Genetic and biochemical analysis of the role of the DEAD -box protein Sub2 in pre -mRNA splicing in Saccharomyces cerevisiae

Accurate removal of introns during pre-mRNA splicing is essential. The splice sites within a transcript are recognized and removed in a two-step trans-esterification reaction by the spliceosome. The spliceosome is a dynamic ribonucleoprotein complex that assembles de novo on each splicing substrate in an ordered series of ATP-dependent interactions between the spliceosome and the pre-mRNA substrate. Through this process, multiple recognition events occur at the intron splice sites, both before and during catalysis. Spliceosomal ATPases have been implicated to play an important role in the fidelity of these recognition events, but their mechanism remains unclear.;To better understand how spliceosomal ATPases affect fidelity during pre-mRNA splicing, I undertook genetic and biochemical analysis of the role of the Sub2 ATPase on early recognition events at the branchpoint sequence (BPS). Sub2 has high homology to the mammalian ATPase UAP56, which associates with the U2AF65 splicing factor and is required for the ATP-dependent U2-BPS basepairing interaction. Genetic and biochemical analysis of sub2 alleles suggests that Sub2 functions to stabilize the ATP-independent BPS binding of BBP and Mud2, the yeast U2AF65 homologue; however, in a subsequent ATP-dependent step, Sub2 likely destabilizes these factors at the BPS, thereby promoting the U2-BPS interaction. Further genetic analysis of SUB2 interactions supports this model. Biochemical analyses of a Deltasub2Deltamud2 extract suggest that the balance of BBP and U2 snRNA binding to the BPS is altered in the absence of Sub2 and Mud2. Taken together these studies suggest that Sub2 may promote accurate BPS recognition by influencing a balance of affinities between BBP, Mud2, and the U2 snRNP at the BPS.;The genetic link between SUB2 and the snRNP biogenesis factor BRR1 implicates a role for Sub2 in snRNP biogenesis. SUB2 and BRR1 exhibit a complex series of genetic interactions, influenced by the levels of each factor. Overexpression of SUB2 partially suppresses the decrease in snRNA levels observed in a brr1-1 strain. Moreover, snRNP levels are similarly reduced in extracts derived from sub2-1 and Deltabrr1 strains. Taken together, these data suggest that Sub2 may play additional roles in the cell related to snRNP biogenesis or stability.