Structure and function of spliceosome

摘要 前体mRNA剪接对于所有真核生物中的基因表达是必不可少的，并且剪接中的错误导致遗传性疾病。 疾病和许多其他疾病。对前mRNA分子机制的深入了解 剪接具有为人类疾病治疗提供有用方法的潜力。内含子的剪接是 通过剪接体催化的两个酯交换反应进行，剪接体是一种大的RNA/蛋白质， 由5个snRNP（U1、U2、U4、U 5、U6）和许多非snRNP相关蛋白因子组成的复合物。的 剪接体在剪接循环中经历显著变化，产生E、A、前B、B、Bact、B*、C、C*、P， 和ILS复合体。A、前B、B、Bact、C、C*、P和ILS复合物的高分辨率cryoEM结构具有 在过去的几年中已经确定，产生了关于剪接机制的有趣假设， 等待通过生物化学和/或遗传分析进行的严格测试。后催化剂的cryoEM结构 我们在3.3 nm分辨率下测定的P复合物提供了关于内含子重要性的有趣假设 二级结构在3' ss识别中的应用。在这项提案中，我们将使用全基因组的化学和酶 来检验这个假设P复合物的结构也导致了关于结构和 DEAD盒解旋酶Prp 22在剪接中的功能，我们将使用生物化学和 基因实验此外，我们刚刚确定的剪接体E复合物的冷冻电镜结构 建议的机制外显子的定义和它的转换内含子的定义，我们将测试使用 生物化学和结构方法。基因组、生物化学和结构方法的结合 在这个建议中使用的，有可能提供一个完整的机械理解的整个剪接 周期

Abstract Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy. The splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNPs (U1, U2, U4, U5, U6) and many non-snRNP related protein factors. The spliceosome undergoes dramatic changes in a splicing cycle, generating the E, A, Pre-B, B, Bact, B*, C, C*, P, and ILS complexes. High resolution cryoEM structures of A, pre-B, B, Bact, C, C*, P, and ILS complexes have been determined in the last few years, generating intriguing hypotheses on the mechanism of splicing that await rigorous testing through biochemical and/or genetic analyses. The cryoEM structure of the post-catalytic P complex we determined at 3.3 Å resolution provided intriguing hypothesis on the importance of intronic secondary structures in 3’ ss recognition. In this proposal, we will use genome-wide chemical and enzymatic probing to test this hypothesis. The structure of the P complex also led to hypotheses on the structure and function of DEAD-box helicase Prp22 in splicing, which we will test using a combination of biochemical and genetic experiments. Furthermore, the cryoEM structure of the spliceosome E complex we just determined suggested mechanism of exon definition and its conversion to intron definition, which we will test using biochemical and structural approaches. The combination of genomic, biochemical, and structural approaches used in this proposal has the potential to provide a complete mechanistic understanding of the entire splicing cycle.