Prp21, a U2-snRNP-associated protein, and Prp24, a U6-snRNP-associated protein, functionally interact during spliceosome assembly in yeast

Earlier studies on genetic suppression of prp24-1 by prp21-2 suggested an a ssociation between yeast Prp21 and Prp24 proteins, which are associated, re spectively, with U2 snRNA and U6 snRNA. Here we report analyses of physical and functional interaction between these factors. Missense mutations in fu nctionally important domains reside in prp21-2 and prp24-1. Two-hybrid assa ys do not detect interaction between wild-type or mutant proteins. Prp21-2 and Prp24-1 protein in prp21-2 or prp24-1 extracts can be heat-inactivated in vitro. In contrast, heat-treated extracts from the revertant strain prp2 1-2 prp24-1 demonstrate allele-specific restoration of splicing. Suppressio n of prp24-1 by prp21-2 does not cause coimmunoprecipitation of U2 and U6 s nRNAs. We demonstrate the presence of Prp21 in the spliceosome assembly int ermediate A2-1, and our data suggest the presence of Prp24 in the same comp lex. Kinetic analysis of assembly in heat-treated revertant extracts reveal a rate-limiting conversion of complex B to A2-1, suggesting transient asso ciation between the mutant proteins at this step. Our data also imply a req uirement for Prp21 during B to A2-1 conversion. We conclude that a transien t yet likely functional association between Prp21 and Prp24 occurs during s pliceosome assembly.