The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress

Abstract

Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase, Rad6. How Rad6-mediated ubiquitination of ribosomes affects global translation, however, is unclear. We therefore performed Ribo-seq and Disome-seq in Saccharomyces cerevisiae, and found that oxidative stress caused ribosome pausing at specific amino acid motifs, and this also led to ribosome collisions. However, these redox pausing signatures were lost in the absence of Rad6 but did not depend on the ribosome-associated quality control (RQC) pathway. We also found that Rad6 is needed to inhibit overall translation in response to oxidative stress and its deletion leads to increased expression of antioxidant genes. Finally, we observed that the lack of Rad6 leads to changes during translation initiation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation.

Context specific ubiquitin modification of ribosomes regulates translation under oxidative stress

“…First, we showed that exposure to either H 2 O 2 or 4-NQO show decreased translation rate compared to untreated rates in wild-type cells (Fig 5A), with a 10-fold decrease in H 2 O 2 , a 4-fold decrease in the lower concentration of 4-NQO, and a more than 20-fold decrease in the higher concentration of 4-NQO. In alignment with our previous findings (Meydan et al ., 2023), H 2 O 2 -induced suppression of active translation was dependent only on Rad6 expression (Fig 5A). Under low concentration of 4-NQO (In 1 μg/mL), deletion of RAD6 had no significant effect on translation suppression, corroborating our findings that the RTU is not induced by 4-NOQ.…”

“…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).…”

“…In yeast, the ISR pathway blocks translation initiation through phosphorylation of the eIF2α translation factor (P-eIF2α) by Gcn2, which decreases the level of competent ternary complex while simultaneously inducing translation of the Gcn4 transcription factor (Pakos-Zebrucka et al, 2016). Previous work by our lab determined loss of the RTU factor, Rad6 decreases dysregulates eIF2α phosphorylation in peroxide stress (Meydan et al, 2023). Additionally, deletion of the RQC factor HEL2 was reported to further activate eIF2α phosphorylation in steady state and MMS stress (Meydan & Guydosh, 2020;Yan & Zaher, 2021).…”

“…To investigate, we mined pre-existing RNA-seq data to quantify differential expression of Gcn4 target transcripts due to different stress and pathway control. Previous work by the lab identified higher expression of Gcn4-target transcripts in H2O2 stress in rad6Δ compared to WT cells (Meydan et al, 2023). While we were not able find RNAseq data for cells treated with 4-NQO stress, we utilized RNAseq data from cells treated with MMS stress in hel2Δ and WT cells as a proxy for RQC induction through mRNA damage (Yan & Zaher, 2021).…”

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

“…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).…”