Revealing molecular determinants of transcript-specific regulation in pre-mRNA splicing via rapid in vivo kinetic rate measurements

通过快速体内动力学速率测量揭示前 mRNA 剪接中转录特异性调节的分子决定因素

• 批准号: 10589841
• 负责人: JEFFREY A PLEISS
• 金额: $ 32.8万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-05 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589841
• 关键词: Acceleration; Alleles; Alternative Splicing; Area; Cell Culture Techniques; Chemicals; Chromatin; Complement; Complex; Coupling; DNA-Directed RNA Polymerase; Data; Deceleration; Detection; Disease; Elements; Environment; Enzymes; Equilibrium; Etiology; Eukaryota; Fission Yeast; Gene Expression; Gene Structure; Genes; Genetic; Genetic Transcription; Goals; Human; Individual; Introns; Kinetics; Knowledge; Label; Mammalian Cell; Measurement; Measures; Metabolic; Methodology; Molecular; Monitor; Mutation; Organism; Pathway interactions; Primer Extension; Process; Property; Protein Isoforms; Proteome; RNA; RNA Splicing; RNA, Messenger, Splicing; Reaction; Regulation; Regulatory Element; Resolution; Role; Saccharomycetales; Site; Spliceosome Assembly Pathway; Spliceosomes; Techniques; Testing; Transcript; Transcription Elongation; Variant; Work; Yeasts; base; experimental study; genetic variant; genome-wide; human disease; improved; in vivo; insight; mRNA Precursor; novel; tool; transcriptome sequencing

ABSTRACT It has long been known that pre-messenger RNA (pre-mRNA) splicing is an essential component of gene expression in eukaryotic organisms, yet the past decade has seen a dramatic increase in our appreciation for its role in regulating gene expression1. Most higher eukaryotes, including humans, regulate alternative splicing as a tool for proteome expansion, and an ever-increasing number of human diseases are associated with mutations in this pathway2,3. The mechanisms by which the spliceosome, which catalyzes pre-mRNA splicing, enacts this regulation is a complex problem whose solution remains poorly understood yet will be critical to understanding the etiology of many diseases. Proper regulation requires the spliceosome to faithfully assemble upon and activate ‘cognate’ splice site sequences in the background of scores of aberrant, ‘near-cognate’ splice sites, yet the spliceosome must balance this high fidelity splice site selection with the need for rapid, efficient splicing. At the simplest level, improved knowledge of how the spliceosome achieves this balance will require understanding both: (1) the landscape of cis-regulatory elements at splice sites that enable them to be distinguished as either ‘cognate’ or ‘non-cognate’; and (2) the mechanisms by which the spliceosome discriminates between such sites. In the work described here, we seek to better understand basic mechanisms of pre-mRNA splicing regulation by leveraging a powerful methodology recently developed in my lab called Multiplexed Primer Extension sequencing, or MPE-seq. Our approach is unique in that it allows for the genome-wide detection of pre-mRNA splicing intermediates. By combining this technique with rapid metabolic RNA labeling techniques developed by others, my group has now determined the in vivo rates of both chemical steps of pre-mRNA splicing across the complement of spliced transcripts in budding yeast. Remarkably, these data reveal a wide variation among the rates, both between the two steps for individual transcripts and between different transcripts. The goals of the work described here are to leverage the information derived from these experiments to push our understanding of the principles that underlie this regulation.

抽象的 人们早就知道前信使RNA（pre-mRNA）剪接是基因的重要组成部分 真核生物中的表达，但在过去的十年里，我们对其的认识急剧增加 1. 调控基因表达的作用大多数高等真核生物，包括人类，将选择性剪接调节为 蛋白质组扩展的工具，越来越多的人类疾病与突变有关 在此途径中2,3。剪接体催化前 mRNA 剪接的机制 监管是一个复杂的问题，其解决方案仍然知之甚少，但对于理解这一问题至关重要 许多疾病的病因。正确的调控需要剪接体忠实地组装并 在大量异常的“近同源”剪接位点背景中激活“同源”剪接位点序列，但 剪接体必须平衡这种高保真剪接位点选择与快速、高效剪接的需求。在 最简单的水平，需要更好地了解剪接体如何实现这种平衡 两者：（1）剪接位点的顺式调控元件的景观，使它们能够被区分为 “同源”或“非同源”； (2)剪接体区分这些位点的机制。 在此描述的工作中，我们寻求更好地了解前 mRNA 剪接调节的基本机制 利用我的实验室最近开发的一种强大的方法，称为多重引物延伸 测序，或 MPE-seq。我们的方法的独特之处在于它允许对前 mRNA 进行全基因组检测 拼接中间体。通过将该技术与快速代谢 RNA 标记技术相结合， 其他人，我的小组现在已经确定了前 mRNA 剪接的两个化学步骤的体内速率 芽殖酵母中剪接转录本的补充。值得注意的是，这些数据揭示了不同国家之间的巨大差异 速率，无论是单个转录本的两个步骤之间还是不同转录本之间。的目标 这里描述的工作是利用从这些实验中获得的信息来推动我们的理解 本条例所依据的原则。