Granules and bodies: specificity in RNA storage and regulation

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Regulation of gene expression is essential for organismal health. In recent years, it has become apparent that much of this regulation occurs post-transcriptionally, through the regulation of mRNA processing, translation and degradation. After their transcription, splicing, capping and polyadenylation, mRNAs are transported in the cytoplasm where they can meet different fates. mRNA can be translated, stored into granules known as stress granules, SGs, when translation needs to be paused (e.g. after stress or viral infections), or transferred to other dense RNA-protein structures known as “processing” or P-bodies, PBs, that contain components of the RNA degradation machinery. While degradation was long thought to be a relatively non-specific process, the identification of RNA silencing pathways driven by endogenously expressed micro RNAs (miRNAs) has revealed an intricate specificity in this process, and revived interest in understanding the cytosolic mRNA cycle and its interaction with granules and bodies.Building on our expertise in interaction proteomics, we propose to perform an exhaustive proteomics characterization of the protein components of different RNA-associated bodies. In-depth proteomics characterization of the composition of these compartments has not been possible in the past, since they are not easily amenable to biochemical purification. Standard biochemical enrichment in buffers of low stringencies followed by high-speed centrifugation also largely preclude the identification of protein-protein interactions by affinity purification coupled to mass spectrometry (AP-MS) for the “insoluble” portion of a given protein (e.g. the population of the translation factors that are associated to stress granules). However, in 2012, a study by Roux et al. (J Cell Biol, PMID 22412018) introduced an attractive approach, BioID, that has the potential to circumvent these issues. The authors fused a protein of interest to a modified bacterial biotin ligase (BirA*) which they introduced in mammalian cells. Following addition of biotin for extended periods, proteins which come in the vicinity of the bait become biotinylated. This permits harsh lysis (which my lab further optimized for compartments of very low solubility) and recovery of the biotinylated proteins on streptavidin columns and identification by mass spectrometry. A graduate student in my group, Christopher Go, with the help of technician Wade Dunham, has demonstrated that this approach could be applied – alongside quantitative proteomics and proper negative controls – to discriminate between components of adjacent cellular compartments, including SGs and PBs (preliminary data). We propose three interrelated specific aims:1) Characterize the composition, at steady-state, of PBs and SGs by systematically analyzing known markers of these “organelles” by BioID coupled to mass spectrometry. This will generate a “parts list”.2) Elucidate the structural organization and mode of recruitment of proteins to PBs and SGs. By depleting or overexpressing PBs or SGs associated proteins and monitoring the recruitment of other factors (microscopically and by proteomics) we will be able to reconstruct the formation of granules and bodies. This will be refined at the molecular level by performing domain-mapping experiments.3) Provide a dynamic view of the formation of the SGs and PBs and further characterize elements of specificity. We will devise approaches to quantitatively monitor the process or assembly (or disassembly of proteins into PBs and SGs), and evaluate the impact of depletion/overexpression of newly identified components on mRNA regulation.Taken together, our studies will set the stage for a much better understanding of RNA regulation in eukaryotes.