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.

项目总结/摘要 G3 BP应激颗粒（SGs）是真核生物应激反应的组成部分。它们是无膜的 细胞器的形成是eIF 2磷酸化和整体翻译抑制的结果。的作用 细胞应激过程中的G3 BP颗粒尚未完全了解。它们由未翻译的mRNA组成 以及来自翻译机制的因素，导致G3 BP SGs通过G3 BP SGs抑制翻译的模型。 从大量细胞质中隔离大分子。此外，G3 BP颗粒剂的生物学功能 可以通过它们的液体性质来调节。该特征可以允许G3 BP SG与 大量的细胞质，并可逆地解离时，细胞从应力恢复。此外，有人提出， SG在长时间的压力下转变为固体状结构对生存是有害的。然而，详细 G3 BP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然是 缺乏我假设G3 BP颗粒通过调节G3 BP的翻译来保护哺乳动物细胞免受应激。 mRNA的表达，并在大量细胞质中保持稳定的液体样相。检测G3 BP颗粒的直接作用 在翻译抑制中，在我的目标1中，我将在以下条件下表征G3 BP颗粒的蛋白质和RNA组成： 通过APEX 2-邻近标记结合RNA测序和质谱分析在人细胞中的外源性应激 光谱法此外，为了解耦应力诱导可能对SG组成的影响，我将 表征与用光遗传学诱导的SG相关的蛋白质/RNA分子。然后，我将执行 多聚体测序中的转录异构体（TrIP-seq）技术，其表征多聚体中的转录异构体的丰度。 与多聚核糖体相关的mRNA，以确定SG募集mRNA及其 翻译.为了测试G3 BP 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 研究了急、慢性应激下G3 BP颗粒形成的液体稳定性和可逆性。 基于微流体的荧光显微镜方法，目前在地板和Wittmann 加州大学旧金山分校的实验室然后，我将通过执行以下操作来评估G3 BP颗粒材料特性的转变： 光漂白后的荧光恢复（FRAP）实验。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 从压力中生存下来。总之，本项目将提供深入了解G3 BP颗粒的生理作用 在应激反应和恢复过程中对生存和生物功能的稳定性有促进作用 细胞对外源胁迫的适应性。