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.

摘要 众所周知，前信使rna(pre-mrna)剪接是基因的重要组成部分。 在真核生物中的表达，然而在过去的十年里，我们对它的欣赏急剧增加 在基因表达调控中的作用1。大多数高等真核生物，包括人类，都将选择性剪接调节为 一种蛋白质组扩张的工具，越来越多的人类疾病与突变有关 在此途径2，3.剪接体，其催化的前-信使核糖核酸剪接，制定机制 监管是一个复杂的问题，其解决方案仍然鲜为人知，但对理解 许多疾病的病原学。适当的调节需要剪接体忠实地组装在和 在数十个异常的、近同源的剪接位点的背景中激活“同源”剪接位点序列，但 剪接体必须在这种高保真的剪接位置选择和快速、高效的剪接的需要之间取得平衡。在… 对剪接体如何达到这种平衡的最简单的、更好的知识需要理解 两者：(1)剪接位点的顺式调控元件的格局，使它们能够被区分为 “同源的”或“非同源的”；以及(2)剪接体区分这些位点的机制。 在这里描述的工作中，我们试图更好地理解前mna剪接调控的基本机制。 通过利用我实验室最近开发的一种名为多路复用初级扩展的强大方法 测序，或MPE-SEQ。我们的方法是独特的，因为它允许在全基因组范围内检测前信使核糖核酸 拼接中间体。通过将该技术与由 其他人，我的团队现在已经确定了前mRNA剪接的两个化学步骤在体内的比率 芽殖酵母中剪接转录本的互补。值得注意的是，这些数据揭示了 不同成绩单之间和不同成绩单之间的比率。世界银行的目标是 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 这项规定所依据的原则。