The molecular grammar of human RNA biology

人类RNA生物学的分子语法

• 批准号: 10622907
• 负责人: Stephen Nicholas Floor
• 金额: $ 36.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10622907
• 关键词: Address; Area; Binding; Biological Assay; Biology; Cells; Cellular biology; Couples; Deaminase; Deamination; Development; Elements; Gene Expression; Genetic; Goals; Heterogeneity; Human; Individual; Measurement; Measures; Mediating; Messenger RNA; Molecular; Molecular Biology; Proteins; RNA; RNA Sequences; RNA Stability; RNA-Binding Proteins; RNA-Protein Interaction; Regulation; Research; Ribonucleoproteins; Ribosomes; Site; Systems Biology; Therapeutic; Training; Translations; Variant; Work; human disease; method development; novel strategies; posttranscriptional; single molecule

Project Summary/Abstract The overarching goal of my research is to define how the molecular grammar of RNA molecules regulates gene expression. To accomplish this goal, I have pursued extensive and interdisciplinary training in the molecular, cellular, and systems biology understanding of human RNA biology. I seek to build upon my documented track record in RNA biology and method development to address pressing questions in RNA biology. This proposal identifies two emphasis areas of importance for inquiry in the next five years. The first emphasis of this proposal builds on my decade of research into the RNA–binding protein DDX3 by identifying and exploring critical gaps in understanding. DDX3 is an essential ATP-dependent RNA–binding protein that couples ATP binding to local RNA duplex unwinding and ribonucleoprotein remodeling. Prior work from my group and others has implicated DDX3 in translational control for mRNA molecules containing a variety of mRNA elements, but the precise mechanism, important mRNA features, and genetic interactions remain incompletely understood. Here, we seek to define how DDX3 interacts with the ribosome to mediate translational control, to use a new assay we developed to define DDX3-dependent translation in an unbiased manner, to define how depletion of DDX3 versus missense variants differ in genetic interactions, and to establish the mechanism leading to changes in RNA levels following DDX3 depletion. The second emphasis of this proposal advances new developments in my group that enable single-molecule measurement of RNA- protein interactions in cells. We evolved a new deamination-based molecular recorder to capture RNA-protein interactions by modifying the sequence of RNA adjacent to an RNA-protein interaction. Through long-read sequencing we can then identify regions in RNA that were bound by a protein tagged with the deaminase. Using this approach, we find unexpected heterogeneity in RNA–binding protein sites on individual mRNA molecules. We propose to build upon these findings, both to understand the mechanistic and functional implications of this heterogeneity and to extend our approach to new RNA–binding proteins. Overall, the proposed research is aligned with my research goal by defining the mechanism of important RNA–binding proteins and by developing new approaches to measure single-molecule RNA biology. I expect the results of the proposed research to advance the understanding of RNA biology with implications for the fundamental understanding of RNA, human disease, and mRNA therapeutics.

项目概要/摘要 我研究的首要目标是定义 RNA 分子的分子语法如何调节 为了实现这一目标，我在基因表达方面进行了广泛的跨学科培训。 我寻求以我对人类 RNA 生物学的分子、细胞和系统生物学的理解为基础。 RNA 生物学和方法开发的记录记录，以解决 RNA 中的紧迫问题 该提案确定了未来五年研究的两个重要重点领域。 该提案的重点是建立在我对 RNA 结合蛋白 DDX3 的十年研究之上，通过识别 并探索理解 DDX3 是一种重要的 ATP 依赖性 RNA 结合蛋白的关键差距。 将 ATP 结合到局部 RNA 双链体解旋和核糖核蛋白重塑。 小组和其他人暗示 DDX3 参与包含多种 mRNA 分子的翻译控制 mRNA 元件，但精确的机制、重要的 mRNA 特征和遗传相互作用仍然存在 在这里，我们试图定义 DDX3 如何与核糖体相互作用来介导。 翻译控制，使用我们开发的新测定法以无偏倚的方式定义依赖于 DDX3 的翻译 方式，以确定 DDX3 的耗竭与错义变体在遗传相互作用中的差异，并 建立导致 DDX3 耗尽后 RNA 水平变化的机制。 该提案推动了我的团队的新发展，使 RNA 的单分子测量成为可能 我们开发了一种新的基于脱氨基的分子记录器来捕获 RNA-蛋白质。 通过修改与 RNA-蛋白质相互作用相邻的 RNA 序列来实现相互作用。 通过测序，我们可以识别 RNA 中与脱氨酶标记的蛋白质结合的区域。 使用这种方法，我们发现单个 mRNA 上的 RNA 结合蛋白位点存在意想不到的异质性 我们建议以这些发现为基础，了解其机制和功能。 这种异质性的影响，并将我们的方法扩展到新的 RNA 结合蛋白。 拟议的研究通过定义重要的 RNA 结合机制与我的研究目标一致 蛋白质和开发测量单分子 RNA 生物学的新方法。 拟议的研究旨在促进对 RNA 生物学的理解，并对基础研究产生影响 了解 RNA、人类疾病和 mRNA 疗法。