Granules and bodies: Toward an integrated view of membraneless organelles

虽然长期以来已知细胞使用脂质膜来组织其复杂的生物化学，但无膜细胞器如应激颗粒（SG）、P体（PB）、核仁、Cajal体等，现在认为是通过蛋白质-蛋白质和蛋白质-核酸相互作用驱动的液-液相分离形成的。要了解这些不同的结构如何获得特异性和功能化，就必须更透彻地了解它们的组成和组织。然而，由于缺乏表征这些结构的膜，历史上使它们的生物化学表征具有挑战性。通过我们的NSERC发现资助，我们已经采用邻近依赖性生物素化方法BioID来定义含RNA的胞质颗粒和体中的组成和特异性（Youn等人，Mol Cell，2018）。SG和PB参与RNA停滞，PB浓缩降解RNA的酶，而动态SG充当RNA和相关蛋白质的分选设施。从靶向PB或SG的四种BioID诱饵开始，我们确认了已知组分的优先标记，并鉴定了我们已经合作跟进的新组分（例如Nishimura等人，NAR，2018; Brandmann等人，EMBO J，2018）。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，这表明我们发现了空间邻近元素，这些元素可以告知形成这些结构的蛋白质的相对组织。我们通过系统地对119种不同的诱饵蛋白进行BioID来更深入地探索这一点，在1，792种蛋白质中生成了7，424种邻近相互作用的大型数据集。重要的是，对猎物之间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可见的SG至关重要的“核心”蛋白。总之，我们的结果为回答该领域中许多剩余的问题奠定了基础，这些问题将在本次更新中得到解决，即：*1）是什么决定了细胞质和细胞核中不同类别的RNA相关体和颗粒的特异性和身份？**2）对于动态结构，触发从亚微观结构到微观结构转变的信号是什么（如特定RNA的募集或翻译后修饰）？**3）是否有一个确定的结构或支架形成不同颗粒和实体的核心，以及如何控制蛋白质向不同实体的募集顺序？**总之，这些研究将揭示真核生物中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.