The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress

Meydan, Sezen; Barros, Géssica C.; Simões, Vanessa; Harley, Lana; Cizubu, Blanche K.; Guydosh, Nicholas R.; Silva, Gustavo M.

doi:10.1016/j.celrep.2023.113359

Cited by 11 publications

(18 citation statements)

References 77 publications

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“…We have previously determined that K63-ub that accumulates in response to oxidative stress modifies ribosomes and pauses translation at the elongation stage (5, 7, 10). Following oxidative stress induction, Ubp2 is required for ubiquitin reversal, however, it remains unclear how Ubp2 regulates translation through its deubiquitinating activity.…”

Section: Resultsmentioning

confidence: 99%

“…As several aspects of translation control can be regulated by ubiquitin (48), DUBs are known to play crucial roles in antagonizing these pathways through the modulation of cycles of ribosomal ubiquitination and deubiquitination. In the RTU, ubiquitination and the activity of Rad6 are required to pause translation and reprogram protein production under stress (10). The ribosomes-associated quality control pathway (RQC) is also controlled by the ubiquitination of ribosomal proteins and has been suggested to rely on the DUBs OTUD3 and USP21 (51).…”

Section: Discussionmentioning

confidence: 99%

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Redox control of the deubiquitinating enzyme Ubp2 regulates translation during stress

Santos,

Cizubu,

Okonkwo

et al. 2024

Preprint

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Protein ubiquitination is essential to govern cellular ability to cope with harmful environments by regulating many aspects of protein dynamics from synthesis to degradation. As important as the ubiquitination process, the reversal of ubiquitin chains mediated by deubiquitinating enzymes (DUBs) is critical for proper recovery from stress and re-establishment of proteostasis. Although it is known that ribosomes are decorated with K63-linked polyubiquitin (K63-ub) chains that control protein synthesis under stress, the mechanisms by which these ubiquitin chains are reversed and regulate proteostasis during stress recovery are still illusive. Here, we showed in budding yeast that the DUB Ubp2 is redox regulated during oxidative stress in a reversible manner, which determines the levels of K63-ub chains present on ribosomes. We also demonstrate that Ubp2 is a processive enzyme whose activity is modulated by a series of repeated domains and the formation of important disulfide bonds. By combining, cellular, biochemical, and proteomics analyses, we showed that Ubp2 is crucial for restoring translation after stress cessation, indicating an important role in determining cellular response to oxidative stress. Our work demonstrates a novel role for Ubp2, revealing that a range of signaling pathways can be controlled by redox regulation of DUB activity in eukaryotes, which in turn will define cellular states of health and diseases.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Redox control of the deubiquitinating enzyme Ubp2 regulates translation during stress

Santos,

Cizubu,

Okonkwo

et al. 2024

Preprint

Self Cite

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“…Tripeptide pause scores were computed as described previously ( Meydan et al, 2023 ). Briefly, this involved dividing the 80S reads per million (rpm) of a three-amino acid motif by the average rpm in the surrounding region (± 50 nt around each motif).…”

Section: Methodsmentioning

confidence: 99%

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo

Gaikwad,

Ghobakhlou,

Zhang

et al. 2024

eLife

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Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met- tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5’- untranslated region (5’ UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative IRES elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met- tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

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“…We sought to determine whether the RQC is induced in a stress-specific manner by testing the ability of the RQC to clear stalled ribosomes using an established reporter system (Matsuo et al, 2017). The inducible dual-luciferase reporter contains either a No-Stall control or 6xCGA polyarginine sequence inserted between Renilla luciferase (Rluc) and Firefly (Fluc) encoding regions (Fig 4C) (Meydan et al, 2023). Stretches of positively charged peptide sequences, especially those encoded by rare codons like CGA, are known to induce ribosome stalling (Barros et al, 2021;Chen et al, 2024;Matsuo et al, 2017).…”

Section: Sensitivity Of the Rqc And Rtu Pathways To Distinct Stressorsmentioning

confidence: 99%

“…K63 polyubiquitin differs from the more common K48-linked polyubiquitin as it does not signal the target for proteasomal degradation (Manohar et al, 2019), but instead has been shown to serve as a regulatory signal in functions such as DNA repair, intercellular trafficking, and immune signaling pathways (Erpapazoglou et al, 2014;Liu et al, 2018;Madiraju et al, 2022). Recent findings suggest Rad6mediated ubiquitination plays a role in mediating ribosome pausing on XIP motifs and contributes to mechanisms of translation repression, but the connection between ribosome ubiquitination and translation control remains unclear (Meydan et al, 2023).…”

Section: Introductionmentioning

confidence: 99%

Context specific ubiquitin modification of ribosomes regulates translation under oxidative stress

Dougherty,

Barros,

Foster

et al. 2024

Preprint

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Cellular exposure to oxidative stress is known to activate several translational control pathways through ribosome ubiquitination. Two such pathways, Redox-control of translation by ubiquitin (RTU) and Ribosome-associated quality control (RQC), modify the ribosome with K63-linked polyubiquitination, but result in two different ribosome fates. RTU responds to peroxide stress exposure by inducing a burst or ribosome polyubiquitination and subsequent pause of translational elongation. Alternatively, RQC leads to ubiquitination of already stalled ribosomes and mediates their clearance through subunit dissociation. Understanding how site-specific ribosome ubiquitination induces translation regulation is difficult due to the simultaneous occurrence of these distinct translational control pathways. Here we develop a targeted proteomics approach to quantify site-specific ubiquitin modification across the ribosome in steady state and stress conditions. We found several sites to be differentially ubiquitinated due to stress, including sites known to be targeted by the RQC. The results indicate that the RTU and RQC target distinct ribosome subpopulations within the cell, and differentially contribute to the cellular stress response in an oxidative stressor-specific manner. These findings significantly contribute to the dissection of the complex coordination of translation in response to stress and shed light on the integration of important quality control pathways during cellular response to stress

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The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress

Cited by 11 publications

References 77 publications

Redox control of the deubiquitinating enzyme Ubp2 regulates translation during stress

Redox control of the deubiquitinating enzyme Ubp2 regulates translation during stress

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo

Context specific ubiquitin modification of ribosomes regulates translation under oxidative stress

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