The Catalytic Mechanism of Nuclear Premessenger RNA Splicing by the Spliceosome

描述(申请人提供)：我们的长期目标是阐明剪接体催化前-mRNA剪接的机制，特别是确定RNA和蛋白质在催化中的结构和功能作用。剪接体催化前mRNA的剪接是真核生物的一个重要特征。在人类中，几乎每个基因的平均10个内含子都需要催化作用，而在其他位置的催化作用扩大了蛋白质组，增加了高等真核生物的复杂性。事实上，至少15%的人类疾病的突变是由剪接错误造成的。尽管前mRNA剪接对真核基因表达如此重要，但几十年来，我们对剪接催化的理解一直停滞不前，特别是与我们对基因表达的另外两个主要阶段-转录和翻译-催化的理解相比。我们甚至还不知道催化是由剪接体的RNA还是蛋白质部分介导的。然而，通过我们的合作努力，我们最近提供了确凿的证据，证明RNA部分催化剪接，定位了两个二价金属离子与剪切的磷酸盐相互作用。值得注意的是，我们的工作还建立了剪接体利用的催化机制与自剪接II类RNA内含子的催化机制没有区别，将与II类内含子的平行延伸到催化核心的原子水平，并为这两种酶具有共同进化起源的假说提供了有力的支持。尽管在该领域取得了这一进展和其他成就，但许多根本问题仍然存在。例如，两个剪接系统中的亲核试剂是如何被激活以进行催化的，剪接体催化核心的结构是如何定位催化的反应位点的，以及蛋白质在介导基于RNA的催化中扮演什么角色？为了回答这些问题，我们的目标是测试核前mRNA剪接催化的金属簇模型，定义剪接体催化核心的结构和动力学，并确定蛋白质在辅助基于RNA的剪接催化中的重要作用。我们将通过继续我们独特和协同的合作来实现这些目标，这种合作允许化学、生物化学和分子遗传学的结合方法。这项工作将改变我们对RNA和蛋白质在剪接体催化中作用的理解，对于理解剪接体的起源和进化具有重要意义。

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the mechanisms by which the spliceosome catalyzes pre-mRNA splicing and in particular to define the structural and functional role of RNA and protein in catalysis. Catalysis of pre-mRNA splicing by the spliceosome is a defining feature of eukaryotes. In humans, catalysis is required to excise ten introns on average for nearly every gene, and catalysis at alternative sites expands the proteome and contributes to the complexity of higher eukaryotes. Indeed, mutations in at least 15% of human diseases result from errors in splicing. Despite this importance of pre-mRNA splicing to eukaryotic gene expression, for decades our understanding of splicing catalysis has languished, particularly in comparison to our understanding of catalysis at the two other major stages of gene expression - transcription and translation. We have not even known whether catalysis is mediated by the RNA or protein parts of the spliceosome. However, through our collaborative efforts, we have recently provided definitive evidence that the RNA parts catalyze splicing, positioning two divalent metal ions for interaction with the scissile phosphates. Strikingly, our work also establishes that the spliceosome utilizes a catalytic mechanism indistinguishable from the catalytic mechanism of self-splicing group II RNA introns, extending the parallels with the group II intron to the atomic level at the catalytic core and providing strog support for the hypothesis that these two enzymes share common evolutionary origins. Despite this advance and other achievements in the field, many fundamental questions remain. For example, how are the nucleophiles in either splicing system activated for catalysis, how does the architecture of the spliceosomal catalytic core position the reactive sites for catalysis, and what is the role of protein in mediating RNA-based catalysis? To answer these questions, we aim to test a metal cluster model for catalysis of nuclear pre-mRNA splicing, to define the architecture and dynamics of the catalytic core of the spliceosome, and to define the essential role of protein in assisting RNA-based splicing catalysis. We will accomplish these aims through a continuation of our unique and synergistic collaboration that allows for a combined approach of chemistry, biochemistry and molecular genetics. This work will transform our understanding of the roles of RNA and protein in spliceosomal catalysis, with significant implications for understanding the origins and evolution of the spliceosome.