The DExD/H-box ATPase Prp2p destabilizes and proofreads the catalytic RNA core of the spliceosome.
After undergoing massive RNA and protein rearrangements during assembly, the spliceosome undergoes a final, more subtle, ATP-dependent rearrangement that is essential for catalysis. This rearrangement requires the DEAH-box protein Prp2p, an RNA-dependent ATPase. Prp2p has been implicated in destabilizing interactions between the spliceosome and the protein complexes SF3 and RES, but a role for Prp2p in destabilizing RNA-RNA interactions has not been explored. Using directed molecular genetics in budding yeast, we have found that a cold-sensitive prp2 mutation is suppressed not only by mutations in SF3 and RES components but also by a range of mutations that disrupt the spliceosomal catalytic core element U2/U6 helix I, which is implicated in juxtaposing the 5' splice site and branch site and in positioning metal ions for catalysis within the context of a putative catalytic triplex; indeed, mutations in this putative catalytic triplex also suppressed a prp2 mutation. Remarkably, we also found that prp2 mutations rescue lethal mutations in U2/U6 helix I. These data provide evidence that RNA elements that comprise the catalytic core are already formed at the Prp2p stage and that Prp2p destabilizes these elements, directly or indirectly, both to proofread spliceosome activation and to promote reconfiguration of the spliceosome to a fully competent, catalytic conformation.
Alissa M
AM
Wlodaver
Jonathan P
JP
Staley
D020134:Catalytic Domain; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017381:Spliceosomes / Q000235:genetics*
D016337:RNA, Catalytic; D012342:RNA, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405484:U2 small nuclear RNA; C405486:U6 small nuclear RNA; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases
DExD/H-box ATPase; Prp2; U2/U6 helix I; spliceosome activation; spliceosome catalytic core