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Abstract：
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.
摘要：
在组装过程中经历大量RNA和蛋白质重排后，剪接体经历了最后的结局，更微妙，对催化至关重要的ATP依赖性重排。这种重排需要DEAH-box蛋白Prp2p,RNA依赖性ATP酶。Prp2p与剪接体和蛋白质复合物SF3和RES之间的不稳定相互作用有关，但是Prp2p在破坏RNA-RNA相互作用中的作用尚未被研究。在萌芽酵母中使用定向分子遗传学，我们已经发现，冷敏感的prp2突变不仅受到SF3和RES成分突变的抑制，而且还受到一系列破坏剪接体催化核心元件U2/U6螺旋I的突变的抑制，这涉及将5'剪接位点和分支位点并置，以及在假定的催化三链体的背景下定位用于催化的金属离子；确实，这种推定的催化三链体中的突变也抑制了prp2突变。值得注意的是,我们还发现prp2突变拯救U2/U6螺旋I中的致死突变。这些数据提供了证据，表明包含催化核心的RNA元件已经在Prp2p阶段形成，并且Prp2p使这些元件不稳定，直接或间接,既要校对剪接体的激活，又要促进剪接体的重新配置，催化构象。
