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.

项目摘要 人类剪接的复杂性令人生畏，但干预用于治疗疾病的剪接却成立 巨大的潜力。基于强大的初步结果，我们提出了三个利用我们的投资领域 小组对剪接的深入了解，以解决关键的开放问题，并探索创新的潜力 工程。第一个区域解决了U2 SNRNP捕获内含子分支的机制 在剪接体组装的早期，一步已被复发性癌症突变改变，并针对自然界 产生抗生素的细菌。使用两个分支争夺认可的新记者，我们有 确定了一种新颖的剪接保真度机制，我们称为“ no-bp衰减”，其中U2复合物因失败而失败 异常的分支选择被破坏。我们将表征这个过程，应用一电池候选人 基于基因的抑制筛选和剪接提取物中的生化测试。第二个调查领域 解决了如何与单个基因和细胞处的转录和细胞生长整合的剪接 级别，如果要成功设计拼接，则需要创新的新兴领域。初步结果 表明酵母细胞具有有限的剪接能力，这会产生至关重要的MRNA的竞争 到细胞功能。我们将使用RNA测序衡量剪接能力和竞争动力学 为了开发一个预测模型，以说明如何在系统级别协调剪接。理解 单个基因对该系统的贡献我们正在采用合成生物学方法。我们有 RNA聚合酶II的工程特定地点暂停，并表明它们改变了剪接效率和 替代剪接，通过未知的机制，我们将剖析。我们还将详细探讨 剪接噪声（随着时间的推移剪接输出的随机变化）在剪接控制稳定的能力上 稳态表达设置（如许多RNA结合蛋白基因所做的那样），并且可以控制Bissable 切换（就像在果蝇性致命基因中一样）。这些实验将定义 简单的剪接调节电路。第三个调查领域的重点是内含子增益的过程 及其在真核基因创造和基因多样化中的作用。我们最近发现的剪接体 剪接后可以将幼虫内含子转换为真正的内含子圆，表明它可以执行反向剪接 体内反应，提出了有关它是否以及如何促进新内含子形成的问题。我们 预测测试生化步骤的建议是剪接体介导的内含子增益所必需的，并且具有 已经设置了实验以记录体内内含子增益。鉴于剪接的基本保护 机械，这项工作有望直接转化为对基因调节机制的新理解 在包括人类在内的真核生物中。剪接缺陷经常被认为是疾病的贡献者，并且 解决剪接缺陷的干预措施越来越成功地治疗途径。