RNA structures critical for dictating the contents, behavior, and function of droplets formed from liquid-liquid phase separation

RNA 结构对于决定液-液相分离形成的液滴的内容、行为和功能至关重要

• 批准号: 10327720
• 负责人: Christine Anne Roden
• 金额: $ 2.53万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10327720
• 关键词: Address; Automobile Driving; Behavior; Binding Proteins; Binding Sites; Biochemical; Biochemistry; Biological; Biophysics; Cell Cycle; Cell Polarity; Cells; Cellular biology; Chemicals; Code; Complex; Diffuse; Diffusion; Gel; Image; In Vitro; Kinetics; Link; Liquid substance; Malignant Neoplasms; Maps; Measurement; Measures; Membrane; Messenger RNA; Modeling; Molds; Mutate; Mutation; Neurodegenerative Disorders; Oils; Pathologic; Pathology; Pathway interactions; Phase; Phenotype; Physical condensation; Play; Porosity; Process; Property; Proteins; RNA; RNA Probes; RNA Sequences; RNA-Binding Proteins; RNA-Protein Interaction; Research; Research Design; Role; Solid; Structure; Surface; Surface Tension; Testing; Time; Translations; Viscosity; Water; Work; base; biophysical properties; crosslink; experience; fungus; human disease; in vitro Assay; in vivo; in vivo imaging; innovation; insight; molecular scale; neuromuscular; physical state; protein complex; scaffold; viscoelasticity

Project Summary: Background: Analogous to the separation of oil from water, a process called liquid-liquid phase separation (LLPS) partitions cellular contents. LLPS is dictated by intrinsic biophysical/biochemical properties of component molecules. Many phase-separated droplets form from RNA-binding proteins in complex with target RNAs. These droplets play roles in neuromuscular/neurodegenerative diseases, and cancer. In pathological contexts, the dynamics of these molecules are disrupted. It is now appreciated that RNA sequence/structure, can dictate assembly and physical state of droplets. This provides a new role for RNA sequence, which is encoding mesoscale biophysical properties of cellular bodies. There is a link between RNA sequence and droplet behaviors but the details are still a mystery. Objective/Hypothesis: To better understand how RNA structure regulates droplets, I will measure and manipulate RNA structure temporally and spatially. I will then use these measurements to gain mechanistic insights into how droplet maturation occurs and how droplets can facilitate or inhibit RNA translation. We hypothesize that sequence directed RNA structures dictates the contents, behavior, and function of condensed droplets and this in turn influences cell biology. Specific Aims: (1) To determine the role of RNA/RNA interactions in droplets. (2) To map RNA structures and RNA protein interaction changes that influence maturation of droplets. (3) To determine how RNA structures at the core and shell of droplets differ and impact functions. Study Design: I will use the model protein called Whi3 from genetically tractable fungi where RNA-structure is known to be critical to droplet formation with target RNAs. I will measure RNA/RNA interaction, RNA structure, and protein binding during the course of droplet maturation. I will then disrupt the structures and interactions identified by mutating RNA sequences, to assess the impact of these features on droplet formation, maturation, and biophysical properties (size, viscosity, diffusivity, etc.). I will next determine how the biophysical properties of the shell influence cell biological phenotypes by measuring how droplets regulate translation efficiency. Innovation: Thus far, a detailed examination of how RNA structure relates to the material and functional state of droplets has not been undertaken. The kinetics of RNA structure and protein binding are unknown, and RNA/RNA interactions in droplets have not been measured. Ultimately, this work will provide insight into the role of RNA structures in driving maturation and physical state in droplets formed from LLPS, and models developed herein will be generalizable to all RNA protein complexes. Therefore, this work contributes to a better understanding of how to manipulate RNA structures in the context of human diseases. !

项目概要： 背景：类似于油与水的分离，一种称为液-液相分离的过程 （LLPS）分配细胞内容物。LLPS由LLPS的固有生物物理/生物化学特性决定。 组成分子。许多相分离的液滴是由RNA结合蛋白与靶蛋白复合形成的 RNA。这些液滴在神经肌肉/神经退行性疾病和癌症中发挥作用。病理 在这种情况下，这些分子的动力学被破坏。现在认识到RNA序列/结构， 可以指示液滴的组装和物理状态。这为RNA序列提供了一个新的角色， 编码细胞体的中尺度生物物理性质。RNA序列与 液滴行为，但细节仍然是个谜。 目的/假设：为了更好地了解RNA结构如何调节液滴，我将测量和 在时间和空间上操纵RNA结构。然后我将使用这些测量来获得 了解液滴成熟如何发生以及液滴如何促进或抑制RNA翻译。我们 假设序列指导的RNA结构决定了其内容、行为和功能， 凝结的液滴，这反过来又影响细胞生物学。 具体目的：（1）确定RNA/RNA相互作用在液滴中的作用。(2)绘制RNA结构图， RNA蛋白质相互作用的变化影响液滴的成熟。(3)为了确定RNA如何在 液滴的核和壳不同并影响功能。 研究设计：我将使用一种名为Whi 3的模型蛋白，它来自基因上易于处理的真菌， 已知对于靶RNA的液滴形成至关重要。我将测量RNA/RNA相互作用，RNA结构， 以及液滴成熟过程中的蛋白质结合。然后我会破坏这些结构和相互作用 通过突变RNA序列鉴定，以评估这些特征对液滴形成的影响， 成熟度和生物物理性质（尺寸、粘度、扩散率等）。接下来我将决定 壳的生物物理性质通过测量液滴如何影响细胞生物学表型 规范翻译效率。 创新：到目前为止，对RNA结构如何与物质和功能状态相关的详细研究 水滴的问题还没有得到解决。RNA结构和蛋白质结合的动力学是未知的， 尚未测量液滴中的RNA/RNA相互作用。最终，这项工作将提供深入了解 RNA结构在驱动由LLPS形成的液滴中的成熟和物理状态中的作用，以及模型 本文开发的方法将可推广到所有RNA蛋白质复合物。因此，这项工作有助于 更好地理解如何在人类疾病的背景下操纵RNA结构。 !