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的代谢，从它在细胞核中的合成，通过它在细胞内稳态中的作用，到在