The role of G3BP granules in mRNA translation regulation and cell adaptation to exogenous stress

G3BP颗粒在mRNA翻译调控和细胞对外源应激适应中的作用

• 批准号: 10677026
• 负责人: Jose Liboy
• 金额: $ 4.36万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10677026
• 关键词: Amyotrophic Lateral Sclerosis; Binding; Binding Proteins; Biological Assay; Biological Process; Biological Testing; Cell Aggregation; Cell Death; Cell Survival; Cells; Cellular Stress; Cessation of life; Chronic stress; Coupled; Cytoplasm; Cytoplasmic Granules; Cytoprotection; Data; Disease Progression; Dissociation; Eukaryotic Cell; Exhibits; Floor; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; G3BP1 gene; Heat-Shock Response; Human; Kinetics; Label; Laboratories; Link; Liquid substance; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Membrane; Messenger RNA; Microfluidics; Modeling; Organelles; Osmosis; Pathologic; Pathology; Peptide Initiation Factors; Phase; Phosphorylation; Physical condensation; Physiological; Physiology; Polyribosomes; Process; Property; Propidium Diiodide; Protein Isoforms; Proteins; RNA; RNA Stability; RNA-Binding Proteins; Recovery; Regulation; Reporting; Role; SH3 Domains; Solid; Stress; Structure; Techniques; Testing; Therapeutic; Transcript; Translating; Translational Regulation; Translational Repression; Translations; Ultraviolet Rays; acute stress; biological adaptation to stress; biophysical properties; design; endoplasmic reticulum stress; experimental study; insight; mRNA Translation; macromolecule; optogenetics; ras GTPase-Activating Proteins; recruit; stress granule; transcriptome sequencing; translation factor

Project Summary/Abstract G3BP stress granules (SGs) are a component of the eukaryotic stress response. They are membrane-less organelles that form as a consequence of eIF2⍺ phosphorylation and global translation inhibition. The role of G3BP granules during cellular stress is not completely understood. They are composed of untranslated mRNAs and factors from the translational machinery leading to the model that G3BP SGs inhibit translation through the sequestration of macromolecules from the bulk cytoplasm. Furthermore, the biological function of G3BP granules may be regulated by their liquid-like properties. This feature may allow G3BP SGs to interact dynamically with the bulk cytoplasm and to reversibly dissociate when cells recover from stress. Moreover, it has been proposed that SG transitioning to solid-like structure during prolonged stress is detrimental to survival. However, detailed characterization of G3BP granules liquid stability and their role in cell survival during stress response is still lacking. I hypothesize that G3BP granules protect mammalian cells against stress by regulating translation of mRNAs and retaining a stable liquid-like phase in the bulk cytoplasm. To test the direct role of G3BP granules in translation inhibition, in my aim 1, I will characterize the protein and RNA composition of G3BP granules under exogenous stress in human cells through APEX2-proximity labeling coupled to RNA sequencing and Mass Spectrometry. Furthermore, to decouple the effects that stress induction may have on SG composition, I will characterize the protein/RNA molecules associated to SGs induced with optogenetics. Then, I will perform the Transcript Isoforms in Polysomes sequencing (TrIP-seq) technique, which characterizes the abundance of mRNAs associated to polysomes, to define the relationship between SG recruitment of mRNAs and their translation. To test the biological function of G3BP SG biophysical properties to cell survival, in my aim 2, I will study the liquid stability and reversibility of G3BP granule formation under acute and chronic stress with a microfluidics-based fluorescence microscopy approach currently developed in the Floor and Wittmann laboratories at UCSF. Then, I will evaluate transitions in the material properties of G3BP granules by performing fluorescence recovery after photobleaching (FRAP) experiments. Finally, I will determine the viability of cells through a propidium iodide-based assay to elucidate the role of liquid stability and kinetics of granule formation to survival from stress. In summary, this project will provide insights into the physiological role of G3BP granules to survival during the stress response and recovery and the biological function of their liquid stability in promoting cellular adaptability under exogenous stress.

项目概要/摘要 G3BP 应激颗粒 (SG) 是真核应激反应的组成部分，它们是无膜的。 由于 eIF2⍺ 磷酸化和全局翻译抑制而形成的细胞器的作用。 细胞应激期间的 G3BP 颗粒尚不完全清楚，它们是由非翻译的 mRNA 组成的。 以及来自翻译机制的因素导致 G3BP SG 通过 从大量细胞质中隔离大分子此外，G3BP 颗粒的生物学功能。 可以通过其类似液体的特性进行调节。此功能可以允许 G3BP SG 与动态交互。 此外，有人提出，当细胞从应激中恢复时，细胞质会可逆地解离。 然而，在长期压力下，SG 转变为固体状结构会对生存造成困扰。 G3BP 颗粒液体稳定性的表征及其在应激反应期间细胞存活中的作用仍不清楚 我发现 G3BP 颗粒通过调节翻译来保护哺乳动物细胞免受应激。 mRNA 并在大量细胞质中保留稳定的液体相 测试 G3BP 颗粒的直接作用。 在翻译抑制中，在我的目标 1 中，我将表征 G3BP 颗粒的蛋白质和 RNA 组成 通过 APEX2 邻近标记结合 RNA 测序和 Mass 检测人类细胞中的外源性应激 此外，为了消除应力诱导可能对 SG 成分的影响，我将 表征与光遗传学诱导的 SG 相关的蛋白质/RNA 分子。 多核糖体测序 (TrIP-seq) 技术中的转录异构体，该技术表征了 与多核糖体相关的 mRNA，以定义 mRNA 的 SG 募集及其作用之间的关系 为了测试 G3BP SG 生物物理特性对细胞存活的生物学功能，在我的目标 2 中，我将 研究急性和慢性应激下 G3BP 颗粒形成的液体稳定性和可逆性 Floor 和 Wittmann 目前开发的基于微流体的荧光显微镜方法 然后，我将通过执行来评估 G3BP 颗粒材料特性的转变。 光漂白（FRAP）实验后的荧光恢复最后，我将确定细胞的活力。 通过基于碘化丙啶的测定来阐明液体稳定性和颗粒形成动力学的作用 总之，该项目将深入了解 G3BP 颗粒的生理作用。 应激反应期间的生存和恢复及其液体稳定性促进的生物学功能 细胞在外源应激下的适应性。