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，从其在细胞核中的合成，通过其在细胞内稳态中的作用，到在细胞中的降解， 溶酶体是一种受RNA结构内在控制的调节过程。我们知道这一点，部分，从 过多的论文详细描述了RNA代谢中的致病缺陷。“结构化”模式 RNA代谢的景观”，使实验调节的过程，需要什么知识， 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它们的局部相互作用。 互动伙伴到目前为止，大多数关于这些RNA调控过程的研究， 例如RNA与RNA结合蛋白的相互作用，使用来自 细胞群。这种细胞系综信息是有用的，然而，这些过程的细胞选择性和 RNA结构变化的动力学在这种结构景观中是未知的。的独特性 单个细胞和亚细胞环境的研究需要在单个细胞水平上进行。 细胞目前还没有实验方法允许跨跨膜RNA分子的结构分析。 单个细胞的自然微环境中的RNA代谢景观。我们建议绘制 通过开发一套敏感的，高灵敏度的， 分辨率的分子方法，产生定量的3-D地图的所有RNA相关的结构， 单个细胞和这些细胞的亚区域。这种方法被称为体内结构分析映射，或VISTA 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， 激活的原位RNA放大器，其产物是RNA结构敏感和信息的。结构性 对单个细胞中所有RNA和RNA相关细胞器的分析将允许确定 细胞内RNA代谢的全貌。拟议研究的目标是创建和理解 单细胞RNA代谢的拓扑图这将使用新开发的VISTA来完成 映射.细胞类型特异性VISTA图，从神经元在其自然微环境中产生，将 为操纵正常细胞生物学以及疾病相关细胞生物学提供了新的见解和机会， RNA病变！ !