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: 10383702
• 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/10383702
• 关键词: Alleles; Alternative Splicing; Area; Cell Culture Techniques; Chemicals; Chromatin; Complement; Complex; Coupling; DNA-Directed RNA Polymerase; Data; 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; Messenger RNA; Metabolic; Methodology; Molecular; Monitor; Mutation; Organism; Pathway interactions; Primer Extension; Process; Property; Protein Isoforms; Proteome; RNA; RNA 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 长期以来，人们已经知道，预上音符RNA（前MRNA）剪接是基因的重要组成部分 在真核生物中的表达，但是过去十年的表达使我们对它的欣赏急剧增加 控制基因表达1。包括人类在内的大多数较高的真核生物都将替代剪接调节为 蛋白质组扩展的工具，以及不断增加的人类疾病与突变有关 在此途径中2,3。剪接催化前mRNA剪接的剪接体的机制，实现了这一点 监管是一个复杂的问题，其解决方案仍然很糟糕，但对于理解至关重要 许多疾病的病因。适当的法规要求剪接体忠实地聚集并 在异常，“近灵”剪接位点的背景中激活“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的选择，并需要快速，有效的剪接。 最简单的水平，提高了对剪接体如何实现这种平衡将需要理解的知识 两者：（1）剪接站点的顺式调节元素的景观，使它们能够被区分为 “ cognate”或“非同义”； （2）剪接体区分此类站点的机制。 在此处描述的工作中，我们试图更好地了解MRNA剪接调节的基本机制 通过利用最近在我的实验室中开发的强大方法，称为多路复用引物扩展 测序或mpe-seq。我们的方法是独特的，因为它允许全基因组检测前MRNA检测 剪接中间体。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两个化学步骤的体内速率 在发芽酵母中的剪接成绩单的补充。值得注意的是，这些数据揭示了 速率，在单个成绩单的两个步骤之间以及不同的成绩单之间。目标的目标 这里描述的工作是利用这些实验得出的信息来推动我们的理解 该法规的基础的原则。