Structure, regulation, and evolution of the splicing machinery

项目摘要 人类剪接的复杂性是令人生畏的，但干预剪接治疗疾病仍然有效。 巨大的潜力基于强有力的初步结果，我们提出了三个调查领域，利用我们的 集团对拼接的深厚知识，以解决关键的开放问题，并探索创新的潜力， 工程.第一个领域涉及U2 snRNP捕获内含子分支点的机制 在剪接体组装的早期，这一步骤被复发性癌症突变改变，并在本质上被 产寄生虫的细菌使用两个分支点竞争识别的新报告子，我们有 确定了一种新的剪接保真度机制，我们称之为“NO-BP衰变”，其中U2复合物由于 异常的分支点选择被破坏。我们将描述这个过程，应用一组候选人 基于基因的抑制筛选和剪接提取物的生化测试。第二个调查领域 阐述了剪接是如何与转录和细胞生长整合在单个基因和细胞中的， 水平，一个需要创新的新兴领域，如果拼接是成功的工程。初步结果 这表明酵母细胞具有有限的剪接能力，这种能力会产生对前mRNA的竞争， to cell细胞function功能.我们将使用RNA测序来测量剪接能力和竞争动力学 开发一个预测模型，解释如何在系统层面协调剪接。了解 我们正在应用合成生物学方法来研究单个基因对这个系统的贡献。我们有 工程化的RNA聚合酶II的位点特异性暂停，并显示它们改变剪接效率， 选择性剪接，通过未知的机制（S），我们将剖析。我们还将详细探讨 拼接噪声（拼接输出随时间的随机变化）对拼接控制稳定的能力的影响 稳态表达设置（如它在许多RNA结合蛋白基因中所做的那样）以及控制一个基因的表达。 开关（就像果蝇的性别致死基因一样）。这些实验将确定 简单的拼接调节电路。第三个研究领域的重点是内含子获得的过程 及其在真核基因创造和基因多样化中的作用。我们最近发现剪接体 在剪接后能将内含子转化为真正的内含子环，表明它能进行反向剪接， 在体内的反应，提出了问题，它是否以及如何可能促进新的内含子的形成。我们 建议测试预测剪接体介导的内含子获得所需的生化步骤，并已 已经建立了实验来记录体内内含子的增加。考虑到剪接的基本守恒性 这项工作有望直接转化为对基因调控机制的新理解 包括人类在内的真核生物。剪接缺陷通常被认为是疾病的贡献者， 解决剪接缺陷的干预是越来越成功的治疗途径。

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.