Understanding the roles of intrinsically disordered proteins in stress granules

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Living organisms are constantly challenged by stressful conditions. To cope, their cells have evolved to utilize specialized subcellular compartments, called membraneless organelles, that rapidly respond to cellular needs by regulating biological processes when stressed. These include stress granules (SGs), which are found in all eukaryotic organisms. Stress stops cells from producing new proteins, and SGs form in the cytoplasm when the proteins and RNAs involved in the process form discrete droplets, much like oil does in water. I previously used a technique called BioID to predict ~140 proteins present in SGs and a related organelle, the processing body (PB; Youn et al. 2018 Mol. Cell). I later curated a comprehensive inventory of SGs and PBs, defining ~300 SG-resident proteins in mammalian cells (Youn et al. 2019 Mol. Cell). Complementing these proteomics studies, advanced microscopy studies have shed light on overall SG organization. Several studies suggest that SGs comprise two phases: a stable core and a labile shell. However, these studies have only examined a few well-known SG proteins, and overall SG organization remains poorly understood. SGs are highly enriched for proteins with intrinsically disordered regions, which may play crucial roles in their organization. These proteins can generate large numbers of interactions, and this high valency is required for membraneless organelle formation. In the mammalian SG proteome, proline rich coiled-coil (PRRC)2A and PRRC2B are prime candidates for high valency interactions because of their largely disordered regions and associations with many SG proteins (Youn et al. 2018 Mol. Cell). To investigate their roles in SG organization, we performed PRRC2A and PRRC2B BioID in human cells during stress and observed distinct interaction profiles during SG assembly. Immunofluorescence-based microscopy also revealed that PRRC2A solely resides in SGs, whereas PRRC2B dually localizes to SGs and a nuclear membraneless organelle. Based on this, we hypothesize that PRRC2A and PRRC2B reside in distinct SG regions and play specific roles in SG organization and interplay with other membraneless organelles. To test this, we will: 1)Determine the distinct SG regions occupied by PRRC2A and PRRC2B 2)Investigate the role of PRRC2A in recruiting other proteins to SGs 3)Elucidate the function of PRRC2B's dual localization to SGs and a distinct nuclear membraneless organelle Our preliminary work sets the stage to elucidate the divergent roles of PRRC2A and PRRC2B in SG organization, function, and interplay with other membraneless organelles. Spearheaded by two graduate students, our findings will provide a better understanding of SGs, knowledge that will be applicable to all eukaryotic systems.