Stress Granule Life Cycle Diagram

Pathway Description:

Stress granules (SGs) are formed in response to acute biotic and abiotic stress. Stressors include, but are not limited to, toxicity exposure, oxidative stress, viral infections, nutrient depletion, and irradiation.¹ The cell responds to these stressors by interrupting normal protein translation. Typically, preinitiation complexes (PICs) are inhibited via phosphorylation of eIF2α by protein kinase R (PKR), protein Kinase RNA-Like ER Kinase (PERK), general control nonderepressible 2 (GCN2), or heme-regulated inhibitor (HRI).¹ Additionally, inactivation of mTOR results in increased activity of eIF4-binding proteins, resulting in interference with assembly of the eIF4 translation complex.² Translating ribosomes subsequently run off, exposing the messenger RNA (mRNA) and 40S subunit, which forms the non-canonical PIC. Canonical nucleating RNA-binding proteins (RBPs), including T-cell restricted intracellular antigen-1/TIA-1-related protein (TIA-1/R) and G3BP stress granule assembly factor 1/2 (G3BP1/2) are recruited to the PIC, though other nucleating proteins can also function in this role. This recruitment, coupled with post-transcriptional modifications of RNA and post-translational modifications of nucleating proteins, facilitates formation of the "SG core" or "SG seed.” This SG seed is hypothesized to be relatively stable and can oligomerize with other SG seeds to form larger SG foci.

SG formation is primarily driven by liquid-liquid phase separation (LLPS),³ and as such, the size, shape, and structure of SGs are influenced by numerous seed characteristics. SG LLPS is strongly influenced by seed composition, which can include an exceedingly diverse range of RNAs and RBPs, adaptor/scaffolding proteins, and enzymes.⁴ The type of stress that induces SG formation also impacts the composition,¹ as do post-transcriptional modifications of RNAs and post-translational modifications of RBPs. Given that SG composition is highly variable, factors such as steric hindrance, electrostatic interactions, and Laplace pressure have additional important effects on SG size and shape; increased local seed concentration and weak low-affinity interactions between seeds drive seed coalescence.

A number of critical proteins have been identified that are crucial to SG recruitment, assembly, and modulation. Poly(A) binding protein cytoplasmic 1 (PABP1), a crucial regulator of mRNA stability and translation initiation, is a primary component of SGs; it is recruited early and is often dynamically active, shuttling in and out of the SG.⁵ Similarly, Ataxin-2 (ATXN2) also promotes mRNA stability and translation and is a core component of SGs.⁶ Ubiquitin associated protein 2 like (UBAP2L) is required for SG assembly, and may act upstream of G3BP1 under certain conditions. It is also responsible for recruitment of component messenger ribonucleoproteins (mRNPs), RBPs, and ribosomal subunits.⁷ Effectors downstream of UBAP2L, such as TIA1 and fragile X mental retardation protein (FMRP) and its associated protein FMR1 interacting protein 2 (NUFIP2) also localize and/or aid in recruiting mRNA and mRNP to the SG.^8,9 RNA binding protein motif 3 (RBM3), an anti-apoptotic protein, also promotes SG formation,¹⁰ while DEAD box 1 (DDX1) binds RNAs and translocates to SGs under varying stress conditions.¹¹ Interestingly, G3BP1/2 are required for SG formation in response to eIF2α/4A inhibition, but not heat or osmotic stress. G3BP1/2 and Caprin1 proteins form a complex, with Caprin promoting G3BP1/2 LLPS.³ In addition to Caprin1, USP10 also binds G3BP1/2, and this binding is mutually exclusive with Caprin-G3BP1/2 complexing; USP10 binding inhibits SG formation, while Caprin binding promotes it.¹² YTHDF1/2/3 bind m6A-modified mRNA. YTHDF1/3 accumulate around G3BP1/2 clusters, while YTHDF2 colocalizes with G3BP1/2 within the SG, further facilitating SG formation.¹³

Disease-linked RBPs translocate from the nucleus to be recruited into the SG via secondary nucleation. These include TAR DNA-binding protein 43 (TDP43), which modulates SG formation through robust interaction with G3BP1/2,14 and the FET (FUS/TLS, EWS and TAF15) family of RBPs.¹⁵ FUS RNA binding protein (FUS) and TATA-box binding protein associated factor 15 (TAF15) translocate to SGs in response to genomic stress.^15,16 Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) translocates and fibrilizes when hyperphosphorylated, driving LLPS to form protein-rich droplets and contribute to SG formation.¹⁷

Upon cessation of cellular stress, ternary complexes are recruited to drive SG disassembly. Autophagic proteins facilitate disassembly via granulophagy, whereby autophagic vesicles envelop and break down SGs. DDX1 can help facilitate this process, though it is not required for it.¹¹ Sequestosome 1 (SQSTM1) promotes SG translocation to autophagic vesicles.^18,19 The ATPase valosin-containing protein (VCP) is activated through phosphorylation by unc-51 like autophagy activating kinase 1/2 (ULK1/2), which in turn promotes granulophagy of SGs.²⁰ PICs are reformed, eIF proteins are recruited, and translation resumes upon complete reassembly of the translational complex.

Selected Reviews:

1. Panas, M. D., Ivanov, P. & Anderson, P. Mechanistic insights into mammalian stress granule dynamics. Journal of Cell Biology. 2016; 215:313–323
2. Sonenberg, N. & Hinnebusch, A. G. Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets. Cell. 2009;136:731–745
3. Li, J., Zhang, Y., Chen, X., et al. Protein phase separation and its role in chromatin organization and diseases. Biomedicine & Pharmacotherapy. 2021;138:111520
4. Jain, S., Wheeler, J. R., Walters, R. W., et al. ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure. Cell. 2016;164:487–98
5. Kedersha, N., Cho, M. R., Li, W., et al. Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules. Journal of Cell Biology. 2000;151:1257–1268
6. Nonhoff, U., Ralser, M., Welzel, F., et al. Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules. Mol Biol Cell. 2007;18:1385–1396
7. Cirillo, L., Cieren, A., Barbieri, S.,et al. UBAP2L Forms Distinct Cores that Act in Nucleating Stress Granules Upstream of G3BP1. Curr Biol. 2020;30:698-707.e6
8. Matheny, T., van Treeck, B., Huynh, T. N., et al. RNA partitioning into stress granules is based on the summation of multiple interactions. RNA. 2021;27:174–189
9. Ozeki, K., Sugiyama, M., Akter, K. A.,et al. FAM98A is localized to stress granules and associates with multiple stress granule-localized proteins. Mol Cell Biochem. 2019; 451:107–115
10. Si, W., Li, Z., Huang, Z., et al. RNA Binding Protein Motif 3 Inhibits Oxygen-Glucose Deprivation/Reoxygenation-Induced Apoptosis Through Promoting Stress Granules Formation in PC12 Cells and Rat Primary Cortical Neurons. Front Cell Neurosci. 2020;14:287
11. Li, L., Garg, M., Wang, Y., et al. DEAD Box 1 (DDX1) protein binds to and protects cytoplasmic stress response mRNAs in cells exposed to oxidative stress. Journal of Biological Chemistry. 2022;298:102180
12. Kedersha, N., Panas, M. D., Achorn, C. A., et al. G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits. Journal of Cell Biology. 2016;212:845–860
13. Fu, Y. & Zhuang, X. m6A-binding YTHDF proteins promote stress granule formation. Nat Chem Biol. 2020;16:955–963
14. Besnard-Guérin, C. Cytoplasmic localization of amyotrophic lateral sclerosis-related TDP-43 proteins modulates stress granule formation. Eur J Neurosci. 2020;52:3995–4008
15. Blechingberg, J., Luo, Y., Bolund, L.,et al. Gene Expression Responses to FUS, EWS, and TAF15 Reduction and Stress Granule Sequestration Analyses Identifies FET-Protein Non-Redundant Functions. PLoS One. 2012;7:e46251
16. Sama, R. R. K., Ward, C. L., Kaushansky, L. J., et al. FUS/TLS assembles into stress granules and is a prosurvival factor during hyperosmolar stress. J Cell Physiol. 2013;228:2222–31
17. Molliex, A., Temirov, J., Lee, J., et al. Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization. Cell. 2015;163:123–133
18. Chitiprolu, M., Jagow, C., Tremblay, V., et al. A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy. Nature Communications. 2018;9:1–18.
19. Sun, D., Wu, R., Zheng, J., et al. Polyubiquitin chain-induced p62 phase separation drives autophagic cargo segregation. Cell Researc. 2018;28:405–415
20. Wang, B., Maxwell, B. A., Joo, J. H.,et al. ULK1 and ULK2 Regulate Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97. Mol Cell. 2019;74:742-757.e8