The Secret Lives of RNA: The In Vivo 3D-Structural Logic of Single Neuron RNA Metabolism

RNA 的秘密生活：单神经元 RNA 代谢的体内 3D 结构逻辑

• 批准号: 10453564
• 负责人: JAMES H EBERWINE
• 金额: $ 113.75万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10453564
• 关键词: 3-Dimensional; Amplifiers; Cell Nucleus; Cells; Cellular biology; Disease; Environment; Goals; Homeostasis; In Situ; Individual; Knowledge; Light; Location; Logic; Lysosomes; Maps; Metabolic; Molecular; Neurons; Normal Cell; Organelles; Paper; Process; Proteins; RNA; RNA analysis; RNA metabolism; RNA-Binding Proteins; Regulation; Resolution; Role; Structural Models; Structure; cell type; in vivo; insight; novel; three dimensional structure

RNA metabolism, from its synthesis in the nucleus, through its role in cellular homeostasis, to degradation in the lysosome, is a regulated process that is inherently controlled by RNA structure. We know this, in part, from the plethora of papers detailing disease-causing deficiencies in RNA metabolism. Modeling of the “structural landscape of RNA metabolism” to enable experimental regulation of the process, requires knowledge of what RNAs are expressed, their 3D-structures, their subcellular location and how they interact with their local interacting partners. To date, most efforts that generate information about these RNA regulatory processes, such as RNA interactions with RNA binding proteins, use purified fractions of cellular homogenates from groups of cells. Such cell-ensemble information is useful, however, the cell-selectivity of these processes and the dynamics of RNA structural changes across this structural landscape is unknown. The uniqueness of individual cells and subcellular environments requires that such studies be performed at the level of single cells. There is currently no experimental approach that allows for structural analysis of RNA molecules across the RNA metabolic landscape within the natural microenvironment of individual cells. We propose to map the structural landscape of RNA metabolism in single cells, in vivo, by developing a suite of sensitive, high- resolution molecular approaches that yields a quantitative 3-D map of all RNA-associated structures within single cells and subregions of these cells. This approach, called In Vivo Structural Analysis mapping, or VISTA mapping, uses a combination of protein, RNA and organelle markers to direct subcellular function of light- activated in situ RNA amplifiers, the product of which is RNA-structure sensitive and informative. The structural analysis of all RNAs and RNA-associated organelles in a single cell will permit a determination the overall logic of the RNA metabolic landscape within a cell. The goal of the proposed studies is to create and understand the “Topological Map of Single Cell RNA Metabolism”. This will be accomplished using newly developed VISTA mapping. Cell-type specific VISTA maps, generated from neurons in their natural microenvironment, will provide novel insights into and opportunities for manipulating normal cell biology as well as disease-associated RNAopathies.! !

RNA代谢，从它在细胞核中的合成，到它在细胞内稳态中的作用，到它在体内的降解 溶酶体是一种受RNA结构控制的调节过程。我们知道这一点的部分原因是 过多的论文详细描述了核糖核酸新陈代谢的致病缺陷。“结构”的模型化 RNA新陈代谢的景观“要实现对该过程的实验性调节，需要了解什么 RNA的表达，它们的3D结构，它们的亚细胞位置以及它们如何与局部的 互动伙伴。到目前为止，大多数产生关于这些RNA调控过程的信息的努力， 例如RNA与RNA结合蛋白相互作用，使用来自 一组细胞。然而，这种细胞集合信息是有用的，然而，这些过程和 RNA结构变化在整个结构图景中的动态尚不清楚。的独特性 单个细胞和亚细胞环境要求在单个细胞水平上进行这种研究 细胞。目前还没有一种实验方法可以对所有的RNA分子进行结构分析 单个细胞自然微环境中的RNA代谢图景。我们建议绘制 体内单细胞RNA代谢的结构图景，通过开发一套敏感的、高密度的 分辨分子方法，产生所有RNA相关结构的定量3-D图谱 单个细胞和这些细胞的亚区。这种方法被称为VIVO结构分析映射，或Vista 作图，使用蛋白质、RNA和细胞器标记的组合来指导光的亚细胞功能- 激活的原位RNA扩增，其产物具有RNA结构敏感性和信息量。结构性的 对单个细胞中的所有RNA和与RNA相关的细胞器的分析将允许确定总体逻辑 细胞内RNA新陈代谢的图景。拟议研究的目标是创建和理解 《单细胞RNA代谢拓扑图》。这将使用新开发的Vista来完成 映射。由神经元在其自然微环境中生成的细胞类型特定的Vista图将 提供对正常细胞生物学以及与疾病相关的操作的新见解和机会 RNAopathies！ 好了！