that involve the region containing the G beta-repeat s.