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

项目总结： 背景：类似于油和水的分离，这一过程称为液-液相分离 (LLP)对细胞内容进行分区。LLP由固有的生物物理/生化特性决定 组成分子。RNA结合蛋白与靶形成络合物，形成许多相分离的液滴 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。在病理上 背景下，这些分子的动力学被打乱了。现在可以理解，RNA序列/结构， 可以决定液滴的组装和物理状态。这为RNA序列提供了一个新的角色，即 编码细胞体的中尺度生物物理属性。在RNA序列和 水滴的行为，但细节仍然是一个谜。 目标/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机械性 深入了解液滴成熟的方式，以及液滴如何促进或抑制RNA翻译。我们 假设序列导向的RNA结构决定了内容、行为和功能 凝聚的液滴，进而影响细胞生物学。 具体目标：(1)确定RNA/RNA相互作用在液滴中的作用。(2)绘制RNA结构图和 RNA与蛋白质的相互作用会影响液滴的成熟。(3)确定RNA的结构如何 液滴的核和壳不同，具有撞击功能。 研究设计：我将使用来自可遗传真菌的名为Whi3的模型蛋白质，其中RNA结构是 已知对与靶RNA形成液滴至关重要。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。然后我将扰乱结构和相互作用 通过突变的RNA序列识别，为了评估这些特征对液滴形成的影响， 成熟度和生物物理性质(大小、粘度、扩散性等)。接下来，我将确定 壳层的生物物理特性通过测量液滴如何影响细胞的生物学表型 规范翻译效率。 创新：到目前为止，对RNA结构与材料和功能状态的关系进行了详细的研究 还没有对水滴进行研究。RNA结构和蛋白质结合的动力学尚不清楚，以及 还没有测量到液滴中的RNA/RNA相互作用。最终，这项工作将提供对 核糖核酸结构在驱动LLP液滴成熟和物理状态中的作用，以及模型 这里开发的将可推广到所有的RNA蛋白质复合体。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。 好了！

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. !