Granules and bodies: Toward an integrated view of membraneless organelles

虽然细胞很早就知道利用脂膜来组织其复杂的生物化学，但现在人们认为无膜细胞器如应激颗粒(SGS)、P小体(PBS)、核仁、Cajal小体等，是在蛋白质-蛋白质和蛋白质-核酸相互作用的驱动下通过液-液相分离形成的。要了解这些不同的结构如何获得专一性和功能化，就必须更彻底地了解它们的组成和组织。然而，缺乏表征这些结构的膜，历史上使它们的生化表征具有挑战性。通过我们的NSERC发现赠款，我们使用了邻近依赖的生物素化方法BioID来定义含RNA的胞浆颗粒和小体的组成和特异性(Youn等人，Mol Cell，2018年)。SGS和PBS与RNA稳定有关，PBS集中降解RNA的酶，动态SGS充当RNA和相关蛋白质的分选设施。从四个针对PBS或SGS的BioID诱饵开始，我们确认了已知成分的优先标签，并确定了我们合作跟进的新成分(例如Nishimura等人，NAR，2018年；Brandmann等人，EMBO J，2018年)。有趣的是，针对相同结构的不同诱饵，例如PB，对不同的猎物有不同的定量富集度，这表明我们正在发现空间邻近元素，这些元素可以信息形成这些结构的蛋白质的相对组织。我们对119种不同的诱饵蛋白进行了系统的BioID，生成了1,792种蛋白质之间7,424个邻近相互作用的大型数据集，从而更深入地探索了这一点。重要的是，对猎物之间的相关模式的分析揭示了这些结构的空间组织，并能够识别对形成显微镜下可见的SGS至关重要的“核心”蛋白质。总之，我们的结果为回答该领域的许多剩余问题奠定了基础，这些问题将在这次更新中得到解决，即：1)是什么决定了细胞质和细胞核中不同类别的RNA相关小体和颗粒的特异性和同一性？2)对于动态结构，什么信号(如特定RNA的招募或翻译后修饰)触发了从亚微观结构到微观结构的转变？3)是否存在形成不同颗粒和小体核心的明确结构或支架，以及如何控制蛋白质向不同小体的募集顺序？综上所述，这些研究将阐明真核生物中RNA调控的机制和无膜细胞器的形成，同时推动定量蛋白质组学方法的技术发展，这是研究无数细胞过程所必需的。

While cells have long been known to use lipid membranes to organize their complex biochemistry, membraneless organelles such as stress granules (SGs), P-bodies (PBs), nucleoli, Cajal bodies, etc., are now thought to be formed through liquid-liquid phase separation driven by protein-protein and protein-nucleic acid interactions. Understanding how these different structures acquire specificity and functionalization necessitates a more thorough understanding of their composition and organization. Yet, the lack of membranes that characterizes these structures has historically made their biochemical characterization challenging. Through our NSERC Discovery Grant, we have employed the proximity-dependent biotinylation approach BioID to define the composition and specificity in RNA-containing cytosolic granules and bodies (Youn et al., Mol Cell, 2018). SGs and PBs are implicated in RNAstasis, with PBs concentrating enzymes that degrade RNAs and the dynamic SGs serving as sorting facilities for RNA and associated proteins. Starting with four BioID baits targeting the PBs or the SGs, we confirmed preferential labeling of known components, and identified new components which we have collaboratively followed up on (e.g. Nishimura et al., NAR, 2018; Brandmann et al., EMBO J, 2018). Interestingly, different baits that targeted the same structure, e.g. the PB, had differential quantitative enrichment of the diverse preys, suggesting that we were uncovering spatial proximity elements that could inform on the relative organization of the proteins that form these structures. We explored this deeper by systematically performing BioID on 119 different bait proteins, generating a large dataset of 7,424 proximity interactions amongst 1,792 proteins. Importantly, analysis of the correlated patterns between the preys revealed the spatial organization of these structures and enabled the identification of "core" proteins that are critical for the formation of microscopically-visible SGs. Together, our results set the stage for answering many of the remaining questions in the field, which will be addressed in this renewal, namely: 1) What dictates the specificity and identity of the different classes of RNA-associated bodies and granules in the cytosol and nucleus? 2) For the dynamic structures, what are the signals (such as recruitment of specific RNA or post-translational modifications) that trigger the transition from submicroscopic to miscroscopic structures? 3) Is there a defined structure or scaffold that forms the core of the different granules and bodies, and how is the order of recruitment of proteins to the different bodies controlled? Taken together, these studies will shed light on mechanisms of RNA regulation in eukaryotes and the formation of membraneless organelles, while pushing the technology development in quantitative proteomics approaches which are necessary for the study of countless cellular processes.