Comparative profiling of stress granule clearance reveals differential contributions of the ubiquitin system

Abstract

Stress granules (SGs) are cytoplasmic condensates containing untranslated mRNP complexes. They are induced by various proteotoxic conditions such as heat, oxidative, and osmotic stress. SGs are believed to protect mRNPs from degradation and to enable cells to rapidly resume translation when stress conditions subside. SG dynamics are controlled by various posttranslational modifications, but the role of the ubiquitin system has remained controversial. Here, we present a comparative analysis addressing the involvement of the ubiquitin system in SG clearance. Using high-resolution immunofluorescence microscopy, we found that ubiquitin associated to varying extent with SGs induced by heat, arsenite, H2O2, sorbitol, or combined puromycin and Hsp70 inhibitor treatment. SG-associated ubiquitin species included K48- and K63-linked conjugates, whereas free ubiquitin was not significantly enriched. Inhibition of the ubiquitin activating enzyme, deubiquitylating enzymes, the 26S proteasome and p97/VCP impaired the clearance of arsenite- and heat-induced SGs, whereas SGs induced by other stress conditions were little affected. Our data underline the differential involvement of the ubiquitin system in SG clearance, a process important to prevent the formation of disease-linked aberrant SGs.

Deubiquitinating enzymes (DUBs): Regulation, homeostasis, and oxidative stress response

“…Stress granules are large aggregate structures of RNA and protein that form as a result of several cellular stresses to regulate translation and to sequester and protect cytosolic proteins from the potentially damaging effects of stress ( 220 ). While ubiquitination is dispensable for their formation, ubiquitin can be found at the surface of stress granules, and regulation of ubiquitin signaling is required for efficient stress granule clearance during stress resolution ( 221 ). Redox-dependent inhibition of DUBs causes an increase in size and stability of stress granules, but during recovery from stress, DUBs are reactivated and remove the ubiquitin signaling from the stress granules, causing them to fragment and dissolve ( 221 ).…”

“…While ubiquitination is dispensable for their formation, ubiquitin can be found at the surface of stress granules, and regulation of ubiquitin signaling is required for efficient stress granule clearance during stress resolution ( 221 ). Redox-dependent inhibition of DUBs causes an increase in size and stability of stress granules, but during recovery from stress, DUBs are reactivated and remove the ubiquitin signaling from the stress granules, causing them to fragment and dissolve ( 221 ). Addition of DUB inhibitors during resolution of oxidative stress caused by sodium arsenite has been shown to increase the retention of stress granules significantly, with 75% of cells having stress granules present after 2 h of recovery, as opposed to 10% of cells in control samples ( 221 ).…”

“…Redox-dependent inhibition of DUBs causes an increase in size and stability of stress granules, but during recovery from stress, DUBs are reactivated and remove the ubiquitin signaling from the stress granules, causing them to fragment and dissolve ( 221 ). Addition of DUB inhibitors during resolution of oxidative stress caused by sodium arsenite has been shown to increase the retention of stress granules significantly, with 75% of cells having stress granules present after 2 h of recovery, as opposed to 10% of cells in control samples ( 221 ). This emphasizes the importance of DUB activity during the recovery from oxidative stress, particularly as redox regulation of the DUBs themselves can therefore determine the duration of stress granule retention, depending on the speed of DUB oxidation reversal.…”

SUMO: Glue or Solvent for Phase-Separated Ribonucleoprotein Complexes and Molecular Condensates?

“…The mRNAs stored in SGs can be either directed toward mRNA decay or their translation can be reinitiated when stress is released and SGs disassemble. The mechanism of SG formation and dissolution are still not fully understood, but there is accumulating evidence that post-translational modifications contribute to these processes (Turakhiya et al, 2018;Hofweber and Dormann, 2019;Hofmann et al, 2021;Tolay and Buchberger, 2021). Recent independent findings by the Hornstein and Müller groups suggest that the SUMO system and the StUbL pathway are critically involved in both assembly and dissolution of SGs (Keiten-Schmitz et al, 2020;Marmor-Kollet et al, 2020).…”

Melatonin: Regulation of Biomolecular Condensates in Neurodegenerative Disorders

“…SG components include RBPs such as TDP-43, FUS, tau, and p53; therefore, mutations and aberrant SG dynamics may significantly contribute to neurodegenerative disorders [ 540 , 837 , 914 ]. Both ubiquitination and SUMOylation have been reported to regulate SG dynamics, where the ubiquitination–protease system [ 915 ] and SUMO-primed ubiquitination facilitate the timely resolution and disassembly of SG upon stress release, preventing aberrant SGs that may result in disease-linked pathological aggregates [ 915 , 916 ]. Consequently, failure to SUMOylate eIF4A2 (or DDX2B), a DEAD-box RNA helicase that acts as a scaffolding protein, impairs stress granule formation [ 917 ].…”