Structure and function of spliceosome

摘要 在所有真核生物中，前mRNA剪接是基因表达所必需的，剪接错误导致基因 疾病和许多其他疾病。深入了解前信使核糖核酸的分子机制 剪接有可能为人类疾病治疗提供有用的方法。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、U5、U6)和许多非SnRNP相关蛋白因子组成的复合体。这个 剪接体在剪接循环中经历戏剧性的变化，产生E，A，前-B，B，Bact，B*，C，C*，P， 和ILS复合体。A、Pre-B、B、BaT、C、C*、P和ILS络合物的高分辨低温电子显微镜结构 在过去的几年中被确定，产生了关于剪接机制的有趣假说 等待通过生化和/或基因分析进行严格的测试。后催化的低温电子显微镜结构 我们在3.3？分辨率下确定的P复合体提供了关于内含子重要性的有趣假说。 3‘s识别中的二级结构。在这个提案中，我们将使用全基因组的化学和酶 试图验证这一假说。P络合物的结构也导致了关于结构和 Dead-box Helicase Prp22在剪接中的功能，我们将使用生物化学和 基因实验。此外，我们还测定了剪接体E复合体的低温电子显微镜结构 外显子定义的建议机制及其到内含子定义的转换，我们将使用 生物化学和结构方法。基因组、生化和结构方法的结合 在本提案中使用的可能提供对整个剪接的完整的机械理解 周而复始。

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.