Functional characterisation of stress granule composition and morphology

有效的细胞应激反应程序将损害降至最低，并允许细胞在不利条件下生存。在真核细胞中，许多应激(如热休克、紫外线(UV)光损伤、氧化应激)会导致蛋白质合成的短暂停止，以减少能量消耗并促进基因表达的重新编程。这种翻译停滞导致含有未翻译信使核糖核蛋白(MRNPs)的应激颗粒(SGS)细胞质焦点的形成。这些病灶的形成是因为无多聚体的mRNP招募具有自我聚集特性的蛋白质，例如T细胞胞内抗原1(TIA1)和RAS GTP酶激活蛋白结合蛋白1和2(G3BP1/2)，这些蛋白质形成SG。从酵母到人类的真核生物中已经证实了SG的形成，因此它代表了一种进化上保守的应激反应途径。然而，mRNP聚集到SGS中的功能意义及其在应对不同类型压力中的作用仍不清楚，并将代表我的NSERC资助研究计划的长期目标。众所周知，SG的形成不是应力诱导的翻译停止所必需的。因此，已经提出SGS的其他功能，包括a)存储和分类未翻译的mRNAs；b)调节胁迫期间的信号级联；c)通过隔离特定因子来中断核质运输。即使这些功能和可能的额外功能可以归因于SGS，它们在应对不同类型的压力中的作用迄今还没有被证明。因此，阐明SGS在各种应激反应途径中的功能将是本实验室的主要研究目标之一。在短期内，我将集中在紫外线诱导的SG的形成和SGS在紫外线损伤后细胞存活中的作用(目标1)。同时，我将使用邻近蛋白质组学方法(目标2)来表征SG组成中的压力依赖的差异。 拟议的研究将回答与SG生物学相关的几个重要问题：a)紫外线激活的信号级联如何决定紫外线照射后形成的SG的独特组成和性质；b)SG的形成如何影响UV损伤后细胞的存活；c)SG组成的特定胁迫差异的程度。这些问题的答案将为正在稳步增长的RNA颗粒生物学领域贡献新的知识，并将为未来研究SG形成对不同胁迫条件的功能后果提供信息。

Effective cellular stress response programs minimize damage and allow cells to survive unfavourable conditions. In eukaryotic cells, many stresses (e.g. heat shock, ultraviolet (UV) light damage, oxidative stress) cause transient cessation of protein synthesis to decrease energy consumption and facilitate reprogramming of gene expression. This translation arrest results in formation of stress granules (SGs) cytoplasmic foci that contain untranslated messenger ribonucleoproteins (mRNPs). These foci form because polysome-free mRNPs recruit proteins with self-aggregating properties, such as the T-cell intracellular antigen 1 (TIA1) and the Ras GTP-ase activating protein binding proteins 1 and 2 (G3BP1/2) that nucleate SG formation. SG formation has been demonstrated for eukaryotes from yeast to humans and thus represents an evolutionarily conserved stress response pathway. However, the functional significance of mRNP aggregation into SGs and its role in responses to different types of stress remain unknown and will represent the long-term objective of my NSERC-funded research program. It is well established that SG formation is not required for stress-induced translation arrest. Thus, other functions have been suggested for SGs, including a) storage and triage of untranslated mRNAs; b) regulation of signaling cascades during stress; c) disruption of nucleocytoplasmic transport through sequestration of specific factors. Even if these and possibly additional functions could be attributed to SGs, their role in response to different types of stress have not been demonstrated to date. Accordingly, elucidating the functions of SGs in various stress response pathways will be one of the major research objectives in my laboratory. In the short term, I will focus on UV light-induced SG formation and the role of SGs in cell survival following UV damage (Aim 1). In parallel, I will characterize stress-dependent differences in SG composition using a proximity proteomics approach (Aim 2). Proposed studies will answer several important questions relating to SG biology: a) how signaling cascades activated by UV light dictate unique composition and properties of SGs formed following UV exposure; b) how SG formation affects cell survival following UV damage; c) the extent of stress-specific differences in SG composition. The answers to these questions will contribute new knowledge to the field of RNA granule biology that is steadily growing and will inform future studies into functional consequences of SG formation in response to different stress conditions.