CHARACTERIZATION AND FUNCTIONAL ORDERING OF SLU7P AND PRP17P DURING THE 2ND STEP OF PRE-MESSENGER-RNA SPLICING IN YEAST

Temperature-sensitive alleles in four genes (slu7-1, prp16-2, prpl7-1, and prp18-1) are known to confer a specific block to the second chemi cal step of pre-mRNA splicing in vivo in the yeast Saccharomyces cerev isiae. Previous studies showed that Prp16p and Prp18p are required sol ely for the second step in vitro. The RNA-dependent ATPase, Prp16p, fu nctions at a stage in splicing when ATP is required, whereas Prp18p fu nctions at an ATP-independent stage. Here we use immunodepletion to sh ow that the roles of Slu7p and Prp17p are also confined to the second step of splicing. We find that extracts depleted of Prp17p require bot h Prp17p and ATP for slicing complementation, whereas extracts deplete d of Slu7p require only the addition of Slu7p. These different ATP req uirements suggest that Prp16p and Prp17p function before Prp18p and Sl u7p. Although SLU7 encodes an essential gene product, we find that a n ull allele of prp17 is temperature-sensitive for growth and has a part ial splicing defect in vitro. Finally, high-copy suppression experimen ts indicate functional interactions between PRP16 and PRP17, PRP16 and SLU7, and SLU7 and PRP18. Taken together, the results suggest that th ese four factors may function within a multi-component complex that ha s both an ATP-dependent and an ATP-independent role in the second step of pre-mRNA splicing.