Characterization of dexD/H-Box RNA Helicases Involved in Fission Yeast Spliceosome

Abstract

The spliceosome is a dynamic macromolecular machine that catalyzes the pre-mRNA splicing reaction. The megadalton-sized spliceosome is composed of four small nuclear RNPs (U1, U2, U5, and U4/U6) and numerous pre-mRNA splicing factors. The formation of an active spliceosome involves a series of regulated steps that requires the assembly and disassembly of large multiprotein/RNA complexes. At least eight DExD/H-box RNA helicases are involved in the splicing reaction (Prp5/Prp11, Sub2/Uap56, Prp28, Brr2, Prp2/Cdc28, Prp16, Prp22 and Prp43), and are highly conserved through yeast to human. Previous studies showed that these RNA helicases are important for rearranging the spliceosome during assembly, activation and disassembly. However, the mechanism(s) of how they regulate and control spliceosomal conformational changes remain elusive. To generate a structural based model for how RNA helicases control spliceosome transitions, we are using the genetically tractable fission yeast, Schizosaccharomyces pombe. To explore RNA helicase function in the splicing reaction we have generated a bank of integrated dominant negative DExD/H RNA helicase alleles that are under the control of a conditional promoter. As expected, cells over-expressing these mutants accumulate pre-mRNA and die. We are now further characterizing the structural and functional consequences to spliceosome complexes containing mutant helicases.