Understanding the roles of intrinsically disordered proteins in stress granules

生物体不断受到压力条件的挑战。为了应对这种情况，它们的细胞已经进化到利用专门的亚细胞区室，即无膜细胞器，在受到压力时通过调节生物过程迅速响应细胞需求。这些包括在所有真核生物中发现的应激颗粒（SGs）。压力会阻止细胞产生新的蛋白质，当蛋白质和rna在细胞质中形成离散的液滴时，SGs就会在细胞质中形成，就像油在水中形成一样。我之前使用了一种名为BioID的技术来预测SGs和相关细胞器（加工体）中存在的约140种蛋白质（PB; Youn et al. 2018 Mol. Cell）。后来，我策划了一份全面的SGs和PBs清单，确定了哺乳动物细胞中约300种SGs驻留蛋白（Youn et al. 2019 Mol. Cell）。补充这些蛋白质组学研究，先进的显微镜研究已经阐明了整个SG组织。一些研究表明，SGs包括两个阶段：一个稳定的核心和一个不稳定的壳。然而，这些研究只研究了少数已知的SG蛋白，并且对SG的整体组织仍然知之甚少。SGs富含具有内在无序区域的蛋白质，这可能在其组织中起着至关重要的作用。这些蛋白质可以产生大量的相互作用，而这种高价是无膜细胞器形成所必需的。在哺乳动物SG蛋白质组中，富含脯氨酸的卷曲线圈（PRRC）2A和PRRC2B是高价相互作用的主要候选者，因为它们的大部分区域无序，并与许多SG蛋白相关（Youn et al. 2018 Mol. Cell）。为了研究它们在SG组织中的作用，我们在应激状态下的人类细胞中进行了PRRC2A和PRRC2B BioID实验，并观察了SG组装过程中不同的相互作用谱。免疫荧光显微镜也显示PRRC2A仅存在于SGs中，而PRRC2B双重定位于SGs和核无膜细胞器。基于此，我们假设PRRC2A和PRRC2B存在于不同的SG区域，在SG组织中发挥特定作用，并与其他无膜细胞器相互作用。为了验证这一点，我们将：1)确定PRRC2A和PRRC2B所占据的不同SG区域2)研究PRRC2A在募集其他蛋白质到SGs中的作用3)阐明PRRC2B在SGs和不同核无膜细胞器的双重定位的功能。我们的初步工作为阐明PRRC2A和PRRC2B在SG组织、功能和与其他无膜细胞器相互作用中的不同作用奠定了基础。在两位研究生的带领下，我们的发现将更好地理解SGs，这一知识将适用于所有真核生物系统。

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.