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

项目概要/摘要 当细胞遇到热时，它们会经历许多典型的细胞内变化： 翻译的减弱、分子伴侣的合成和细胞内蛋白质的形成 集料.这些聚集体长期以来被认为是错误折叠蛋白质的结果，然而，最近的工作 已经表明，这些组件可能是生物分子凝聚的适应性结果。它有 目前尚不清楚生物分子凝聚如何帮助细胞在热应激下存活。在此，我提议 研究热诱导缩合和分子伴侣翻译之间的联系。 以前的工作已经证明，聚（A）结合蛋白（Pab 1）在热休克过程中浓缩， 酵母和破坏Pab 1缩合损害细胞生长在压力，表明压力触发 Pab 1的浓缩是适应性应激反应的一部分。这一发现促使我调查为什么 Pab 1在应激期间对细胞生长很重要。Pab 1通过结合转录物作为翻译抑制因子 具有富含A的5'非翻译区（5' UTR），包括其自身的转录物。有趣的是， 在应激期间产生的分子伴侣具有富含A的5 'UTR，并且这些分子伴侣继续 再溶解Pab 1。我的初步工作表明，可溶性Pab 1可以抑制内源性 转录本与富含A的5 'UTR，而Pab 1缩合抑制这种作用。这表明， Pab 1的热触发冷凝促进高水平应力转化的机制- 诱导的分子伴侣，其重新溶解Pab 1的能力导致足够的翻译后被抑制 已经产生了伴侣。 为了验证这个模型，我将使用体外细胞培养技术来探索Pab 1翻译抑制的分子基础。 翻译测定和荧光各向异性。我还将对富含A的5 'UTR进行生物信息学分析， 识别保守的序列特征并测试它们是否有助于Pab 1抑制。接下来我会 设计酵母菌株来干扰体内Pab 1的凝聚，以测试这种变化如何影响 分子伴侣最后，Pab 1凝聚并不能完全解释热休克转录物是如何被激活的。 特别是在全球翻译衰减的中间翻译，所以我将研究富含A的5 'UTR 可以促进选择性翻译，使用类似的体外和体内方法。我提出的模型 通过冷凝直接环境感知适应性细胞应激反应，并帮助塑造我们的 了解细胞如何在其他情况下对热作出反应，例如发烧时的免疫细胞。 这项研究将在芝加哥大学与D.艾伦·德拉蒙德将建立我的 在体外，体内和计算生物学方面的技能，为我作为一名独立调查员的职业生涯做好准备。

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.