The Catalytic Mechanism of Nuclear Premessenger RNA Splicing by the Spliceosome

描述（由申请人提供）：真核基因，包括大多数人类基因，被许多内含子中断。在这些基因转录后，内含子在剪接体催化的两个磷酰基转移反应中被切除，剪接体是由蛋白质和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，从5'外显子切割内含子。在第二个反应中，释放的5'外显子的新形成的3'羟基攻击3'剪接位点，切除内含子并连接侧翼外显子。剪接体的RNA成分参与识别内含子和催化内含子切除。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在催化中的作用，包括结构和功能方面。虽然还原论的方法已经揭示了剪接体RNA的催化活性，但这些反应是低效的，并且不完全表征。因此，前体mRNA剪接的机械理解需要对剪接体本身进行研究。有趣的是，第二组内含子剪接的途径与剪接体没有区别，两种酶都有共同的RNA特征。最近的一个II组内含子的晶体结构揭示了两个绑定的金属，这表明金属配体的剪接体和II组内含子和剪接体的催化机制。事实上，使用最先进的化学方法，我们以前的研究已经暗示了剪接和我们的工作的两个步骤中的基于金属的催化，并且其他人已经暗示了剪接体RNA作为催化金属配体。此外，我们最近在体外发现的保真度机制，对剪接的化学施加高严格性，现在提供了一种策略，以放松这些限制，并更广泛地研究催化。我们的近期目标是研究金属在催化前体mRNA剪接中的作用，这些金属的配体的身份以及金属结合和催化所需的RNA结构。具体而言，我们的目标是（i）调查的作用，金属和金属配体的外显子连接，（ii）调查的作用，金属和金属配体的5'剪接位点切割和（iii）调查的作用，RNA三级相互作用，促进催化。我们建议通过独特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。我们将利用模式生物芽殖酵母，这使得生物化学和遗传学研究的前mRNA剪接。考虑到剪接体和II组内含子的催化机制之间的潜在相似性，这项工作将有重要的意义，了解剪接体的进化起源。鉴于至少有15%的人类疾病是由拼接错误引起的，这项工作也将阐明对人类福祉至关重要的机器的内部运作。 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。我们治疗这些疾病的能力在很大程度上取决于我们对剪接机制复杂性的理解。在试图确定剪接体的基本催化元素的过程中，该项目将为必须在健康人体细胞中正常发挥作用的基本机制提供新的见解，从而阐明疾病的可能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.