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: 10018804
• 负责人: JAMES H EBERWINE
• 金额: $ 113.43万
• 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/10018804
• 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，从其在细胞核中的合成，从其在细胞稳态中的作用到降解 溶酶体是一个固有地由RNA结构控制的调节过程。我们部分地知道这一点 大量论文详细介绍了RNA代谢中引起疾病​​的缺陷。 “结构性建模 RNA代谢的景观”以实现对过程的实验调节，需要了解什么 表示RNA，其3D结构，亚细胞位置以及它们与当地的互动方式 互动合作伙伴。迄今为止，生成有关这些RNA调节过程的信息的大多数努力， 例如与RNA结合蛋白的RNA相互作用，使用来自细胞匀浆的纯化部分 细胞组。但是，这种细胞收集信息很有用，但是，这些过程的细胞选择性和 该结构景观中RNA结构变化的动力学尚不清楚。独特性 单个细胞和亚细胞环境要求在单个水平上进行此类研究 细胞。目前尚无实验方法可以对RNA分子进行结构分析 单个细胞自然微环境中的RNA代谢景观。我们建议映射 通过开发一套敏感，高 - 分辨率分子方法，该方法得出所有与RNA相关结构的定量3-D图 这些细胞的单细胞和子区域。这种方法称为体内结构分析映射或远景 映射，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 被激活的原位RNA放大器，其乘积是RNA结构敏感和信息性的。结构 分析单个单元中所有RNA和RNA相关细胞器将允许确定整体逻辑 细胞内的RNA代谢景观。拟议研究的目的是创建和理解 “单细胞RNA代谢的拓扑图”。这将使用新开发的Vista来完成 映射。细胞型特异性远景图，由自然微环境中的神经元产生，将 提供有关操纵正常细胞生物学以及疾病相关的新见解和机会 rnaopathies。 呢