Stress granules have gained considerable exposure and interest in recent years. These micron-sized entities, composed of RNA and protein, form following a stress exposure and have been linked to ...several pathologies. Understanding stress granule function is paramount but has been arduous due to the membraneless nature of these organelles. Several new methodologies have recently been developed to catalogue the protein and RNA composition of stress granules. Collectively, this work has provided important insights to potential stress granule functions as well as molecular mechanisms for their assembly and disassembly. This chapter reviews the latest advancements in the understanding of stress granule dynamics and discusses the various protocols developed to study their composition.
Stress granules are non-membrane bound RNA-protein (RNP) assemblies that form when translation initiation is limited and contain a biphasic structure with stable core structures surrounded by a less ...concentrated shell. The order of assembly and disassembly of these two structures remains unknown. Time course analysis of granule assembly suggests that core formation is an early event in granule assembly. Stress granule disassembly is also a stepwise process with shell dissipation followed by core clearance. Perturbations that alter liquid-liquid phase separations (LLPS) driven by intrinsically disordered protein regions (IDR) of RNA binding proteins in vitro have the opposite effect on stress granule assembly in vivo. Taken together, these observations argue that stress granules assemble through a multistep process initiated by stable assembly of untranslated mRNPs into core structures, which could provide sufficient high local concentrations to allow for a localized LLPS driven by IDRs on RNA binding proteins.
Stress granules are assemblies of untranslating messenger ribonucleoproteins (mRNPs) that form from mRNAs stalled in translation initiation. Stress granules form through interactions between ...mRNA-binding proteins that link together populations of mRNPs. Interactions promoting stress granule formation include conventional protein–protein interactions as well as interactions involving intrinsically disordered regions (IDRs) of proteins. Assembly and disassembly of stress granules are modulated by various post-translational modifications as well as numerous ATP-dependent RNP or protein remodeling complexes, illustrating that stress granules represent an active liquid wherein energy input maintains their dynamic state. Stress granule formation modulates the stress response, viral infection, and signaling pathways. Persistent or aberrant stress granule formation contributes to neurodegenerative disease and some cancers.
The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP ...granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.
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•SG assembly is driven by the collective interactions of a core protein-RNA network•The central node G3BP encodes a molecular switch that regulates RNA-dependent LLPS•Interplay between 3 distinct IDRs in G3BP tunes the intrinsic propensity for LLPS•Extrinsic factors regulate SG assembly through positive or negative cooperativity
The molecular basis for stress granule assembly is a network of protein-RNA interactions with G3BP1 as the central node and molecular switch that triggers phase separation of granule components in response to free cytosolic RNA concentrations.
Stress granules (SGs) are membraneless organelles that form in eukaryotic cells after stress exposure 1 (reviewed in 2–4). Following translation inhibition, polysome disassembly releases 48S ...preinitiation complexes (PICs). mRNA, PICs, and other proteins coalesce in SG cores 1, 5–7. SG cores recruit a dynamic shell, whose properties are dominated by weak interactions between proteins and RNAs 8–10. The structure and assembly of SGs and how different components contribute to their formation are not fully understood. Using super-resolution and expansion microscopy, we find that the SG component UBAP2L 11, 12 and the core protein G3BP1 5, 11–13 occupy different domains inside SGs. UBAP2L displays typical properties of a core protein, indicating that cores of different compositions coexist inside the same granule. Consistent with a role as a core protein, UBAP2L is required for SG assembly in several stress conditions. Our reverse genetic and cell biology experiments suggest that UBAP2L forms granules independent of G3BP1 and 2 but does not interfere with stress-induced translational inhibition. We propose a model in which UBAP2L is an essential SG nucleator that acts upstream of G3BP1 and 2 and facilitates G3BP1 core formation and SG assembly and growth.
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•UBAP2L displays properties of a core component of stress granules•UBAP2L and G3BP1 form stress granule cores that do not always colocalize•UBAP2L forms small RNP granules independent of G3BP1•UBAP2L functions upstream of G3BP1 in stress granule assembly
Stress granules (SGs) are formed by a stable core and a dynamic shell. Cirillo, Cieren, et al. demonstrate that UBAP2L is an SG core component that does not always colocalize with the previously identified core protein G3BP1. UBAP2L is required for SG formation in several stress conditions and forms SGs independent and upstream of G3BP proteins.
Stress granules (SGs) are evolutionary conserved aggregates of proteins and untranslated mRNAs formed in response to stress. Despite their importance for stress adaptation, no complete proteome ...composition has been reported for plant SGs. In this study, we addressed the existing gap. Importantly, we also provide evidence for metabolite sequestration within the SGs.
To isolate SGs we used Arabidopsis seedlings expressing green fluorescent protein (GFP) fusion of the SGs marker protein, Rbp47b, and an experimental protocol combining differential centrifugation with affinity purification (AP). SGs isolates were analysed using mass spectrometry-based proteomics and metabolomics.
A quarter of the identified proteins constituted known or predicted SG components. Intriguingly, the remaining proteins were enriched in key enzymes and regulators, such as cyclin-dependent kinase A (CDKA), that mediate plant responses to stress. In addition to proteins, nucleotides, amino acids and phospholipids also accumulated in SGs.
Taken together, our results indicated the presence of a preexisting SG protein interaction network; an evolutionary conservation of the proteins involved in SG assembly and dynamics; an important role for SGs in moderation of stress responses by selective storage of proteins and metabolites.
Ribonucleoprotein (RNP) granules are RNA–protein assemblies that are involved in multiple aspects of RNA metabolism and are linked to memory, development, and disease. Some RNP granules form, in ...part, through the formation of intermolecular RNA–RNA interactions. In vitro, such trans RNA condensation occurs readily, suggesting that cells require mechanisms to modulate RNA-based condensation. We assess the mechanisms of RNA condensation and how cells modulate this phenomenon. We propose that cells control RNA condensation through ATP-dependent processes, static RNA buffering, and dynamic post-translational mechanisms. Moreover, perturbations in these mechanisms can be involved in disease. This reveals multiple cellular mechanisms of kinetic and thermodynamic control that maintain the proper distribution of RNA molecules between dispersed and condensed forms.
Intermolecular RNA–RNA interactions contribute to the formation, content, and biophysical properties of many RNP granules.Cells utilize both genetically programmed and promiscuous RNA–RNA interactions in RNP granules.RNA–protein interactions modulate protein dynamics in RNP granules.Cells have evolved mechanisms to regulate RNA condensation through RNA chaperones.RNA chaperones can be influenced by post-translational modifications (PTMs).
RNA-protein (RNP) granules have been proposed to assemble by forming solid RNA/protein aggregates or through phase separation into a liquid RNA/protein phase. Which model describes RNP granules in ...living cells is still unclear. In this study, we analyze P bodies in budding yeast and find that they have liquid-like properties. Surprisingly, yeast stress granules adopt a different material state, which is reminiscent of solid protein aggregates and controlled by protein disaggregases. By using an assay to ectopically nucleate RNP granules, we further establish that RNP granule formation does not depend on amyloid-like aggregation but rather involves many promiscuous interactions. Finally, we show that stress granules have different properties in mammalian cells, where they show liquid-like behavior. Thus, we propose that the material state of RNP granules is flexible and that the solid state of yeast stress granules is an adaptation to extreme environments, made possible by the presence of a powerful disaggregation machine.
The supramolecular structure of the A- and B-type granules of wheat starch was compared. Polarized light microscopy, X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) were ...used to study the granular, crystalline, and short-range structures. The A- and B-type granules displayed a typical A-type crystalline structure with the degrees of crystallinity of 31.95% and 29.38% respectively. In addition, the B-type granules had some V-type crystallites. The nanostructure and fractals were characterized by small angle X-ray scattering (SAXS), which showed that the average thickness of the lamellae of the A-type granules was larger, while the B-type granules possessed a higher degree of ordering in the lamellar regions. A second order reflection was found in both A- and B-type granules, which was proposed due to the crystalline lamellae of the semicrystalline lamellae. The A- and B-type granules had mass and surface fractal structures respectively.
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► Aggregation structure of the A- and B-type granules of wheat starch was compared. ► A-type granules had higher crystallinity and greater birefringence intensity. ► Double helices in the B-type granules were oriented more uniformly. ► Scattering objects of the B-type granules were more smooth.
Lysosome‐related organelles (LROs) comprise a diverse group of cell type‐specific, membrane‐bound subcellular organelles that derive at least in part from the endolysosomal system but that have ...unique contents, morphologies and functions to support specific physiological roles. They include: melanosomes that provide pigment to our eyes and skin; alpha and dense granules in platelets, and lytic granules in cytotoxic T cells and natural killer cells, which release effectors to regulate hemostasis and immunity; and distinct classes of lamellar bodies in lung epithelial cells and keratinocytes that support lung plasticity and skin lubrication. The formation, maturation and/or secretion of subsets of LROs are dysfunctional or entirely absent in a number of hereditary syndromic disorders, including in particular the Hermansky‐Pudlak syndromes. This review provides a comprehensive overview of LROs in humans and model organisms and presents our current understanding of how the products of genes that are defective in heritable diseases impact their formation, motility and ultimate secretion.
Lysosome‐related organelles (LROs) are cell type‐specific subcellular structures that derive from both the secretory and endolysosomal pathways and that play key roles in numerous physiological systems, in most cases following stimulus‐dependent secretion of their contents. The biogenesis and/or secretion of LRO subsets are disrupted in hereditary syndromic disorders such as the Hermansky‐Pudlak syndromes and familial hemophagocytic lymphohistiocytosis. We comprehensively review LROs in humans and model organisms and the mechanisms by which products of disease genes regulate their formation, motility and ultimate secretion.
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