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 当细胞遇到热量时，它们会经历一些典型的细胞内变化：全球 翻译的减弱、分子伴侣的合成和胞内蛋白的形成 集合体。这些聚集体长期以来被认为是蛋白质错误折叠的结果，然而，最近的研究 提出这些组装可能是生物分子缩合的适应性结果。它有 目前尚不清楚生物分子缩合是如何帮助细胞在热应激中生存的。在此，我建议 研究分子伴侣的热诱导缩合和翻译之间的联系。 以前的工作已经证明，聚(A)结合蛋白(Pab1)在热休克过程中发生缩合。 酵母菌和破坏Pab1凝聚会在应激期间损害细胞生长，这表明应激触发 Pab1的缩合是适应性应激反应的一部分。这一发现促使我调查为什么 在应激过程中，Pab1对细胞生长起着重要的作用。Pab1通过结合转录本发挥翻译抑制因子的作用 带有富含A的5‘非翻译区(5’UTRs)，包括它自己的转录本。有趣的是， 在应激过程中产生的分子伴侣具有富含A的5‘UTRs，这些分子伴侣继续 重新增溶Pab1。我的初步工作表明，可溶性Pab1可以抑制内源性的翻译 转录与富含A的5‘UTRs，而Pab1缩合抑制这一作用。这表明了一种自动调节 Pab1的热引发缩合促进应力的高水平转化的机制- 诱导伴侣蛋白，其重新溶解Pab1的能力导致在足够的时间后抑制翻译 人们已经培育出了伴侣。 为了验证这一模型，我将使用体外实验来探索Pab1翻译抑制的分子基础 翻译分析和荧光各向异性。我还将对富含A的5‘UTRs进行生物信息学分析，以 确定保守的序列特征，并测试它们是否对Pab1抑制起作用。接下来，我会 设计酵母菌株扰乱体内Pab1缩合，以测试这种变化如何影响Pab1的生产 分子伴侣。最后，Pab1凝聚不能完全解释热休克转录本是如何 特别是在全球翻译减弱的情况下，所以我将研究富含A的5‘UTRs是如何 可以促进选择性翻译，使用类似的体外和体内方法。我建议的模型连接 通过凝聚到适应性细胞应激反应的直接环境感知，并帮助塑造我们的 了解细胞在其他环境中对热的反应，例如发烧时的免疫细胞。 这项研究将在芝加哥大学与D·艾伦·德拉蒙德博士一起完成，并将建立我的 在体外、体内和计算生物学方面的技能，为我作为一名独立研究员的职业生涯做好了准备。