Heterogenous cytoplasmic RNA-protein aggregates enhance cellular survival under stress

项目摘要/摘要 基因表达程序的可塑性使细胞能够对压力做出反应和适应。在适应的同时 涉及转录和转录后变化，翻译调控在 需要快速反应的情况。细胞对低氧、饥饿、氧化应激、 热休克或病毒感染，通过关闭翻译和将mRNA隔离成应激颗粒来实现； 动态的、非膜结合的、相分离的细胞质细胞器。细胞形成压力的能力 颗粒对于癌细胞在化疗后存活的能力尤为重要。 然而，压力颗粒并不是单一的。G3BP是应激颗粒的核心核蛋白，至少有 三个已知的变异体--G3BP1、G3BP2a和G3BP2b。G3BP变异体可以形成同源或异源多聚体 应激颗粒的背景，也被证明具有不同的组织特异性。然而， 颗粒成核蛋白之间这种变异的功能后果还没有研究过。这个 由于缺乏这方面的知识，我们无法理解细胞对各种侮辱的反应 不完整。G3BP变异体也有不同的颗粒形成能力，表明不止一个 细胞应力颗粒间的异质性维度。此外，我们的数据表明，G3BP变种 在功能上无法相互补偿，表明应激反应存在差异。因此， 我们假设应力颗粒成分的异质性对于细胞能够 对不同的压力有不同的反应和适应，不同成分的颗粒对细胞的作用不同 信使核糖核酸对压力的反应决定命运。我们将通过以下具体目标来解决这一假设：(1)确定 应激颗粒异质性在细胞抵抗外源压力中的作用：我们将研究能够 只产生有限数量的或只有一个颗粒亚型(S)给广泛的应激源和监控 用灵敏的流式细胞术进行绝对和相对适合度分析以及(2)阐明其作用机制(S) 应激源反应中依赖颗粒的差异适应度：结合使用分子生物学和 高通量测序技术，我们将测量转录本对有核颗粒的定位 特定的G3BP变异体。最近开发的一种光遗传工具将使我们能够在 无应激，从而独立地测量颗粒对细胞质翻译的影响。我的目标 是成为一名首席研究员，从事一项专注于破译的独立研究计划 细胞对压力的反应。加州大学旧金山分校是追求跨学科方法的理想环境 回答这些基本的生物学问题。拟议工作的完成将扩大我们的 了解细胞对应激反应的潜在机制，为应激反应提供信息 病理学和治疗方案。

Project Summary/Abstract Plasticity of gene expression programs allows cells to respond and adapt to stress. While adaptation involves both transcriptional and post-transcriptional changes, translational regulation is particularly crucial in situations that demand a rapid response. Cells respond to stress such as hypoxia, starvation, oxidative stress, heat shock or viral infections by shutting down translation and sequestering mRNA into stress granules; dynamic, non-membrane bound, phase-separated cytoplasmic organelles. The ability of cells to form stress granules is particularly important in the ability of cancer cells to survive chemotherapeutic treatments. However, stress granules are not monotypic. G3BP, a core nucleator protein of stress granules, has at least three known variants - G3BP1, G3BP2a and G3BP2b. G3BP variants can form homo or hetero-multimers in the context of stress granules, and have also been shown to have different tissue specificities. However, the functional consequences of this variation amongst granule nucleating proteins has not been studied. The absence of this knowledge renders our understanding about the cellular response to various insults incomplete. G3BP variants also have differential granule-forming capacities, indicating more than one dimension of heterogeneity amongst cellular stress granules. Further, our data suggests that G3BP variants are unable to compensate for each other functionally, indicating divergence in the stress response. Therefore, we hypothesize that the heterogeneity in stress granule composition is important for the cell to be able to respond and adapt to diverse stresses, and that granules of different compositions have different effects on the mRNA fates in response to stress. We will address this hypothesis via the following specific aims: (1) Identify role of stress granule heterogeneity in cellular resistance to exogenous stresses: We will subject cells that can produce only a limited number of or only a single granule subtype(s) to a wide panel of stressors and monitor absolute and relative fitness using a sensitive flow cytometry assay and (2) Elucidate the mechanism(s) of granule-dependent differential fitness in response to stressors: Using a combination of molecular biology and high-throughput sequencing technologies, we will measure localization of transcripts to granules nucleated by specific G3BP variants. A recently developed optogenetic tool will allow us to induce granule formation in the absence of stress, thereby independently measuring the effect of granules on cytoplasmic translation. My goal is to become a principal investigator pursuing an independent research program focused on deciphering cellular responses to stress. UCSF is an ideal environment to pursue an interdisciplinary approach to answering these fundamental biological questions. Completion of the proposed work will expand our knowledge of the mechanisms underlying the cellular response to stress, informing on responses to pathologies and therapeutic regimens.