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

项目摘要 这项提议的首要目标是开发一种操纵单个mRNA的策略。 并用它来研究转录后基因调控，这对细胞 在正确的时间和地点限制蛋白质的合成。它成为一个领先的研究焦点。 因为它在学习、记忆、发育和其他基础生物学中的重要性 流程。尽管经过了几十年的研究，转录后基因的时空动力学 人们对监管知之甚少。这是由于缺乏控制基因的实验工具。 在亚细胞间隔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、神经元生长锥体或 树枝状刺。在这项工作中，我们建议开发策略来调控基因表达 亚细胞内单个基因的表达水平。为了实现这一目标，我们将创造 控制活细胞中单个RNA的光遗传和化学遗传工具。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拴在靶RNA上。因为 蛋白质控制着RNA的代谢，这使得我们可以调节单个mRNAs的命运或修饰 通过精确操纵激光照射或给药，细胞中任何位置的编码蛋白质 小分子。第二，我们将使用化学修饰的光敏引导RNA来 最近开发的可编程RNA靶向CRISPR-Cas13技术。我们计划 建立一种光诱导的RNA敲除方法和RNA结合蛋白来调节任何 内源RNA。我们将利用这项技术来同步研究衰变机理。 诱导RNA的快速降解。与先前开发的单个mRNA结合 在我们的实验室中，我们将研究翻译和衰变之间的相互作用 机械制造。通过受控的RNA编辑，我们将可视化新合成的 神经元树突中单个mRNAs的蛋白质。 基因表达调控在所有生物学问题中都起着核心作用。该工具是 这里提出的PI表示当一个人在以下情况下可以操纵的最终时空精度 基因在哪里表达。它可与RNA干扰技术相媲美，添加了 具有亚细胞分辨、激活、抑制和信使核糖核酸编辑能力的优势。这个 分子生物试剂和显微工具将广泛应用于科学研究领域 社区。这将使我们能够解决以前无法回答的问题。

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.