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

项目摘要/摘要 G3BP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 由于eif2⍺磷酸化和整体翻译抑制而形成的细胞器。的作用 G3BP颗粒在细胞应激过程中的作用尚不完全清楚。它们由未翻译的mRNAs组成 以及来自翻译机制的因素导致G3BP SGS通过 从大块细胞质中隔离大分子。此外，G3BP颗粒的生物学功能 可能受其类液体性质的调节。此功能可能允许G3BP SGS与 当细胞从应激中恢复时，细胞质变得可逆地解体。此外，还提出了 在长时间的应激过程中，SG向类固体结构过渡，不利于生存。然而，详细的 G3BP颗粒液体稳定性的表征及其在应激反应中对细胞存活的作用 缺乏。我假设G3BP颗粒通过调节G3BP颗粒的翻译来保护哺乳动物细胞免受应激 MRNAs，并在胞质中保持稳定的液状相。检测G3BP颗粒的直接作用 在翻译抑制方面，在我的目标1中，我将表征G3BP颗粒在 APEX2-邻近标记结合RNA测序和质量分析研究外源应激在人细胞中的作用 光谱分析。此外，为了消除压力诱导对SG成分可能产生的影响，我将 描述与光遗传学诱导的SGS相关的蛋白质/RNA分子。然后，我将表演 多聚体测序中的转录异构体(TRIP-SEQ)技术，该技术表征了 与多聚体相关的mRNAs，以确定SG招募mRNAs与其 翻译。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，我将 急、慢性应激条件下G3BP颗粒形成的液体稳定性和可逆性 目前在Floor和Wittmann开发的基于微流体的荧光显微镜方法 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G3BP颗粒材料性质的转变 光漂白后荧光恢复(FRAP)实验。最后，我将确定细胞的存活能力 通过基于碘化丙啶的测定来阐明液体稳定性的作用和颗粒形成的动力学 为了从压力中生存。综上所述，该项目将提供对G3BP颗粒的生理作用的见解 对生存期间应激反应和恢复的生物学功能及其对液体稳定性的促进作用 外源压力下的细胞适应性。

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.