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: 10686023
• 负责人: Caitlin Wong
• 金额: $ 4.77万
• 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/10686023
• 关键词: 5' Untranslated Regions; Affect; Behavior; Binding; Bioinformatics; Body Temperature; Cell Communication; Cell Survival; Cells; Cellular Stress; Chicago; Computational Biology; Environment; Exposure to; Fever; Flow Cytometry; Fluorescence Anisotropy; Growth; Heat Stress Disorders; 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 Protein I; Production; Proteins; RNA-Binding Proteins; Recovery; 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; biological adaptation to stress; career; cell growth; design; environmental change; fitness; in vivo; misfolded protein; protein aggregation; protein folding; protein function; protein misfolding; response; skills; stoichiometry; thermophilic organism; 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.

项目概要/摘要 当细胞遇到热量时，它们会经历许多典型的细胞内变化： 翻译减弱、分子伴侣合成和细胞内蛋白质形成 长期以来，这些聚集体被认为是蛋白质错误折叠的结果，然而，最近的研究发现。 表明这些组装可能是生物分子凝聚的适应性结果。 目前尚不清楚生物分子凝聚如何帮助细胞抵御热应激。 研究热诱导凝结与分子伴侣翻译之间的联系。 先前的研究表明，多聚腺苷酸结合蛋白 (Pab1) 在热激过程中会凝结 酵母和破坏 Pab1 凝结会损害应激期间的细胞生长，表明应激触发 Pab1 的凝结是适应性应激反应的一部分，这一发现促使我研究原因。 Pab1 对于应激期间的细胞生长很重要。Pab1 通过结合转录本充当翻译抑制因子。 具有富含 A 的 5' 非翻译区域 (5'UTR)，包括其自己的转录本。 应激期间产生的分子伴侣具有富含 A 的 5’UTR，这些分子伴侣会继续 重新溶解 Pab1。我的初步工作表明可溶性 Pab1 可以抑制内源性翻译。 转录物具有富含 A 的 5’UTR，而 Pab1 缩合会抑制这种效应。 Pab1 的热触发凝结促进应力高水平转换的机制 诱导伴侣，其重新溶解 Pab1 的能力导致在足够的时间后抑制翻译 伴侣已经产生。 为了测试这个模型，我将利用体外实验探究 Pab1 翻译抑制的分子基础 我还将对富含 A 的 5'UTR 进行生物信息学分析。 接下来，我将识别保守的序列特征并测试它们是否有助于 Pab1 抑制。 设计酵母菌株来扰乱体内 Pab1 凝结，以测试这种变化如何影响生产 最后，Pab1 缩合并不能完全解释热激转录本的原理。 特别是在全球翻译衰减期间翻译，所以我将研究富含 A 的 5'UTRs 如何 使用类似的体外和体内方法可以促进选择性翻译。 通过凝结到适应性细胞应激反应来直接环境感知，并帮助塑造我们的 了解细胞在其他情况下如何对热做出反应，例如发烧时的免疫细胞。 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、体内和计算生物学方面的技能，为我作为一名独立研究者的职业生涯做好了准备。