Molecular mechanisms of stress granule assembly and disassembly

Abstract

Stress granules (SGs) are membrane-less ribonucleoprotein (RNP)-based cellular compartments that form in the cytoplasm of a cell upon exposure to various environmental stressors. SGs contain a large set of proteins, as well as mRNAs that have been stalled in translation as a result of stress-induced polysome disassembly. Despite the fact that SGs have been extensively studied for many years, their function is still not clear. They presumably help the cell to cope with the encountered stress, and facilitate the recovery process after stress removal upon which SGs disassemble. Aberrant formation of SGs and impaired SG disassembly majorly contribute to various pathological phenomena in cancer, viral infections, and neurodegeneration. The assembly of SGs is largely driven by liquid-liquid phase separation (LLPS), however, the molecular mechanisms behind that are not fully understood. Recent studies have proposed a novel mechanism for SG formation that involves the interplay of a large interaction network of mRNAs and proteins. Here, we review this novel concept of SG assembly, and discuss the current insights into SG disassembly.

Keywords: Liquid-liquid phase transition; Protein synthesis; RNA granules; Stress granules; Stress response.

Figure 1:

(A) generic model of a central SG protein, depicting an oligomerization domain (OD) or dimerization domain (DD), intrinsically disordered regions (IDRs) or low-complexity domains (LCDs), and a substrate binding domain (SBD), such as RNA-recognition motif (RRM) or RGG/RG domain. (B) Different types of protein-protein (pp), protein-RNA (pR), and RNA-RNA (RR) interaction that are implicated in SG assembly. The schematic was created in ©BioRender (biorender.com) and does not resemble actual sizes and ratios.

Figure 2:

Localization of key motifs and domains, as well as predicted disordered regions calculated using the PONDR-VSL2 algorithm of (A) G3BP1, (B) FXR1, (C) Caprin1, (D) UBAP2L, and (E) TIA1.

Figure 3:

Methodology to study SGs. (A) siRNAs screen to identify genes whose knockdown impairs assembly (or disassembly) of SGs. (B) Use of molecular crowding agents (e.g. B-isox, PEG) causes LLPS within a cell lysate, and allows separation of soluble and non-soluble fractions. (C) Both live imaging and fixed cell imaging present a microscopy-based approach to study LLPS and SGs. (D) An affinity purification approach (e.g. via GFP-tagged G3BP) can be utilized in combination with mass spectrometry analysis to identify proteins that are enriched in SGs. (E) Bioinformatic studies that can predict whether a particular protein is localized to SGs. (F) Interactome analyses using proximity labeling studies reveals a wide network of SG-associated proteins. (G) Yeast-two hybrid screen can be used to identify protein-protein interactions with SG-associated proteins as bait. (H) The Corelet system visualizes condensate formation in live cells using imaging as a readout. The schematic was created in ©BioRender (biorender.com) and does not resemble actual sizes and ratios.

Figure 4:

Novel model depicting a SG protein and RNA interaction network: Macromolecules with more than three interaction domains (v ≥ 3) are defined as nodes, and constitute the essential drivers for LLPS. Proteins that lack interactions with other particles are bystanders (v = 0), whereas v = 1 proteins are called caps (modified from Sanders et al., 2020). This model depicts a mechanistic framework for network-based cellular condensation. The schematic was created in ©BioRender (biorender.com) and does not resemble actual sizes and ratios.

Figure 5:

Speculative model Upon polysome disassembly, G3BP-USP10 complexes bind to dissociated 40S subunits. Prior to its dissociation from G3BP, USP10 mediates deubiquitination of specific ribosomal proteins to prevent their degradation through the lysosome. Binding of UBAP2L and caprin1 to G3BP leads to increased valency, which promotes LLPS and subsequent SG formation. The schematic was created in ©BioRender (biorender.com); it is not time-resolved and does not resemble actual sizes and ratios.

Figure 6:

Model of SG assembly and disassembly. Upon RNA influx, key SG node and bridge proteins form a multi-interaction network, which causes LLPS and the assembly of SGs. Following stress removal, PTMs potentially lead to the loss of protein-protein, protein-RNA, and RNA-RNA interactions, leading to a decrease in valence and allowing subsequent SG disassembly. The schematic was created in ©BioRender (biorender.com); it is not time-resolved and does not resemble actual sizes and ratios.