Investigating the adaptive role of heat-induced biomolecular condensates in translational regulation

研究热诱导生物分子缩合物在翻译调节中的适应性作用

• 批准号: 10475632
• 负责人: Caitlin Wong
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10475632
• 关键词: 5' Untranslated Regions; Affect; Behavior; Binding; Bioinformatics; Body Temperature; Cell Survival; Cells; Cellular Stress Response; Chicago; Computational Biology; Environment; Exposure to; Fever; Flow Cytometry; Fluorescence Anisotropy; Global Change; Growth; Heat Stress Disorders; Heat shock proteins; Heat-Shock Response; High temperature of physical object; Immune; Impairment; In Vitro; Luciferases; Measures; Mediating; Methods; Modeling; Molecular Chaperones; Molecular Probes; Nature; Orthologous Gene; Pathogenicity; Pathway interactions; Physical condensation; Physiological; Play; Poly(A)-Binding Proteins; Production; Proteins; RNA-Binding Proteins; Recovery; Regulation; Repression; Research; Research Personnel; Resources; Role; Saccharomyces cerevisiae; Saccharomycetales; Shapes; Signal Transduction; Stress; System; Temperature; Testing; Transcript; Translating; Translation Initiation; Translational Regulation; Translational Repression; Translations; Universities; Work; Yeasts; attenuation; base; biological adaptation to stress; career; cell growth; design; environmental change; fitness; in vivo; misfolded protein; protein aggregation; protein folding; protein function; protein misfolding; response; stoichiometry; translation assay

Project Summary/Abstract When cells encounter heat, they undergo a number of archetypal intracellular changes: global attenuation of translation, synthesis of molecular chaperones, and the formation of intracellular protein aggregates. These aggregates were long thought to be the result of misfolded proteins, however, recent work has suggested that these assemblies may be the adaptive result of biomolecular condensation. It has remained unclear how biomolecular condensation functions to help cells survive heat stress. Here, I propose to investigate a connection between heat-induced condensation and translation of molecular chaperones. Previous work has demonstrated that poly(A)-binding protein (Pab1) condenses during heat shock in yeast and disrupting Pab1 condensation impairs cellular growth during stress, indicating that stress-triggered condensation of Pab1 is a part of the adaptive stress response. This finding motivated me to investigate why Pab1 is important for cell growth during stress. Pab1 acts as a translational repressor by binding transcripts with A-rich 5’ Untranslated Regions (5’UTRs), including its own transcript. Interestingly, the transcripts of molecular chaperones produced during stress have A-rich 5’UTRs, and these molecular chaperones go on to re-solubilize Pab1. My preliminary work shows that soluble Pab1 can repress translation of endogenous transcripts with A-rich 5’UTRs, while Pab1 condensation inhibits this effect. This suggests an autoregulatory mechanism through which heat-triggered condensation of Pab1 facilitates high level translation of stress- induced chaperones, whose capacity to re-solubilize Pab1 leads to repressed translation after sufficient chaperones have been produced. To test this model, I will probe the molecular basis of Pab1 translational repression using in vitro translation assays and fluorescence anisotropy. I will also carry out a bioinformatic analysis of A-rich 5’UTRs to identify sequence features that are conserved and test whether they contribute to Pab1 repression. Next, I will design yeast strains to perturb Pab1 condensation in vivo to test how this change affects the production of molecular chaperones. Finally, Pab1 condensation does not completely explain how heat shock transcripts are specifically translated in the midst of global translational attenuation, so I will investigate how A-rich 5’UTRs can promote selective translation, using similar in vitro and in vivo methods. My proposed model connects direct environmental sensing by condensation to the adaptive cellular stress response and helps shape our understanding of how cells respond to heat in other contexts, such as immune cells in fever. This research will be done at the University of Chicago with Dr. D. Allan Drummond and will build my skillset in in vitro, in vivo, and computational biology, preparing me for a career as an independent investigator.

项目摘要/摘要 当细胞遇到热时，它们会发生多种原型细胞内变化：全球 翻译的衰减，分子伴侣的合成和细胞内蛋白的形成 聚合。这些聚集体长期以来被认为是错误折叠蛋白的结果，但是最近的工作 这些组件可能是生物分子凝结的适应性结果。它有 尚不清楚生物分子凝结功能如何帮助细胞存活热应激。在这里，我建议 研究热诱导的凝结与分子链烷的翻译之间的联系。 先前的工作表明，聚（A）结合蛋白（PAB1）在热休克中的冷凝物 酵母和破坏PAB1凝结会损害压力期间的细胞生长，表明应力触发 PAB1的凝结是自适应应激反应的一部分。这一发现促使我调查了为什么 PAB1对于压力期间的细胞生长很重要。 PAB1通过结合转录本作为翻译复制品 具有富含5'非翻译区域（5'UTRS）的A，包括其自己的成绩单。有趣的是，成绩单 在压力期间产生的分子伴侣具有富含A的5英尺，这些分子伴侣继续进行 重新启动PAB1。我的初步工作表明可溶性PAB1可以抑制内源性的翻译 具有富含A 5'UTR的转录本，而PAB1冷凝抑制了这种效果。这表明自动调节 热触发PAB1最爱的凝结的机制高水平压力 - 应力 - 诱导的伴侣，其重新溶解PAB1的能力在充分后导致压抑翻译 伴侣已经产生。 为了测试该模型，我将使用体外探测PAB1平移表达的分子基础 翻译测定和荧光各向异性。我还将对A-RICH 5'UTRS进行生物信息学分析 我将确定保守的序列特征，并测试它们是否有助于PAB1表达。接下来，我会的 设计酵母菌菌株在体内扰动PAB1凝结以测试这种变化如何影响 分子伴侣。最后，PAB1冷凝不能完全解释热激电笔录 在全球翻译衰减中专门翻译 可以使用类似的体外和体内方法来促进选择性翻译。我建议的模型连接 通过对自适应细胞应力反应的凝结来直接环境敏感性，并有助于塑造我们的 了解细胞在其他情况下如何反应热量，例如发烧中的免疫细胞。 这项研究将在芝加哥大学与D. Allan Drummond博士一起完成，并将建立我的 体外，体内和计算生物学的技能，为我做好了独立研究者的准备。