Understanding the roles of intrinsically disordered proteins in stress granules

生命有机体不断受到压力条件的挑战。为了应对，它们的细胞已经进化成利用被称为无膜细胞器的特殊亚细胞间隔，当受到压力时，通过调节生物过程来快速响应细胞需求。这些包括应激颗粒(SGS)，它存在于所有真核生物中。压力会阻止细胞产生新的蛋白质，当参与这一过程的蛋白质和RNA形成离散的液滴时，SGS就会在细胞质中形成，就像油在水中一样。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~140个蛋白质(PB；Youn等人)。2018年摩尔单元格)。后来，我策划了一份SGS和PBS的全面清单，定义了哺乳动物细胞中约300个SG驻留蛋白(Youn等人)。2019摩尔。单元格)。作为这些蛋白质组学研究的补充，高级显微镜研究揭示了整个SG的组织结构。一些研究表明，SGS由两个阶段组成：稳定的核心和不稳定的外壳。然而，这些研究只研究了几个广为人知的SG蛋白，总体上SG的组织结构仍然知之甚少。SGS高度富含具有内在无序区域的蛋白质，这些区域可能在其组织中发挥关键作用。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，因为它们在很大程度上是无序的区域并且与许多SG蛋白质结合(Youn等人。2018年摩尔单元格)。为了研究它们在SG组织中的作用，我们在应激过程中对人类细胞进行了PRRC2A和PRRC2B BioID，并观察了SG组装过程中不同的相互作用曲线。免疫荧光显微镜还显示，PRRC2A仅存在于SGS中，而PRRC2B双重定位于SGS和一个核无膜细胞器。在此基础上，我们推测PRRC2A和PRRC2B位于不同的SG区域，在SG的组织和与其他无膜细胞器的相互作用中发挥着特定的作用。为了验证这一点，我们将：1)确定PRRC2A和PRRC2B所占据的不同的SG区域；2)研究PRRC2A在招募其他蛋白质到SGS中的作用；3)阐明PRRC2B与SGS和一个独特的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的组织、功能以及与其他无膜细胞器的相互作用中的不同作用奠定了基础。在两名研究生的带头下，我们的发现将提供对SGS的更好理解，即适用于所有真核系统的知识。

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.