Developing optogenetic and chemogenetic approaches to control RNA metabolism in live cells

开发光遗传学和化学遗传学方法来控制活细胞中的 RNA 代谢

• 批准号: 10582002
• 负责人: Bin Wu
• 金额: $ 14.62万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582002
• 关键词: Address; Anterior; Biological; Biological Process; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; Dendrites; Dendritic Spines; Development; Fibroblasts; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genetic Translation; Goals; Growth Cones; Guide RNA; Lasers; Learning; Light; Lighting; Memory; Messenger RNA; Metabolism; Methods; Microscopy; Molecular; Neurons; Oocytes; Play; Post-Transcriptional Regulation; Protein Biosynthesis; Proteins; RNA; RNA Decay; RNA Degradation; RNA Editing; RNA Interference; RNA metabolism; RNA-Binding Proteins; Reagent; Research; Resolution; Role; Technology; Therapeutic; Time; Translations; Work; dimer; knock-down; nervous system disorder; neurodevelopment; neuromechanism; neuronal growth; optogenetics; programs; small molecule; spatiotemporal; temporal measurement; tool; translation assay

Project Summary The overarching goal of this proposal is to develop a strategy to manipulate single mRNAs in live cell and use it to study post-transcriptional gene regulation, which is essential for cells to restrict proteins synthesis at the right time and place. It becomes a leading research focus because of its importance in learning, memory, development and other fundamental biological processes. Despite decades of research, the spatiotemporal dynamics of post-transcriptional regulation is poorly understood. This is due to the lack of experimental tools to control gene expression with high temporal resolution in subcellular compartments, such as leading edges of moving fibroblasts, anterior or posterior poles of developing oocytes, neuronal growth cones or dendritic spines. In this work, we propose to develop strategies to regulate gene expression at the single mRNA level in subcellular compartments. To achieve this goal, we will create optogenetic and chemigenetic tools to control single RNAs in live cells. First, we will use light- induced or chemical-induced dimerizer to tether protein factors onto target RNAs. Because proteins control the RNA metabolism, this allows us to regulate the fate of single mRNAs or modify the coded protein anywhere in a cell by precisely manipulating laser illumination or administering small molecules. Second, we will use chemically-modified light-sensitive guide RNA for the recently developed programmable RNA-targeting CRISPR-Cas13 technology. We plan to develop a light inducible RNA knock-down method and RNA binding proteins to modulate any endogenous RNA. We will use the technology to study the decay mechanism by synchronously induction of rapid RNA degradation. Combined with previously developed single mRNA translation assay in our lab, we will investigate the interplay between translation and decay machineries. By controlled RNA editing, we will visualize the distribution of newly synthesized proteins from single mRNAs in neuronal dendrites. Gene expression regulation plays a central role in all biological problems. The tool that the PI proposed here represents the ultimate spatiotemporal precision that one can manipulate when and where a gene is expressed. It is comparable to RNA interference technology, with added advantages of subcellular resolution, activating, repressive, and mRNA editing capability. The molecular biological reagents and microscopy tools will be applicable to a broad range of scientific community. This will allow us to address questions that cannot be answered before.

项目概要 该提案的总体目标是开发一种操纵单个 mRNA 的策略 在活细胞中并用它来研究转录后基因调控，这对于细胞 在正确的时间和地点限制蛋白质的合成。成为前沿研究热点 因为它在学习、记忆、发育和其他基本生物方面的重要性 流程。尽管经过了数十年的研究，转录后的时空动态 监管了解甚少。这是由于缺乏控制基因的实验工具 亚细胞区室中具有高时间分辨率的表达，例如 移动的成纤维细胞、发育中的卵母细胞的前极或后极、神经元生长锥或 树突棘。在这项工作中，我们建议制定调控基因表达的策略 亚细胞区室中的单一 mRNA 水平。为了实现这一目标，我们将创建 用于控制活细胞中单个 RNA 的光遗传学和化学遗传学工具。首先，我们将使用光- 诱导或化学诱导的二聚体将蛋白质因子束缚到靶RNA上。因为 蛋白质控制 RNA 代谢，这使我们能够调节单个 mRNA 的命运或修改 通过精确操纵激光照明或施用细胞中任何位置的编码蛋白质 小分子。其次，我们将使用化学修饰的光敏向导RNA 最近开发了可编程RNA靶向CRISPR-Cas13技术。我们计划 开发光诱导 RNA 敲低方法和 RNA 结合蛋白来调节任何 内源性RNA。我们将利用该技术同步研究衰减机制 诱导RNA快速降解。与先前开发的单一 mRNA 相结合 在我们实验室的翻译分析中，我们将研究翻译和衰变之间的相互作用 机械。通过受控 RNA 编辑，我们将可视化新合成的分布 来自神经元树突中单个 mRNA 的蛋白质。 基因表达调控在所有生物学问题中发挥着核心作用。该工具 这里提出的 PI 代表了人们可以操纵的最终时空精度 以及基因表达的地方。它可与RNA干扰技术相媲美，并添加了 亚细胞分辨率、激活、抑制和mRNA编辑能力的优势。这 分子生物试剂和显微镜工具将适用于广泛的科学领域 社区。这将使我们能够解决以前无法回答的问题。