Regulatory mechanisms of protein and RNA phase transitions

蛋白质和RNA相变的调控机制

• 批准号: 9910707
• 负责人: Wilton Thomas Snead
• 金额: $ 6.49万
• 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/9910707
• 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。分子分为不同体积对于细胞寿命至关重要。 由液体样，分离蛋白和RNA组成的生物分子冷凝物是重要的中心 在各种情况下的分区化。相分开的结构在病理中也起着核心作用 导致疾病的聚集。尽管相位分离在生理学和 病理学，是在细胞中何时何地形成何时何地的调节机制。 我们的小组发现，生物分子过渡在多核中起着重要的生理作用 真菌（Zhang等人，分子细胞2015； Langdon等，Science 2018）。具体而言，RNA结合蛋白 WHI3形成不同的功能性液滴，具有不同的RNA转录本，可调节核周期或 细胞极性。细胞如何控制时空中不同液滴的组装和模式？最近的 报告表明，膜表面为促进蛋白质相提供了强大的平台 分离（Case等人，Science 2019; Huang等，Science 2019）。但是，尚无研究检查 膜在控制基于RNA的相变的作用。在我的初步研究中，我发现WHI3 液滴稳定地与活细胞中的内膜相关。此外，我发现膜促进阶段 与溶液中的自由放扩向蛋白相比，在体外分离WHI3的体外浓度大大降低。 这些发现表明，内膜表面调节时空中的WHI3/RNA相分离。 有趣的是，我还发现WHI3分区强烈地与接触膜之间的接口，这表明 细胞器或质膜之间的膜接触区域可能调节WHI3/RNA 相分离。 WHI3如何招募到恩膜膜？我的初步发现表明 内膜相关的分子伴侣分量与WHI3结合并调节液滴特性。 重要的是，已知分子伴侣会影响液滴行为，并可能定义出现 液滴的身份和功能。综上所述，我的发现表明（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 确定总体功能。我提议的工作的目的是阐明膜和 相关的伴侣在空间和时间的调节和模式相分离中。第一个特定目标 将检查膜表面和界面如何控制生物分子冷凝物的组装。这 第二个特定目标将评估膜相关的伴侣如何调节新兴特性和 生物分子冷凝物的功能。这项工作将创建创新的生物物理工具来研究 蛋白质/RNA相位在体外和活细胞中。这项研究的总体结果将更深入 了解控制阶段分离的关键调节平台。因此，我的工作将有助于揭示 细胞如何建立和维护控制生长和分裂的基本隔室。