Mechanisms of Spliceosome Assembly and Regulation

项目摘要/摘要 RNA剪接是人类基因表达的关键特征，也是基因扩增的主要贡献者 通过选择性剪接获得信息。拼接是由一台大型的动态细胞机器执行的，这台机器被称为 剪接体。剪接体由小核核糖核蛋白(SnRNP)组成，这些核糖核蛋白组装在 前体转录本(前-mRNAs)去除内含子并将外显子拼接在一起。这一过程必须发生 正是为了保存信使核糖核酸携带的遗传信息。对拼接至关重要的是正确的 RNA键的断裂和形成位点[5‘和3’剪接位点(SS)及其分支]的鉴定 地点(BS)]。许多不同的ATPase有助于SS和BS识别以及载波的保真度 对剪接体进行了广泛的成分和构象改造。最近，生化研究 通过对酵母和人类的数十种不同结构的测定，剪接的结构发生了变化 用冷冻-EM技术剪接体。尽管发生了这场结构性革命，但许多人仍不清楚 剪接反应。我实验室的研究目标是阐明剪接体的机制 使用各种技术进行生物化学深度的组装和调节。我们经常使用单分子 荧光显微镜解开复杂和非均相的反应途径 拼接机械。在最近的工作中，我们研究了5‘SS和BS的识别、组装和 U6SnRNP的动力学，以及开发的荧光标记、纯化和抑制RNA的方法 和RNPs。我们未来五年的愿景是融合从剪接体结构中获得的见解 通过单分子、生物化学、计算和遗传实验来解决我们在 有关拼接的知识。这些差距包括RNP折叠和组装的基本原则， 调控剪接的机制，以及缺乏特异和有效的化学抑制物 剪接体。作为这一愿景的一部分，我们将使用多学科方法回答以下问题： 1)RNA和蛋白质如何共折叠组装U6 SnRNP？ 2)剪接体在其活性部位的创建过程中如何重塑？ 3)调控蛋白如何促进弱5‘SS的剪接？ 4)在小分子抑制剂的剪接过程中，我们如何阻止ATPase依赖的转换？ 5)如何定量分析、比较和整合剪接体的冷冻-EM结构？

PROJECT SUMMARY/ABSTRACT RNA splicing is a key feature of human gene expression and a major contributor to expansion of genetic information by alternative splicing. Splicing is carried out by a large and dynamic cellular machine called the spliceosome. Spliceosomes are composed of small nuclear ribonucleoproteins (snRNPs) that assemble on precursor transcripts (pre-mRNAs) to remove introns and splice together exons. This process must occur precisely in order to preserve the genetic information carried in the mRNA. Critical for splicing is the correct identification of the sites of RNA bond cleavage and formation [the 5' and 3' splice sites (SS) and the branch site (BS)]. A number of different ATPases contribute to the fidelity of SS and BS recognition as well as carry out extensive compositional and conformational remodeling of the spliceosome. Recently, biochemical studies of splicing have been transformed by determination of dozens of different structures of yeast and human spliceosomes by cryo-EM. Despite this structural revolution, much remains unknown about central features of the splicing reaction. The goal of my laboratory’s research is to elucidate mechanisms of spliceosome assembly and regulation in biochemical depth using a variety of techniques. We often use single molecule fluorescence microscopy to deconvolute the complex and heterogeneous reaction pathways employed by the splicing machinery. In recent work, we have studied mechanisms of 5'SS and BS recognition, assembly and dynamics of the U6 snRNP, and developed methods for fluorescently-labeling, purifying, and inhibiting RNAs and RNPs. Our vision for the next five years is to merge the insights obtained from structures of spliceosomes with single molecule, biochemical, computational, and genetic experiments to address outstanding gaps in our knowledge of splicing. These gaps include fundamental principles of RNP folding and assembly, the mechanisms of regulated splicing, and the scarcity of specific and effective chemical inhibitors of the spliceosome. As part of this vision, we will answer the following questions using multi-disciplinary approaches: 1) How do RNA and protein co-fold to assemble the U6 snRNP? 2) How is the spliceosome remodeled during creation of its active site? 3) How do regulatory proteins promote splicing at weak 5'SS? 4) How can we block ATPase-dependent transitions during splicing with small molecule inhibitors? 5) How do we quantitatively analyze, compare, and integrate cryo-EM structures of spliceosomes?