The Catalytic Mechanism of Nuclear Premessenger RNA Splicing by the Spliceosome

真核基因，包括大多数人类基因，被许多内含子中断。转录后， 这样的基因，内含子在剪接体催化的两个磷酰基转移反应中被切除， 由蛋白质和RNA组成的大分子机器。在第一个反应中，内含子的2'羟基 腺苷攻击5 ′剪接位点，从5 ′外显子上切割内含子。在第二个反应中，新的- 释放的5'外显子形成的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的机制， 催化前体mRNA剪接，特别是确定RNA在催化中的作用，无论是在结构上， 功能术语。虽然还原论方法已经揭示了剪接体RNA的催化活性， 这些反应是低效的并且不完全表征。因此，机械地理解 前mRNA剪接需要研究剪接体本身。有趣的是，第二组内含子通过一个 这两种酶具有共同的RNA特征。最近的一 第II组内含子的晶体结构显示两个结合的金属，提示剪接体中的金属配体 以及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，我们以前的研究涉及金属基催化的剪接和 我们的工作和其他人的工作已经暗示剪接体RNA作为催化金属配体。此外，我们最近 在体外发现了对剪接的化学作用施加高度严格性的保真度机制， 一个策略，以放宽这些限制，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， 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， 金属和金属配体在外显子连接中的作用，（ii）研究金属和金属配体在5'剪接位点中的作用 切割和（iii）研究RNA三级相互作用在促进催化中的作用。我们建议 通过独特的合作实现这些目标，这种合作允许化学， 生物化学和分子遗传学。我们将利用模式生物芽殖酵母，它允许两者 前体mRNA剪接的生物化学和遗传学研究。考虑到两者之间的潜在相似性， 剪接体和第二组内含子的催化机制，这项工作将有重要的意义， 了解剪接体的进化起源。至少有15%的人类疾病是由 从拼接的错误，这项工作也将照亮一个机器的内部工作，这是必不可少的井- 人类的存在。

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.