The Catalytic Mechanism of Nuclear Premessenger RNA Splicing by the Spliceosome

描述(申请人提供)：真核基因，包括大多数人类基因，被许多内含子打断。在这些基因转录后，内含子在剪接体催化的两个磷酸转移反应中被切除，剪接体是一个由蛋白质和RNA组成的大分子机器。在第一个反应中，内含子腺苷的2‘羟基攻击5’剪接位点，将内含子从5‘外显子中切割出来。在第二个反应中，新形成的5‘外显子的3’羟基攻击3‘剪接位点，切除内含子并连接两侧的外显子。剪接体的RNA组分与识别内含子和催化内含子切除有关。我们的长期目标是确定剪接体催化前mRNA剪接的机制，特别是确定RNA在结构和功能方面的催化作用。虽然还原论方法揭示了剪接体RNA的催化活性，但这些反应效率低下，特征不完全。因此，对前信使核糖核酸剪接的机制理解需要对剪接体本身进行研究。有趣的是，第二组内含子通过与剪接体难以区分的途径进行剪接，两种酶都有共同的RNA特征。最近一个第二类内含子的晶体结构揭示了两个结合的金属，这表明剪接体中有金属配体，并且第二类内含子和剪接体都有催化机制。事实上，使用最先进的化学方法，我们之前的研究表明，在剪接和我们的工作中，都有基于金属的催化作用，而其他人的研究表明，剪接体RNA是催化金属配体。此外，我们最近在体外发现的对剪接化学施加高度严格的保真机制现在提供了一种放松这些限制和更广泛地研究催化的策略。我们的近期目标是研究金属在催化前mRNA剪接中的作用，确定这些金属的配体的同一性，以及金属结合和催化所需的RNA结构。具体地说，我们的目标是(1)研究金属和金属配体在外显子连接中的作用；(2)研究金属和金属配体在5‘剪接位点切割中的作用；(3)研究RNA三级相互作用在促进催化作用中的作用。我们建议通过一种独特的合作来实现这些目标，这种合作允许结合化学、生物化学和分子遗传学的方法。我们将利用模式生物发芽酵母，它允许对前mRNA剪接进行生化和遗传学研究。考虑到剪接体和第II组内含子的催化机制之间的潜在相似性，这项工作将对理解剪接体的进化起源具有重要意义。鉴于至少15%的人类疾病是由拼接错误引起的，这项工作也将揭示对人类福祉至关重要的机器的内部工作原理。 公共卫生相关性：据估计，至少15%的人类疾病是由剪接缺陷引起的。我们治疗这种疾病的能力在很大程度上将取决于我们对剪接机械的复杂性的理解。在试图确定剪接体的基本催化元件的过程中，该项目将为必须在健康的人类细胞中正常发挥作用的基本机制提供新的见解，从而阐明可能的疾病机制以及潜在的治疗策略。

DESCRIPTION (provided by applicant): Eukaryotic genes, including most human genes, are interrupted by numerous introns. After transcription of such genes, the introns are excised in two phosphoryl transfer reactions catalyzed by the spliceosome, a macromolecular machine composed of both protein and RNA. In the first reaction, the 2' hydroxyl of an intronic adenosine attacks the 5' splice site cleaving the intron from the 5' exon. In the second reaction, the newly- formed 3' hydroxyl of the liberated 5' exon attacks the 3' splice site, excising the intron and ligating the flanking exons. The RNA components of the spliceosome have been implicated in both recognizing introns and catalyzing intron excision. Our long-term objective is to determine the mechanism by which the spliceosome catalyzes pre-mRNA splicing and in particular to define the role of RNA in catalysis, both in structural and functional terms. While reductionist approaches have revealed catalytic activities of the spliceosomal RNAs, these reactions are inefficient and incompletely characterized. Consequently, a mechanistic understanding of pre-mRNA splicing requires an investigation of the spliceosome itself. Interestingly, group II introns splice by a pathway indistinguishable from the spliceosome, and both enzymes share common RNA features. A recent crystal structure of a group II intron reveals two bound metals, suggesting metal ligands in the spliceosome and a mechanism for catalysis by both group II introns and the spliceosome. Indeed, using state-of-the-art chemical approaches, our previous studies have implicated metal-based catalysis in both steps of splicing and our work and that of others has implicated spliceosomal RNAs as catalytic metal ligands. Further, our recent discovery of fidelity mechanisms in vitro that impose high stringency on the chemistry of splicing now provides a strategy to relax these constraints and to more broadly investigate catalysis. Our near-term goal is to investigate the roles of metals in catalyzing pre-mRNA splicing, the identity of the ligands for such metals and the RNA structure required for metal binding and catalysis. Specifically, we aim (i) to investigate the role of metals and metal ligands in exon ligation, (ii) to investigate the role of metals and metal ligands in 5' splice site cleavage and (iii) to investigate the role of RNA tertiary interactions in promoting catalysis. We propose to accomplish these aims through a unique collaboration that allows a combined approach of chemistry, biochemistry and molecular genetics. We will utilize the model organism budding yeast, which allows for both biochemical and genetic studies of pre-mRNA splicing. Considering the potential similarity between the catalytic mechanisms of the spliceosome and group II introns, this work will have important implications for understanding the evolutionary origins of the spliceosome. Given that at least 15% of human diseases result from errors in splicing, this work will also illuminate the inner workings of a machine that is essential to the well- being of humans. PUBLIC HEALTH RELEVANCE: It has been estimated that at least 15% of human diseases result from defects in splicing. Our ability to treat such diseases will depend, in large part, on our understanding of the complexities of the splicing machinery. In its attempt to identify the essential catalytic elements of the spliceosome, this project will provide novel insights into the fundamental mechanisms that must function properly in healthy human cells and thereby illuminate possible mechanisms for disease as well as potential strategies for treatment.