Regulatory mechanisms of protein and RNA phase transitions

蛋白质和RNA相变的调控机制

• 批准号: 10319595
• 负责人: Wilton Thomas Snead
• 金额: $ 6.98万
• 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: 2020-01-06 至 2023-01-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319595
• 关键词: Amyotrophic Lateral Sclerosis; Behavior; Binding; Biophysical Process; Cell Cycle; Cell Polarity; Cell membrane; Cell physiology; Cells; Clinical; Complex; Data; Diffuse; Diffusion; Disease; Endoplasmic Reticulum; Growth; Human; In Vitro; Knowledge; Lead; Life; Liquid substance; Membrane; Mission; Molecular; Molecular Chaperones; Neurodegenerative Disorders; Nuclear; Organelles; Outcome; Outcomes Research; Pathologic; Pathology; Pattern; Phase; Phase Transition; Physiological; Physiology; Play; Process; Property; Proteins; Public Health; Quantitative Microscopy; RNA; RNA-Binding Proteins; Reporting; Research; Role; Science; Structure; Surface; Testing; Time; Transcript; United States National Institutes of Health; Work; base; biophysical properties; biophysical tools; cell growth; disability; experimental study; fungus; innovation; live cell imaging; live cell microscopy; novel strategies; overexpression; reconstitution; recruit; screening; two-dimensional

Project Summary. Compartmentalization of molecules into distinct volumes is essential for cellular life. Biomolecular condensates, composed of liquid-like, phase-separated protein and RNA, are important centers of compartmentalization in diverse contexts. Phase-separated structures also play central roles in pathological aggregates that cause disease. Despite the critical importance of phase separation in physiology and pathology, the regulatory mechanisms that govern when and where condensates form in cells are unknown. Our group discovered that biomolecular phase transitions play essential physiological roles in a multinucleate fungus (Zhang et al., Molecular Cell 2015; Langdon et al., Science 2018). Specifically, the RNA-binding protein Whi3 forms distinct, functional droplets with different RNA transcripts that regulate either the nuclear cycle or cell polarity. How do cells control assembly and patterning of different droplets in space and time? Recent reports demonstrated that membrane surfaces provide a powerful platform for promoting protein phase separation (Case et al., Science 2019; Huang et al., Science 2019). However, no studies have examined the role of membranes in controlling RNA-based phase transitions. In my preliminary studies, I found that Whi3 droplets stably associate with endomembranes in live cells. Moreover, I found that membranes promote phase separation of Whi3 in vitro at substantially lower concentration compared to free-diffusing protein in solution. These findings suggest that endomembrane surfaces regulate Whi3/RNA phase separation in space and time. Intriguingly, I also found that Whi3 partitions strongly to interfaces between contacting membranes, suggesting that regions of membrane contact between organelles or with the plasma membrane may regulate Whi3/RNA phase separation. How is Whi3 recruited to endomembranes? My preliminary findings reveal that an endomembrane-associated molecular chaperone component binds to Whi3 and tunes droplet properties. Importantly, molecular chaperones are known to influence droplet behavior, potentially defining the emergent identities and functions of droplets. Taken together, my findings suggest that (i) endomembranes promote and regulate protein/RNA phase transitions and (ii) membrane-associated chaperones control droplet properties to determine overall function. The objective of my proposed work is to elucidate the role of membranes and associated chaperones in regulating and patterning phase separation in space and time. The first specific aim will examine how membrane surfaces and interfaces control assembly of biomolecular condensates. The second specific aim will evaluate how membrane-associated chaperones regulate the emergent properties and functions of biomolecular condensates. This work will create innovative biophysical tools for the study of protein/RNA phase transitions in vitro and in live cells. The overall outcome of this research will be a deeper understanding of the key regulatory platforms that control phase separation. As such, my work will help reveal how cells build and maintain the fundamental compartments that control growth and division.

项目摘要。将分子分隔成不同的体积对于细胞生命是必不可少的。 生物分子凝聚体是生物体内的重要中心，它由液态的、相分离的蛋白质和RNA组成 在不同的背景下进行划分。相分离的结构也在病理学上起着重要作用。 导致疾病的聚集体。尽管相分离在生理学和生物学中至关重要， 在病理学上，控制细胞中何时何地形成冷凝物的调节机制是未知的。 我们的研究小组发现，生物分子的相变在多核细胞中起着重要的生理作用。 真菌（Zhang等人，Molecular Cell 2015; Langdon等人，Science 2018）。具体来说，RNA结合蛋白 Whi 3与不同的RNA转录物形成不同的功能性液滴，这些RNA转录物调节核周期或 细胞极性细胞如何控制不同液滴在空间和时间中的组装和图案化？最近 报道表明，膜表面提供了一个强有力的平台， 分离（Case等人，Science 2019; Huang等人，Science 2019）。然而，没有研究检查 膜在控制RNA相变中的作用。在我的初步研究中，我发现Whi 3 液滴稳定地与活细胞中的内膜结合。此外，我发现膜促进相 与溶液中的自由扩散蛋白相比，在体外以显著更低的浓度分离Whi 3。 这些发现表明内膜表面调节Whi 3/RNA在空间和时间上的相分离。 有趣的是，我还发现Whi 3强烈地分配到接触膜之间的界面，这表明 细胞器之间或与质膜的膜接触区域可以调节Whi 3/RNA 相分离Whi 3如何被招募到内膜中？我的初步调查结果显示 内膜相关的分子伴侣组分与Whi 3结合并调节液滴性质。 重要的是，已知分子伴侣影响液滴行为，潜在地定义了出现的 液滴的特性和功能。总之，我的研究结果表明，（i）内膜促进和 调节蛋白质/RNA相变和（ii）膜相关的伴侣控制液滴特性， 决定整体功能。我的工作目标是阐明膜的作用， 相关的伴侣蛋白在调节和图案化空间和时间的相分离。第一个具体目标 将研究膜表面和界面如何控制生物分子凝聚物的组装。的 第二个具体的目标将评估膜相关伴侣如何调节涌现特性， 生物分子凝聚物的功能。这项工作将创造创新的生物物理工具，用于研究 在体外和活细胞中的蛋白质/RNA相变。这项研究的总体成果将是一个更深层次的 了解控制相分离的关键监管平台。因此，我的工作将有助于揭示 细胞如何建立和维持控制生长和分裂的基本隔间。