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: 10805200
• 负责人: Bin Wu
• 金额: $ 1.2万
• 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/10805200
• 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; 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; Visualization; Work; genetic approach; knock-down; mRNA Translation; nervous system disorder; neurodevelopment; neuromechanism; neuronal growth; optogenetics; posttranscriptional; 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.

项目摘要 该提案的总体目标是制定操纵单个mRNA的策略 在活细胞中，并使用它来研究转录后基因调控，这对于细胞至关重要 限制蛋白质在正确的时间和地点合成。它成为领先的研究重点 由于它在学习，记忆，发展和其他基本生物学方面的重要性 过程。尽管进行了数十年的研究，但文章后的时空动力学 监管知之甚少。这是由于缺乏控制基因的实验工具 在亚细胞室中具有高时间分辨率的表达，例如 移动成纤维细胞，发育卵母细胞的前或后杆，神经元生长锥或 树突状刺。在这项工作中，我们建议制定策略以调节基因表达 亚细胞室中的单个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的光遗传学和化学工具。首先，我们将使用光 诱导或化学诱导的二聚体对链球蛋白因子的二聚体到靶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地方的编码蛋白 小分子。其次，我们将使用化学改性的光敏感指南RNA进行 最近开发的可编程RNA靶向CRISPR-CAS13技术。我们计划 开发一种可诱导的RNA敲低方法和RNA结合蛋白来调节任何 内源性RNA。我们将使用该技术同步研究衰减机制 诱导快速RNA降解。与先前开发的单个mRNA结合 在我们实验室中的翻译测定法，我们将研究翻译与衰减之间的相互作用 机械。通过受控的RNA编辑，我们将可视化新合成的分布 来自神经元树突中的单个mRNA的蛋白质。 基因表达调节在所有生物学问题中均起着核心作用。工具是 此处提出的PI代表了最终的时空精度 以及表达基因的地方。它可与RNA干扰技术相媲美，并增加 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范围 社区。这将使我们能够解决以前无法回答的问题。