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。将分子分割成不同的体积对细胞生命至关重要。 生物分子凝聚体是由液状、相分离的蛋白质和核糖核酸组成的重要中心 在不同的情况下进行划分。相分离结构在病理学中也起着核心作用 导致疾病的聚集体。尽管相分离在生理学和 病理上，控制细胞中何时何地形成凝聚体的调节机制尚不清楚。 我们的研究小组发现，生物分子相变在多核生物中扮演着重要的生理角色。 真菌(张等人，分子细胞2015年；兰登等人，科学2018年)。具体来说，RNA结合蛋白 WHI3形成不同的功能液滴，具有不同的RNA转录本，调节核周期或 细胞的极性。细胞如何控制不同液滴在空间和时间上的组装和图案？近期 研究表明，膜表面为促进蛋白质相的形成提供了强有力的平台 分离(Case等人，《科学》2019；Huang等人，《科学》2019)。然而，还没有研究检查过 膜在控制基于RNA的相变中的作用。在我的初步研究中，我发现Whi3 液滴稳定地与活细胞的内膜联系在一起。此外，我发现膜促进了相态 与在溶液中自由扩散的蛋白质相比，在体外以低得多的浓度分离Whi3。 这些发现表明，内膜表面调节着Whi3/RNA在空间和时间上的相分离。 有趣的是，我还发现Whi3强烈地分割到接触膜之间的界面上，这表明 细胞器之间或与质膜接触的膜区域可能调节Whi3/RNA 相分离。Whi3是如何被招募到内膜的？我的初步调查结果显示， 膜内相关的分子伴侣组件与WHI3结合并调节液滴特性。 重要的是，分子伴侣已知会影响液滴的行为，潜在地定义了紧急情况 液滴的特性和功能。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的作用和 在调节和构图空间和时间的相分离中的伴随伴侣。第一个具体目标 将研究膜表面和界面如何控制生物分子冷凝物的组装。这个 第二个具体目标将评估膜相关伴侣如何调节突现特性和 生物分子凝聚体的功能。这项工作将创造创新的生物物理工具来研究 蛋白质/RNA在体外和活细胞中的相变。这项研究的总体结果将是更深层次的 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。