Stress granule homeostasis is modulated by TRIM21-mediated ubiquitination of G3BP1 and autophagy-dependent elimination of stress granules

Autophagy. 2023 Jul;19(7):1934-1951. doi: 10.1080/15548627.2022.2164427. Epub 2023 Jan 24.


Eukaryotic stress granules (SGs) are highly dynamic assemblies of untranslated mRNAs and proteins that form through liquid-liquid phase separation (LLPS) under cellular stress. SG formation and elimination process is a conserved cellular strategy to promote cell survival, although the precise regulation of this process is poorly understood. Here, we screened six E3 ubiquitin ligases present in SGs and identified TRIM21 (tripartite motif containing 21) as a central regulator of SG homeostasis that is highly enriched in SGs of cells under arsenite-induced oxidative stress. Knockdown of TRIM21 promotes SG formation whereas overexpression of TRIM21 inhibits the formation of physiological and pathological SGs associated with neurodegenerative diseases. TRIM21 catalyzes K63-linked ubiquitination of the SG core protein, G3BP1 (G3BP stress granule assembly factor 1), and G3BP1 ubiquitination can effectively inhibit LLPS, in vitro. Recent reports suggested the involvement of macroautophagy/autophagy, as a stress response pathway, in the regulation of SG homeostasis. We systematically investigated well-defined autophagy receptors and identified SQSTM1/p62 (sequestosome 1) and CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2) as the primary receptors that directly interact with G3BP1 during arsenite-induced stress. Endogenous SQSTM1 and CALCOCO2 localize to the periphery of SGs under oxidative stress and mediate SG elimination, as single knockout of each receptor causes accumulation of physiological and pathological SGs. Collectively, our study broadens the understanding in the regulation of SG homeostasis by showing that TRIM21 and autophagy receptors modulate SG formation and elimination respectively, suggesting the possibility of clinical targeting of these molecules in therapeutic strategies for neurodegenerative diseases.Abbreviations: ACTB: actin beta; ALS: amyotrophic lateral sclerosis; BafA1: bafilomycin A1; BECN1: beclin 1; C9orf72: C9orf72-SMCR8 complex subunit; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; Co-IP: co-immunoprecipitation; DAPI: 4',6-diamidino-2-phenylindole; FTD: frontotemporal dementia; FUS: FUS RNA binding protein; G3BP1: G3BP stress granule assembly factor 1; GFP: green fluorescent protein; LLPS: liquid-liquid phase separation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NBR1: NBR1 autophagy cargo receptor; NES: nuclear export signal; OPTN: optineurin; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; SG: stress granule; TAX1BP1: Tax1 binding protein 1; TOLLIP: toll interacting protein; TRIM21: tripartite motif containing 21; TRIM56: tripartite motif containing 56; UB: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Keywords: Autophagy receptor; CALCOCO2; G3BP1; SQSTM1; TRIM21; stress granule; ubiquitination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis Regulatory Proteins / metabolism
  • Arsenites* / metabolism
  • Arsenites* / toxicity
  • Autophagy / physiology
  • C9orf72 Protein / genetics
  • Calcium / metabolism
  • Carrier Proteins / metabolism
  • DNA Helicases* / metabolism
  • Homeostasis
  • Poly-ADP-Ribose Binding Proteins / genetics
  • Poly-ADP-Ribose Binding Proteins / metabolism
  • RNA Helicases / metabolism
  • RNA Recognition Motif Proteins / genetics
  • RNA Recognition Motif Proteins / metabolism
  • Sequestosome-1 Protein / metabolism
  • Stress Granules
  • Ubiquitination
  • Ubiquitins / metabolism


  • Sequestosome-1 Protein
  • DNA Helicases
  • arsenite
  • Arsenites
  • C9orf72 Protein
  • Calcium
  • RNA Helicases
  • RNA Recognition Motif Proteins
  • Poly-ADP-Ribose Binding Proteins
  • Carrier Proteins
  • Apoptosis Regulatory Proteins
  • Ubiquitins

Grants and funding

This work was supported by the National Natural Science Foundation of China [32070697 to Y.F.L.]; the Young Scientists Fund of the National Natural Science Foundation of China [32100624 to Y.J.K]; National Natural Science Foundation of China [31770831 to Y.F.L.]; National Natural Science Foundation of China [31670756 to Y.B.]; ShanghaiTech University start-up fund [2015F0202-000 to Y.F.L.].