Granules and bodies: Toward an integrated view of membraneless organelles

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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.