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Structure and function of spliceosome

剪接体的结构和功能

基本信息

批准号：
10219306
负责人：
RUI ZHAO
金额：
$ 42.97万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2023-07-31
项目状态：
已结题

项目摘要

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.

抽象的 mRNA 前体剪接对于所有真核生物的基因表达至关重要，剪接错误会导致遗传紊乱和许多其他疾病。全面了解pre-mRNA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（一种大的 RNA/蛋白质）催化的两个酯交换反应进行由五个 snRNP（U1、U2、U4、U5、U6）和许多非 snRNP 相关蛋白因子组成的复合物。这剪接体在剪接周期中经历巨大的变化，产生E、A、Pre-B、B、Bact、B*、C、C*、P、和 ILS 综合体。 A、pre-B、B、Bact、C、C*、P 和 ILS 复合物的高分辨率冷冻电镜结构具有在过去几年中得到了确定，产生了关于剪接机制的有趣假设等待通过生化和/或遗传分析进行严格测试。后催化的冷冻电镜结构我们以 3.3 Å 分辨率测定的 P 复合物提供了关于内含子重要性的有趣假设 3' ss 识别中的二级结构。在本提案中，我们将使用全基因组化学和酶促进行探索以检验这一假设。 P复合体的结构也引发了关于其结构和结构的假设 DEAD-box 解旋酶 Prp22 在剪接中的功能，我们将使用生化和基因实验。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外显子定义机制及其向内含子定义的转换，我们将使用它进行测试生物化学和结构方法。基因组、生物化学和结构方法的结合本提案中使用的方法有可能提供对整个拼接的完整机制理解循环。