Multiple functions of Saccharomyces cerevisiae splicing protein Prp24 in U6 RNA structural rearrangements

U6 spliceosomal RNA has a complex secondary structure that includes a highl y conserved stemloop near the 3' end. The 3' stem is un unwound when U6 RNA base-pairs with U4 RNA during spliceosome assembly, but likely reforms whe n U4 RNA leaves the spliceosome prior to the catalysis of splicing. A mutat ion in yeast U6 RNA that hyper-stabilizes the 3' stem confers cold sensitiv ity and inhibits U4/U6 assembly as well as a later step in splicing. Hel-e we show that extragenic suppressors of the 3' stem mutation map to the gene coding for splicing factor Prp24. The suppressor mutations are located in the second and third of three RNA-recognition motifs (RRMs) in Prp24 and ar e predicted to disrupt RNA binding. Mutations in U6 RNA predicted to destab ilize a novel helix adjacent to the 3' stem also suppress the 3' stem mutat ion and enhance the growth defect of a suppressor mutation in RRM2 of Prp24 . Both phenotypes are reverted by a compensatory mutation that restores pai ring in the novel helix. These results are best explained by a model in whi ch RRMs 2 and 3 of Prp24 stabilize an extended intramolecular structure in U6 RNA that competes with the U4/U6 RNA interaction, and thus influence bot h association and dissociation of U4 and U6 RNAs during the splicing cycle.