Structure, regulation, and evolution of the splicing machinery

熔接机械的结构、调节和演变

• 批准号: 10406517
• 负责人: Manuel Ares
• 金额: $ 51.89万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10406517
• 关键词: Address; Alternative Splicing; Antibiotics; Area; Awareness; Bacteria; Basic Science; Biochemical; Candidate Disease Gene; Cell physiology; Cells; Complex; Defect; Disease; Drosophila genus; Engineering; Eukaryota; Evolution; Fruit; Gene Expression Regulation; Gene Proteins; Genes; Genetic Transcription; Health; Human; Individual; Intervention; Introns; Investigation; Knowledge; Lethal Genes; Malignant Neoplasms; Measures; Mediating; Mutation; Nature; Noise; Output; Pathway interactions; Process; RNA Polymerase II; RNA Splicing; RNA-Binding Proteins; Reaction; Recurrent Malignant Neoplasm; Regulation; Reporter; Role; Site; Spinal Muscular Atrophy; Spliceosome Assembly Pathway; Spliceosomes; Structure; System; Testing; Time; Translating; U2 Small Nuclear Ribonucleoprotein; Variant; Work; Yeasts; base; cell growth; experimental study; improved; in vivo; innovation; mRNA Precursor; novel; novel strategies; predictive modeling; repaired; sex; success; synthetic biology; transcriptome sequencing; tumor progression

PROJECT SUMMARY The complexity of human splicing is daunting, yet intervention in splicing for treatment of diseases holds huge potential. Based on strong preliminary results, we propose three areas of investigation that leverage our group’s deep knowledge of splicing to address critical open questions, and to explore the potential for innovative engineering. The first area addresses the mechanism by which U2 snRNP captures the intron branchpoint early in spliceosome assembly, a step altered by recurrent cancer mutations and targeted in nature by antibiotic-producing bacteria. Using new reporters in which two branchpoints compete for recognition, we have identified a novel splicing fidelity mechanism we call “NO-BP decay,” in which U2 complexes that fail due to aberrant branchpoint selection are destroyed. We will characterize this process, applying a battery of candidate gene-based suppressor screens and biochemical tests in splicing extracts. The second area of investigation addresses how splicing is integrated with transcription and cell growth at the individual gene and cellular levels, an emerging area in need of innovation if splicing is to be successfully engineered. Preliminary results indicate that yeast cells have a limited capacity for splicing that creates competition for pre-mRNAs that is critical to cell function. We will measure both splicing capacity and the dynamics of competition, using RNA sequencing to develop a predictive model that explains how splicing is coordinated at a systems level. To understand the contribution of individual genes to this system we are applying synthetic biology approaches. We have engineered site-specific pauses of RNA polymerase II and shown that they alter splicing efficiency and alternative splicing, by unknown mechanism(s) that we will dissect. We will also explore in detail the role of splicing noise (stochastic variations in splicing output over time) on the ability of splicing to control stable homeostatic expression settings (as it does in many RNA binding protein genes) as well as to control a bistable switch (as it does in the Drosophila Sex lethal gene). These experiments will define the operational principles of simple splicing regulatory circuits. The third area of investigation is focused on the process of intron gain and its roles in eukaryotic gene creation and gene diversification. Our recent discovery that the spliceosome can convert the lariat intron to a true intron circle after splicing indicates that it can carry out reverse splicing reactions in vivo, raising questions about whether and how it might promote formation of new introns. We propose to test biochemical steps predicted to be necessary for spliceosome-mediated intron gain, and have already set up experiments to document intron gain in vivo. Given the fundamental conservation of the splicing machinery, this work promises to translate directly into new understanding of the mechanisms of gene regulation in eukaryotes, including humans. Defects in splicing are frequently recognized as contributors to disease, and interventions that address splicing defects are increasingly successful pathways to treatment.

项目概要 人类剪接的复杂性令人望而生畏，但干预剪接以治疗疾病仍然有效 基于巨大的潜力，我们提出了三个利用我们的研究领域的研究。 小组对拼接的深入了解，以解决关键的开放性问题，并探索创新的潜力 第一个领域涉及 U2 snRNP 捕获内含子分支点的机制。 剪接体组装的早期，这是复发性癌症突变的一步，并且具有自然靶向性 使用两个分支点竞争识别的新生产者，我们有 确定了一种新的剪接保真度机制，我们称之为“NO-BP 衰减”，其中 U2 复合物由于以下原因而失败 我们将通过应用一系列候选点来描述这个过程。 剪接提取物中基于基因的抑制筛选和生化测试第二个研究领域。 解决剪接如何与个体基因和细胞的转录和细胞生长整合 如果要成功设计剪接的初步结果，这是一个需要创新的新兴领域。 表明酵母细胞的剪接能力有限，这会产生对前 mRNA 的竞争，这一点至关重要 我们将使用 RNA 测序来测量剪接能力和竞争动态。 开发一个预测模型来解释拼接如何在系统级别进行协调。 单个基因对该系统的贡献我们正在应用合成生物学方法。 设计了 RNA 聚合酶 II 的位点特异性暂停，并表明它们改变了剪接效率 选择性剪接，通过我们将剖析的未知机制，我们还将详细探讨其作用。 拼接噪声（拼接输出随时间的随机变化）对拼接控制稳定能力的影响 稳态表达设置（正如许多 RNA 结合蛋白基因中所做的那样）以及控制双稳态 开关（如果蝇性致死基因中的作用）。这些实验将定义开关的操作原理。 简单的剪接调控电路的第三个研究领域集中于内含子增益的过程。 及其在真核基因创建和基因多样化中的作用。我们最近发现剪接体。 剪接后能将套索内含子转化为真正的内含子环，表明其可以进行反向剪接 体内反应，引发了关于它是否以及如何促进新内含子形成的问题。 测试预计剪接体介导的内含子增益所必需的生化步骤，并已 考虑到剪接的基本保守性，已经建立了记录内含子增益的实验。 机器，这项工作有望直接转化为对基因调控机制的新理解 在真核生物中，包括人类，剪接缺陷经常被认为是导致疾病的因素，并且 解决剪接缺陷的干扰是越来越成功的治疗途径。