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The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress

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. HIGHLIGHTS Rad6 is required for sequence-specific ribosome pausing under oxidative stress. Rad6 affects translation independently of the RQC pathway. Cells lacking Rad6 show dysregulated translational repression upon oxidative stress. Loss of Rad6 leads to altered activation of the ISR pathway.


INTRODUCTION
Eukaryotic organisms frequently encounter harmful environmental conditions throughout the course of their lifetime. In these environments, the timely and precise regulation of gene expression is essential to support cellular stress defense, adaptation, and survival (Advani and Ivanov, 2019). Cellular stress caused by the accumulation of reactive oxygen species (ROS) affects important biomolecules such as nucleic acids, proteins, and lipids, and is associated with several pathologies, such as cancer, and cardiovascular and neurodegenerative diseases (Finkel and Holbrook, 2000;Liguori et al., 2018;Sharifi-Rad et al., 2020). ROS, including hydrogen peroxide (H2O2), can form as a result of metabolic processes but also from exposure to a range of chemicals and pollutants (Picazo and Molin, 2021;Sharifi-Rad et al., 2020). Thus, oxidative stress occurs when ROS production overloads the cellular antioxidant defense. To prevent the detrimental effects of ROS, cells evoke an intricate regulatory network of gene expression both at the transcriptional and translational level (Gasch et al., 2000;Gerashchenko et al., 2012;Picazo and Molin, 2021;Shenton et al., 2006). Although the transcriptional response to oxidative stress has been more extensively studied (Beckhouse et al., 2008;Gasch et al., 2000;Levings et al., 2021;Ma, 2010;Marguerat et al., 2014), a genomewide understanding of how cells regulate translation in response to oxidative stress is only beginning to be elucidated (Gerashchenko and Gladyshev, 2014;Gerashchenko et al., 2012;Marguerat et al., 2014;Rubio et al., 2021;Shenton et al., 2006).
In response to oxidative stress, eukaryotic cells activate a series of pathways that reprogram translation by shutting down protein production globally while favoring the translation of essential proteins for cell survival (Picazo and Molin, 2021). Some of these pathways are determined by the levels and availability of translation factors or dictated by cis regulatory mRNA sequences (Grant, 2011;Picazo and Molin, 2021). However, multiple steps of translation (initiation, elongation, and termination) can be controlled under stress (Grant, 2011), increasing the complexity of the system. Although an extensive number of studies have focused on the regulation of translation initiation (Bresson et al., 2020;Harding et al., 2000;Shenton et al., 2006), mechanisms of elongation regulation during stress are still not well understood.
We previously discovered in the budding yeast S. cerevisiae a new mechanism responsible for controlling translation elongation during oxidative stress via ubiquitination of ribosomes (Back et al., 2019;Silva et al., 2015;Zhou et al., 2020). We named this pathway redox control of translation by ubiquitin (RTU) . A key regulator of the RTU is the E2 ubiquitin conjugase Rad6 that rapidly modifies ribosomal proteins with K63-linked polyubiquitin chains in response to H2O2 (Back et al., 2019;Silva et al., 2015;Simoes et al., 2022;Zhou et al., 2020). Ubiquitinated ribosomes arrest at the pre-translocation stage of translation elongation (Zhou et al., 2020) and are proposed to participate in the shutdown of protein production during stress (Back et al., 2019;Zhou et al., 2020). Furthermore, we recently showed that deletion of RAD6 prevents K63-linked ubiquitination of ribosomes, and leads to continued protein production under oxidative stress and dysregulated levels of antioxidant proteins (Simoes et al., 2022). Rad6 is a multifunctional and highly conserved protein, in which mutations to its human homolog UBE2A are associated with the X-linked intellectual disability type Nascimento (Bruinsma et al., 2016;Nascimento et al., 2006). However, an understanding of the means by which Rad6 controls the translational landscape by modifying ribosomes and the crosstalk of the RTU with other pathways of translation control remains elusive.
To characterize the translational landscape mediated by Rad6 we made use of next generation sequencing of ribosome protected mRNA fragments, also known as Riboseq or ribosome profiling. Ribo-seq has uncovered many new aspects of translation dynamics and regulation in a diverse range of organisms and conditions (Ingolia et al., 2009;Ingolia et al., 2019). Moreover, we recently improved our disome profiling (Disomeseq) approach to allow us to identify and quantify the endogenous mRNA sequences occupied by collided ribosomes (disomes) in yeast (Meydan and Guydosh, 2020). Using this method, we were able to show that collisions are widespread events connected with quality control and stress response pathways (Meydan and Guydosh, 2020). We also showed that although most collisions do not activate mRNA decay pathways, they may have an important signaling role in co-translational events (Meydan and Guydosh, 2020).
Here, we used both Ribo-seq and Disome-seq combined with RNA-seq to understand the role of Rad6 in translational control during stress. We found that upon hydrogen peroxide treatment, ribosomes from wild-type (WT) cells are preferentially paused on isoleucine-proline enriched sequences. Surprisingly, this redox pausing signature is largely abolished upon deletion of RAD6. Furthermore, we showed that the RTU functions independently of the RQC pathway, which is known for detecting and rescuing collided ribosomes. The RQC also relies on K63-linked ubiquitination of ribosomes but deletion of its main E3 ubiquitin ligase HEL2 did not abolish the burst of K63-linked ubiquitination, or the translation pause signature during stress. Finally, we showed that lack of Rad6 affects translation rates and activates additional translation programs, including the ISR through a non-canonical mechanism. Therefore, this study uncovers a novel mechanism of translational control and positions Rad6 as new remodeler of the translation landscape through a selective ribosome pausing mechanism.

Rad6 is required for redox pausing of ribosomes
Rad6-mediated ubiquitination was suggested to affect translation during oxidative stress by arresting translation elongation at the pre-translocation stage (Back et al., 2019;Zhou et al., 2020). To further understand the impact of Rad6-mediated ubiquitination on ribosome pausing at a genome-wide level, we conducted Ribo-seq experiments in wildtype (WT) and rad6 cells incubated with 0.6 mM hydrogen peroxide (H2O2, "peroxide" hereafter) for 30 min ( Figure 1A and S1A) (Ingolia et al., 2009;McGlincy and Ingolia, 2017). This peroxide concentration and the treatment time were optimized based on the peak accumulation of K63-linked polyubiquitin chains after the addition of peroxide to the media (Silva et al., 2015). Supporting the establishment of our system, RNA-seq experiments showed that the peroxide treatment resulted in significantly upregulated expression of genes involved in the oxidative stress response in both WT and rad6 cells ( Figures 1B-C). These results show that both strains respond to this peroxide treatment, allowing us to expand our analysis of the stress response to the translational level.
To understand the effect of Rad6 on translation elongation and to globally quantify ribosome pausing differences, we computed "average pause scores" for every combination of tri-amino acid motif ( Figure 1D and Methods). First, we observed that amino-acid sequences such as PPD, PPE and RKK caused the strongest pausing in untreated WT cells ( Figure 1E, left panel). However, the relative level of pausing at these sequences was not as high as in stressed cells. Instead, we found that peroxide treatment in WT cells caused reprogramming of ribosome arrest and resulted in elevated pausing at specific sequence motifs, which we define as "redox pausing" ( Figure 1E, left panel).
These redox pausing signatures were enriched in sites that have proline (Pro) and histidine (His) codons at the ribosomal A-site. We also observed that stalling at isoleucine (Ile) codons at the ribosomal P-sites increased upon peroxide treatment, especially in combination with A-site Pro codons (Figures 1E-F). Since we did not detect substantial enrichment for residues that specifically mapped to the E-site (i.e. the amino acid corresponding to the penultimate, C-terminal position of the nascent peptide), we designated this stalling motif as "XIP", where X refers to any amino acid composition.
Given prior evidence that Rad6-mediated ubiquitination could modulate translation elongation (Simoes et al., 2022), we hypothesized that loss of accumulated ubiquitination by Rad6 under oxidative stress could affect ribosome stalling. To test this hypothesis, we performed our pausing analysis in rad6 cells. Strikingly, we found that redox pausing signatures were lost in cells lacking Rad6 ( Figure 1E, right panel). The previously identified XIP redox pausing motifs were the most susceptible to the loss of Rad6 ( Figure   1F). Consistently, analysis of individual or averaged ribosome occupancy at XIP sites also revealed considerable loss of redox pausing in the absence of Rad6 ( Figures 1G-H). In the untreated cells, ribosome stalling signatures looked similar between the two strains except for increased stalling in rad6 cells at A-site Trp codons, a result that is specific to the SUB280 background used here ( Figure S1B, see below for further discussion). As expected, plots of average ribosome occupancy centered at XIP motifs were consistent with the pause score analysis and revealed a peroxide-induced stalling peak that was absent in untreated WT cells and in the rad6 strain ( Figure 1I, top and bottom panel, respectively).
Upon arresting at XIP motifs, ribosomes could resume translation or be rescued by quality control systems to make them available for new rounds of translation. To determine whether these XIP motifs lead to ribosome rescue, we developed an inducible dual luciferase reporter, in which we can insert sequences between Renilla Luciferase (Rluc) and Firefly Luciferase (Fluc) coding regions that are expected to stall translation and lead to ribosome rescue. As a proof of principle, we determined that several sequences identified with a high pause score in our Ribo-seq data indeed prevent the synthesis of Fluc ( Figure S1C). Because of the global translation repression that occurs under oxidative stress ( Figure S1D), this method did not allow us to measure dynamic changes in the Fluc/Rluc ratios during the short time window following peroxide treatment used in this study. However, we still observed a reduction in the Fluc/Rluc ratio when 3xKIP was inserted in the absence of peroxide, consistent with an interpretation that there could be a loss of ribosomes between Fluc and Rluc in WT cells due to rescue or drop off. This trend was unchanged in rad6 cells, suggesting that Rad6 does not promote ribosome rescue (Figure 2A). Because oxidative stress induced by peroxide was previously associated with increased translation of 5 and 3 untranslated regions (UTRs) (Gerashchenko and Gladyshev, 2014;Gerashchenko et al., 2012), we also tested whether Rad6 impacts these translation events outside of main open reading frames. Metagene analysis, performed by averaging data from genes aligned by their start or stop codons, showed modest changes in the occupancy of 5 and 3 UTRs with peroxide treatment in WT cells, as expected (Figures S1E-F). The absence of Rad6 did not affect these trends (Figures S1E-F), suggesting that Rad6 does not strongly impact translation of UTR regions under oxidative stress but mainly affects elongation in the main ORF.
To confirm our observation that Rad6 has a specific role in modulating redox pausing, we conducted additional Ribo-seq experiments in which we expressed both the WT Rad6 and the catalytically dead mutant Rad6 (Rad6 C88A ) in a rad6 background.
Expression of WT Rad6 in rad6 cells restored peroxide-induced stalling at the XIP motif and other sites, whereas the ubiquitination-deficient mutant Rad6 C88A did not (Figures 2B-C). These results suggest that Rad6 catalytic activity is essential to regulate redox pausing.
To confirm the generality of our results, we also considered the effect of the yeast strain background. The yeast strains used in this study (SUB280 background) were constructed to express a single ubiquitin gene episomally (Finley et al., 1994). To test whether the unique properties of this strain were related to the observed redox pausing signatures, we repeated experiments in the S288C background. Experiments in S288C did not show an increase in A-site pausing at Trp codons in the rad6 cells ( Figure S1B), suggesting that this particular effect is not a robust feature of redox pausing. However, they did recapitulate the previously observed redox pausing signatures at A-site Pro codons, including XIP motifs ( Figure 2D). These data therefore show that redox pausing is a consistent mechanism of translational control in response to stress and that Rad6 plays a key role in this translation phenotype.

Redox pausing signatures are not mediated by the RQC pathway
We next investigated whether Rad6 activity plays any role in the well-established RQC pathway. Stalled ribosomes can physically block upstream ribosomes from translating, resulting in the formation of a ribosome collision complex called a disome, where the two ribosomes interact. The RQC pathway is a cellular mechanism that detects disomes and promotes their removal from mRNAs. Therefore, we first evaluated whether peroxide treatment would produce disomes that could be rescued by the RQC. We, and others, have previously established the Disome-seq technique in yeast to find the genome-wide distribution of collided ribosomes in the cell (Guydosh and Green, 2014;Meydan and Guydosh, 2020;Zhao et al., 2021). Disome-seq in WT cells also showed evidence of redox pausing signatures, such as the XIP motif and generally mirrored our Ribo-seq data ( Figure S2A-B). This suggests that stalled ribosomes formed during oxidative stress also collide with each other.
Previous studies showed that an E3 ubiquitin ligase, Hel2, triggers the RQC pathway by ubiquitinating collided ribosomes stalled at positively charged amino acid sequences, such as poly-Arg or poly-Lys (Brandman et al., 2012;Houston et al., 2022;Ikeuchi et al., 2019;Matsuo et al., 2017;Matsuo et al., 2020). Ubiquitination leads to ribosome rescue but, in the absence of Hel2, collided ribosomes bypass these stallinducing sequences and continue translating. Since the RQC pathway regulates translation arrest via Hel2-mediated ubiquitination of ribosomes, it raises the question of whether the E3 Hel2 and E2 Rad6 cooperate in the same pathway of translational control.
To further investigate whether Hel2 would be involved in rescuing disomes formed in response to stress, we performed a Ribo-seq experiment in hel2 cells. During peroxide treatment, XIP redox pausing signatures were still present in cells lacking Hel2, which suggests a separation of functions ( Figure 3A). Consistent with the idea that Hel2 does not work with Rad6 to regulate translation under oxidative stress, we also showed that loss of Hel2 did not affect the burst of K63 ubiquitination induced by peroxide treatment ( Figure 3B), further supporting that Rad6 activity is independent of Hel2 and the RQC. To explore the activity of Hel2 and Rad6 in rescuing stalled ribosomes, we inserted a known RQC-targeted stalling sequence consisting of 6 consecutive Arg codons (6XCGA) into our Renilla-Firefly luciferase reporter for ribosome rescue. This sequence is particularly problematic for the ribosome to translate due to I-C wobble codon-anticodon pairing (Letzring et al., 2013). Ribosomes stalled at 6XCGA are known to be rescued by RQC (Letzring et al., 2013), and as expected, we observed more ribosomes bypassing this stall-inducing site in the absence of Hel2 ( Figures 3C and S2C). However, deletion of RAD6 did not result in a significant increase in Fluc/Rluc signal of the 6XCGA reporter ( Figure 3C and S2C). These results suggest that Rad6 does not influence ribosome stalling and rescue in the same way as Hel2. Collectively, our findings further indicate that Hel2 operates on a subpopulation of arrested ribosomes that likely does not include those (i.e. XIP motifs) that are enhanced by oxidative stress.

Rad6 is required for translational repression during oxidative stress
Having established that deletion of Rad6 affects ribosome stalling in a way that is different from Hel2, we further explored mechanisms that could be responsible for the redox-induced pausing of ribosomes. We first hypothesized that differential availability of tRNAs could stall ribosomes at XIP motifs during peroxide treatment. It was previously shown that peroxide treatment causes degradation of Pro-tRNA AGG , resulting in a ribosome stalled with an empty A-site as it waits for binding of prolyl-tRNA (Wu et al., 2019). One possibility for the loss of redox pausing in rad6 cells could be that Rad6 indirectly or directly mediates the degradation of prolyl-tRNAs. Loss of Rad6 then might stabilize prolyl-tRNAs and thereby alleviate ribosome stalling at Pro codons. To test this possibility, we conducted northern blotting experiments with a probe that matched Pro-tRNA AGG . We found that the peroxide concentration that we used for our Ribo-seq, Disome-seq and RNA-seq experiments (0.6 mM) did not result in tRNA degradation ( Figure 4A, lane 2). Only at higher concentrations of peroxide (9.8 mM, as used in (Wu et al., 2019), and 98 mM), we were able to detect the appearance of a tRNA fragment ( Figure 4A, lanes 3-4, indicated by an arrow). We also observed that the magnitude and concentration dependence of prolyl-tRNA degradation induced by peroxide was the same in the rad6 strain ( Figure 4A, lanes 5-8), further suggesting that loss of redox pausing in the absence of Rad6 is not due to changes in tRNA stability.
We next assessed the overall rate of translation in rad6 cells to see if the absence of redox pausing was related to the cell s ability to make proteins. We evaluated changes in translation rates by incorporation of a methionine analog, Homopropargylglycine (HPG). This assay captures the totality of all effects on translation, including changes to both initiation and elongation, and provides an opportunity to evaluate mechanistic models. One possibility is that in the absence of Rad6, ribosomes would no longer undergo redox pausing and could therefore generate more protein during stress. Consistent with this model, the drop in the rate of translation in rad6 cells due to oxidative stress was significantly less than in WT cells ( Figures 4B-C, S3A). While translation in WT cells was severely inhibited by peroxide treatment, cells lacking Rad6 were significantly less sensitive to this translation repression. In addition, the effect of Rad6 on peroxide-induced translational repression was reproducible in the S288C background ( Figure S3B-C). These findings are also in agreement with previous data showing higher puromycin incorporation in rad6 cells in the presence of peroxide compared to WT cells (Simoes et al., 2022). Overall, our results show that Rad6 is necessary for the oxidative stress-induced translational repression.

Rad6 activity induces eIF2α phosphorylation
To understand the physiological impact of dysregulated translation in the absence of Rad6, we next explored by RNA-seq how peroxide affects the transcriptome in WT and rad6 cells. In the absence of oxidative stress (untreated), rad6 cells had significantly upregulated expression of multiple genes involved in metabolic processes (such as GDB1, GPH1, PKP1, DAK2), the heat shock response (such as HSP26, HSP30, HSP78), and oxidative stress response (such as GRX1, SOD2, TSA2, PRX1) compared to WT cells ( Figure S4A). This suggests that even without oxidative stress, the lack of Rad6 causes a mild stress response. We next looked at the gene expression changes upon peroxide treatment. In rad6 cells, we found that the expression of oxidative stress response genes is upregulated beyond that found in the equivalently treated WT cells (Figures 1B,5A and S4B), consistent with the previous observation of increased ROS in rad6 cells under peroxide (Simoes et al., 2022). When we limited our analysis to 21 genes coding for known antioxidant enzymes, we observed overactivated expression of these redox genes in rad6 cells in both untreated and peroxide-treated samples ( Figures   5B and S4B). In addition, there were fewer ribosomal protein transcripts in rad6 cells, and this downregulation was exacerbated with peroxide treatment ( Figure S4C).
Decreased level of ribosomal protein transcripts is a hallmark of TOR inactivation, which is likely being driven by ROS in these cells (Picazo and Molin, 2021). We also observed that the translation efficiency of redox genes (ribosome footprints per transcript) was significantly increased by loss of Rad6 in peroxide-treated cells ( Figure S4D). These results support a model that in the absence of Rad6, the increased ROS in the cell drives a distinct response at the RNA and translational level.
Since rad6 cells seem to display a higher basal level of stress, we reasoned that loss of Rad6 could lead to the specific activation of the ISR pathway (also known as general amino acid control pathway in yeast) that is known to be induced by many stresses, including peroxide treatment (Grant, 2011;Picazo and Molin, 2021). Oxidative stress-induced ISR results in phosphorylation of eIF2 (eIF2 -P) by the Gcn2 kinase and its coactivators Gcn1 and Gcn20 (Mascarenhas et al., 2008). This leads to repression of overall translation while activating the transcription of stress response genes. In WT cells, we observed the expected induction of the ISR (increased eIF2 -P) during peroxide treatment, reaching its maximum level at 0.6 mM ( Figure 5C). Surprisingly, however, phosphorylation of eIF2 in rad6 cells remained low in response to oxidative stress (Figures 5C and S4E,and Discussion). Expression of Rad6 WT , but not its ubiquitination deficient mutant (Rad6 C88S ), in rad6 cells restored eIF2 -P at 0.6 mM peroxide ( Figure   5D). Even longer incubation times with peroxide did not result in increased levels of eIF2 -P in rad6 cells expressing the mutant Rad6 C88S ( Figure 5E). These observations could be explained by a model where lack of Rad6 affects the expression of the ISR machinery and thereby impairs regulation of eIF2 phosphorylation. To test this possibility, we checked if the components of the ISR machinery, Gcn1, Gcn2 and Gcn20, are properly expressed in the absence of Rad6. The Ribo-seq and RNA-seq data showed no difference in RNA levels and translation efficiency (TE) of the genes coding for Gcn1/2/20 proteins ( Figure S4F), suggesting that the absence of Rad6 does not directly affect the expression of ISR machinery components. Another possibility is that loss of Rad6 activates the TOR pathway, which in turn inhibits Gcn2 activity (Cherkasova and Hinnebusch, 2003;Kubota et al., 2003). However, our RNA-seq data showed that the TOR pathway is likely inhibited in rad6 cells ( Figure S4C), suggesting that loss of ISR activation in rad6 cells is not due to TOR activation.

Lack of Rad6 confers constitutive GCN4 translation
Since we observed a lack of peroxide-induced eIF2 -P in rad6 cells ( Figure 5C), we expected the ISR, and its associated effects on the translation of the GCN4 gene, would not occur. However, we noticed that translation of the GCN4 gene was increased in this strain (while transcript level remained constant) and this was true both in the presence and absence of peroxide ( Figure 6A). This is an unexpected effect because the accumulation of eIF2 -P is the typical driver of increased translation of the GCN4 mRNA, which encodes a transcription factor that upregulates many ISR genes (Hinnebusch, 2005).
We therefore explored alternative mechanisms that could increase translation of GCN4. The GCN4 gene has 4 upstream open reading frames (uORFs) ( Figure 6B). After translating uORF1, many ribosomes are not fully recycled and the 40S subunits remain on the mRNA (Hinnebusch, 2005). These 40S subunits then resume scanning and rebind the ternary complex (eIF2-GTP-tRNAi-Met), which allows them to reinitiate translation at uORF2, uORF3, or uORF4. Termination and recycling after these uORFs are generally more efficient, which prevents the ribosomes from reinitiating again and translating the GCN4 main ORF. During stress conditions, eIF2 -P reduces ternary complex (TC) levels, and thereby increases the odds that 40S subunits bypass uORF2, uORF3 and uORF4 and instead reinitiate translation at the GCN4 main ORF. In addition, leaky scanning, where ribosomes skip the uORFs and instead initiate at the downstream GCN4 main ORF, or other initiation defects can also lead to increased GCN4 translation. Since eIF2 is not phosphorylated in rad6 cells with oxidative stress, the observed high translation efficiency of GCN4 could be due either due to initiation defects or leaky scanning. To monitor GCN4 main ORF expression in relationship to its uORFs, we used a GCN4-lacZ reporter assay, which includes the natural context of GCN4 with all 4 uORFs ( Figure 6B).
Consistent with Ribo-seq and RNA-seq experiments ( Figure 6A), the GCN4-lacZ reporter showed that rad6 cells have higher Gcn4 levels compared to WT cells ( Figure 6B, left bar chart). As a positive control for our reporter, we treated the cells with 3-Amino-1,2,4triazole (3-AT), which mimics amino acid starvation by inhibiting histidine (His) biosynthesis. 3-AT increases ribosome stalling at His codons and thereby activates Gcn2, leading to phosphorylation of eIF2 and higher Gcn4 translation (Klopotowski and Wiater, 1965). Expectedly, the GCN4-lacZ reporter showed increased expression upon 3-AT treatment in both WT and rad6 , but not gcn2 cells ( Figure S5A). As a separate control, we also included a construct with only uORF1, which exhibited increased expression of GCN4 for both strains, due to the lack of uORF2-4 that would otherwise repress GCN4 translation (Fig. 6B, middle bar chart, note change in axis). To test if the translation of GCN4 is activated by leaky scanning in rad6 cells, we used another reporter where uORF1 is repositioned downstream and extended to overlap with the beginning of the GCN4 main ORF. Under normal conditions, uORF1 should prevent scanning ribosomes from reaching the GCN4 main ORF. The only way scanning ribosomes could reach the GCN4 main ORF would be if they scanned past uORF1 via "leaky scanning." We did not see any difference in GCN4-lacZ expression from the uORF1-extended reporter between WT and rad6 cells ( Figure 6B, right bar chart), suggesting that lack of Rad6 does not increase leaky scanning.
Mutations that cause GCN4 translation to become constitutively active lead to a phenotype traditionally referred to as "Gcd -" (Hinnebusch, 2005). This phenotype, which describes the rad6 cells, could occur when TC assembly is impaired (lowering the availability of TC) or when the rate of TC re-loading onto 40S subunits after uORF1 is reduced. Lower overall translation level (Figures 4B and S3A) in rad6 cells in the absence of peroxide is indeed consistent with an interpretation where TC availability is low, which also results in constitutive translation of GCN4. One explanation for reduced TC availability would be lower expression of genes that maintain the TC. While we did not notice a change in the expression of these "Gcd" genes in our RNA-seq and Ribo-seq data ( Figure S5B), other genes or other mechanisms (such as proteolysis) could mediate this effect.
Our data suggested this activation of GCN4 translation in the absence of eIF2 -P may also be true in the presence of peroxide. We showed that rad6 cells that are peroxide-treated had a lower level of eIF2 -P compared with similarly treated WT cells ( Figure 5C). However, the translational efficiency of GCN4 was higher in these peroxidetreated rad6 cells (Figures 6A, pink bar higher than blue bar) and this was also true for the GCN4-lacZ reporter (S5C, gray bar with peroxide higher than black bar with peroxide).
Moreover, RNA-seq data showed that known Gcn4-target mRNAs (Rawal et al., 2018) were upregulated at the transcriptional level in rad6 cells upon peroxide treatment compared with WT cells ( Figure 6C). This indicates that the production of Gcn4 without eIF2 -P leads to the expected functional outcome. Our results therefore suggest a model where loss of ubiquitination by Rad6 causes dysregulation of eIF2 phosphorylation but that constitutive translation of GCN4 in rad6 cells offers some compensation. The interplay of these effects may lead to altered fitness of rad6 cells during oxidative stress.

DISCUSSION
Our work has revealed an oxidative stress response pathway that regulates translation through the E2 ubiquitin conjugase, Rad6. We found that Rad6 plays a key role in redox pausing of ribosomes as well as oxidative stress-induced translation inhibition and eIF2 phosphorylation, all contributing to the maintenance of cellular homeostasis during oxidative stress.
Cells induce complex regulatory programs in order to survive challenging conditions induced by oxidative stress. One such strategy is to inhibit global protein synthesis to direct cellular resources for the specific expression of survival genes.

Although inhibition of translation initiation by eIF2 phosphorylation plays a key role in
repressing translation during oxidative stress, previous data showed that even in the absence of Gcn2 (the sole eIF2 kinase in yeast), translation continues to be inhibited upon oxidative stress (Shenton et al., 2006). These data hinted that in addition to eIF2 -P, other pathways can also repress translation during and after initiation. We previously showed that Rad6-mediated K63-linked polyubiquitin chains change the conformation of the ribosome, and this conformation could block translation elongation (Zhou et al., 2020).
Consistent with this idea, we found that oxidative stress induced by peroxide treatment leads to redox pausing at specific sequence motifs, particularly XIP, and this was dependent on Rad6 (Figures 1E-I and 2B-D). In addition, the redox pausing signatures were lost in the cells that express the catalytically dead Rad6 (Figures 2B-C), showing that it is the ubiquitination event that influences redox pausing. Strikingly, Rad6 was also necessary for a substantial portion of translational repression that is induced by oxidative stress (Figures 4B-C and S3), further supporting a potential role for redox pausing in the inhibition of protein synthesis. Although the mechanisms of redox pausing are not entirely clear, we ruled out changes to tRNA stability due to peroxide treatment (Thompson et al., 2008;Wu et al., 2019) by showing that the peroxide concentration used herein does not lead to Pro-tRNA AGG degradation ( Figure 4A). Altered modifications or the aminoacylation status of tRNA could possibly underlie redox pausing signatures. In particular, oxidative stress is known to affect tRNA modifications (Gu et al., 2014) and these changes in tRNA modifications influence ribosome pausing at the corresponding codons. For example, a yeast tRNA-Leu modification (m 5 C) was shown to be promoted by peroxide treatment, which leads to biased translation of genes containing Leu codons (Chan et al., 2012). Therefore, it is possible that increased or decreased modification of other tRNAs by peroxide could contribute to the P-site pausing signatures, such as Ile, observed in our data. These pausing events could be exacerbated when combined with a poor peptidyl-transfer substrate at the A-site, such as Pro. Interestingly, Pro acts as a scavenger of ROS (Ben Rejeb et al., 2014;Liang et al., 2013;Szabados and Savoure, 2010;Takagi, 2008) and therefore overall Pro amino acid levels (and tRNA aminoacylation) may also be affected by oxidative stress. In S. pombe, peroxide treatment was shown to cause ribosome pausing and collisions at Trp codons due to decreased levels of charged Trp-tRNA (Rubio et al., 2021). It is therefore possible that lower levels of charged Pro-tRNA, either due to diminished levels of Pro amino acids or an aminoacylation defect, could contribute to the A-site Pro redox pausing that we observed.
At least two models could explain how redox pausing events are eliminated in the absence of Rad6: (1) Ribosome ubiquitination by Rad6 alters a step in the elongation cycle. For example, it could change the rate-limiting step so that the time spent stalled on redox pausing motifs no longer makes up a significant part of the translation cycle. Loss of Rad6 would therefore lead to slower elongation under oxidative stress without any strong sequence-specific signature. However, this seems unlikely since the translation rate is faster, rather than slower, than expected (Figures 4B-C and S3) in rad6 cells under oxidative stress. Alternatively, Rad6-mediated ubiquitination could cause ribosome pausing and collisions during oxidative stress (i.e. at XIP motifs) and loss of Rad6 would eliminate these redox pausing events. Our findings support this second model, since translation is consistently faster in peroxide-treated rad6 cells compared to WT (Figures 4B-C and S3). (2) Rad6 could target transient redox pausing events (i.e. at XIP motifs) for ubiquitination, making them longer-lived and visible in our Ribo-seq data. We have shown that ribosomal ubiquitination during oxidative stress occurs independently of active translation (Back et al., 2019), supporting the fact that ribosomes paused at problematic sequences could be the preferred target of ubiquitination. Our study therefore establishes that Rad6-mediated ubiquitination affects elongation and offers models to guide future investigation of this phenomenon s effect in the cell.
Even in the absence of oxidative stress, Rad6 is strongly associated with polysomes (Simoes et al., 2022), suggesting that Rad6 constitutively binds to ribosomes and ubiquitinates them, consistent with the notion that redox inhibition of the deubiquitinating enzyme Ubp2 seems to drive ubiquitin chain accumulation (Silva et al., 2015). The fate of these ubiquitinated ribosomes, however, remains unclear. One possibility is that they are rescued, perhaps by proteins in the RQC pathway. However, our rescue-based reporter experiments showed that lack of Rad6 did not affect the Fluc/Rluc ratio in the reporter containing the redox pausing motif XIP (Figure 2A), suggesting that ubiquitination of ribosomes by Rad6 does not lead to rescue. This finding is also consistent with the previous results showing that K63-ubiquitinated ribosomes are detected in polysomes and are therefore engaged in translation (Silva et al., 2015). The other, and more plausible scenario is that ubiquitin marks are removed from the ribosome once the oxidative stress insult is no longer present. We favor this possibility since the deubiquitinase, Ubp2, removes K63 ubiquitin on the ribosome and the activity of this enzyme is mediated by peroxide levels in the cell (Silva et al., 2015).
Although lack of Hel2 did not affect redox pausing signatures and K63-linked ubiquitination ( Figures 3A-B), it is possible that oxidative stress induced by other ROS could lead to ribosome collisions that are targeted by RQC. For example, it was shown that the treatment of cells with 4-nitroquinoline 1-oxide (4-NQO), which is proposed to produce superoxide and hydroxyl radicals after enzymatic process (Arima et al., 2006), increases oxidation of guanines and leads to ribosome stalling that is detected by the RQC pathway (Yan et al., 2019). Therefore, different ROS, their modes of production, abundance, and subcellular location could potentially engage unique pathways of translation control mediated by Hel2 and Rad6 pathways.
In addition to RQC, we also examined the relationship of Rad6 with the ISR.
Peroxide was known to trigger the ISR and we and others have shown that the ISR can also be activated by ribosome collisions in yeast (Meydan and Guydosh, 2020;Yan and Zaher, 2021), which induces eIF2 phosphorylation. Our data in rad6 cells show that the ISR pathway is functional and can be activated by starvation of histidine ( Figure S5A).
Our observation that Rad6-mediated stalling is induced by peroxide treatment therefore offers an explanation for how oxidative stress leads to eIF2 phosphorylation. The loss of redox pausing in the absence of Rad6 may account for the lack of eIF2 phosphorylation. In combination with the loss of redox pausing, lower levels of oxidative stress-induced eIF2 -P could also help explain the lack of translational repression in peroxide-treated rad6 cells ( Figures 4B-C and S3). Interestingly, although the effect of Rad6 on redox pausing was reproducible in both SUB280 and S288C strains, we observed impaired induction of eIF2 phosphorylation by peroxide only in the SUB280 strain ( Figure S4E). This suggests that other cellular inputs contribute to eIF2 phosphorylation in addition to Rad6-mediated redox pausing and these other inputs could be more dominant in the S288C strain. Consistent with this idea that ISR regulation may vary somewhat between strains, it has been reported that Gcn4 translation is not activated by peroxide in the S288C strain (Picazo and Molin, 2021). In addition, S288C has a unique genetic background that affects mitochondria physiology and cellular redox biology (Young and Court, 2008), which could influence redox experiments in this strain.
Despite impaired eIF2 phosphorylation, Gcn4 is still constitutively translated ( Figure 6) in rad6 cells (Gcdphenotype) even in the absence of oxidative stress, which is potentially indicative of an initiation defect (Hinnebusch, 2005). An initiation defect would also be consistent with the slower growth of rad6 cells (Simoes et al., 2022) and overall lower translation rate in the absence of peroxide (Figures 4B-C and S3). One possibility for the Gcdphenotype could be that the lack of Rad6 affects the expression of "Gcd" genes and decreases their availability, resulting in uORF bypassing and increased translation of GCN4. However, we found that the expression of these genes was not affected in rad6 cells ( Figure S5B) and therefore other mechanisms drive the constitutive translation of GCN4. We previously observed increased presence of subunits of the translation initiation factor eIF3 associated with the WT compared to cells unable to produce K63-linked ubiquitin chains (Back et al., 2019). It is therefore possible that ubiquitination of translation factors by Rad6 may regulate initiation. Finally, the lack of Rad6 could lead to increased GCN4 translation by promoting reinitiation over recycling at uORF1. Although the exact mechanisms by which Rad6 affect GCN4 translation are yet to be identified, the potential role of Rad6 in translation initiation expands its functions beyond translation elongation. It remains unclear whether translational control at initiation and elongation mediated by Rad6 occurs independently and whether both contribute to cellular fitness during oxidative stress.
Rad6 is conserved from yeast to human cells and mutations in its human homolog, UBE2A, are linked to intellectual disability type Nascimento due to loss of UBE2A activity (Czeschik et al., 2013;Nascimento et al., 2006). UBE2A was also shown to modulate neuronal function in flies by interacting with the E3 ubiquitin ligase Parkin and thereby inducing mitophagy (Haddad et al., 2013). We previously showed that Rad6 carrying the corresponding disease mutations in its human homolog leads to dysregulated K63 ubiquitination response during oxidative stress in yeast (Simoes et al., 2022), further supporting the idea that the role of Rad6 in cellular homeostasis is conserved across species. Our studies in yeast, therefore, reveal crucial insights into the cellular response to UBE2A deficiency and could be important for delineating the disease mechanisms.

DECLARATIONS OF INTEREST
The authors declare no competing interests. oxidative stress profiling datasets. We thank Alan Hinnebusch for kindly providing GCN4

EXPERIMENTAL MODEL AND SUBJECT DETAILS
The yeast strains used in this study are listed in Table S1. SUB280 strain derivatives were grown in synthetic defined (SD) medium composed of D-Glucose (BD Difco, #215510), yeast nitrogen base (BD Difco, #291940) and drop-out amino acid medium without Leu and Trp (Sigma, #Y0750). SUB280 rad6 RAD6 and SUB280 rad6 RAD6(C88A) cells were grown in SD media supplemented with drop-out amino acid supplements without Leu, Trp and Ura (Sigma, #Y1771). S288C strain derivatives were grown in SD complete media by using drop-out amino acid supplements without Leu and Trp (Sigma, #Y0750) and supplementing it back with L-leucine (Sigma, #L800) and Tryptophan (Sigma, #T8941). Starter cultures were grown at 30°C overnight and then diluted to an OD600 of 0.001 (rad6 cells) or 0.0001 (WT cells) and were grown to a final OD600 between 0.5 and 0.6 for ~16 hours. Unless noted otherwise, the cultures were treated with freshly diluted H2O2 (peroxide) (Sigma, #216763), achieving a final concentration of 0.6 mM, for 30 minutes, filtered and frozen in liquid nitrogen for Ribo-seq, Disome-seq and RNA-seq experiments.

Ribo-seq, Disome-seq and RNA-seq experiments
Ribo-seq, Disome-seq and RNA-seq experiments were performed based on published protocols (Guydosh and Green, 2014;McGlincy and Ingolia, 2017;Meydan and Guydosh, 2020). Frozen yeast cell pellets and frozen droplets of lysis buffer (20 mM Tris pH 8.0, 140 mM KCl, 1.5 mM MgCl2, 1% Triton X-100 and 0.1 mg/mL cycloheximide [Sigma, #C7698]) were lysed using a Retsch Cryomill (Retsch 20.749.0001). The resulting powder of frozen cell and lysis buffer mixture was thawed at room temperature, transferred to a 50 mL falcon tube to spin at 3000 g for 5 min at 4 C. The supernatant was then spun at 21000 g for 10 min at 4 C. The absorbance of the supernatant (cell lysate) at 260 nm was recorded and total "OD" of the lysate was calculated as the product of the volume (in mL) multipled with A260 reading. A fraction of the lysate equivalent to OD=45 was flash frozen in liquid nitrogen. Prior to RNase I digestion, lysates were thawed, diluted with an equal volume of lysis buffer and then digested with ~26 U of RNase I (Ambion, #AM2294) per OD for 1 h at room temperature (22 C) with gentle agitation at 700 rpm. Monosome (for Ribo-seq) and disome (for Disome-seq) fractions were separated by loading the lysates onto a 10%-50% sucrose gradient, prepared in gradient buffer (final concentration: 20 mM Tris pH 8.0, 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT), and spun at 40,000 rpm for 3 hours at 4 C using a SW 41 Ti Swinging-Bucket Rotor (Beckman Coulter). Sucrose gradient fractionation was performed by using a Brandel Density Gradient Fractionation System. The peaks corresponding to monosomes and disomes were collected and RNA was purified by using the SDS, hot acid phenol-chloroform extraction method. For RNAseq, the total RNA was isolated directly from the frozen cell pellets by the SDS, hot acid phenol-chloroform extraction method and fragmented in a buffer (pH 9.2) containing 12 mM Na2CO3, 88 mM NAHCO3, 2 mM EDTA for 35 minutes at 95 C. The total RNA was cleaned up using the Oligo Clean & Concentrator kit (Zymo Research, #D4060).
Monosome/disome footprints and total RNA isolated as described above were run on a 15% TBE-Urea polyacrylamide gel (Bio-Rad, #3450091) for the size selection process.
For Ribo-seq, Disome-seq and RNA-seq, RNA fragments between 25-34 nt, 54-68 nt and 50-70 nt were excised from the gel, respectively. We used the 50 nt band from a small RNA marker (Abnova, #R0007) for RNA-seq experiments and other RNA size markers used for size selection are listed in Table S2. The excised gel pieces were frozen on dry ice for 30 minutes and thawed in RNA extraction buffer (0.3 M NaOAc, 1 mM EDTA, 0.25% SDS) overnight at 20 C with gentle agitation (700 rpm). Next day, RNA was precipitated and the pellet was resuspended in 10 mM Tris pH 8.

Next generation sequencing library preparation
Library preparation was conducted by following published protocol (McGlincy and Ingolia, 2017). The RNA fragments from Ribo-seq, Disome-seq and RNA-seq experiments were first dephosphorylated using PNK (NEB, #M0201L) and ligated to preadenylated linkers containing a 5 nt-long random Unique Molecular Index ('UMI ) and a 5 nt barcode that is unique for each sample (listed in Table S2). The linkers that were pre-adenylated using a 5 DNA adenylation mix (NEB, #E2610L) were ligated to dephosphorylated RNAs using T4 truncated RNA ligase 2 (K227Q) (NEB, #M0351L).  Table S2) containing a random 2 nt UMI. At this step, ribosomal RNA (rRNA) was removed from Disome-seq and RNA-seq samples by using Qiagen FastSelect (Qiagen, #334215). The cDNAs obtained from this reaction were resolved on a 10% TBE-Urea gel (Bio-Rad, #3450089) and cDNAs were extracted using DNA gel extraction buffer (0.3 M NaCl, 1 mM EDTA, 10 mM Tris pH 8) with gentle agitation (700 rpm) overnight at 20 C. The next day, DNA was precipitated and the pellet was resuspended in 10 mM Tris pH 8. The footprints were circularized using CircLigase ssDNA Ligase (Biosearch Technologies, #CL4115K). For Ribo-seq samples, rRNA removal was performed at this stage by oligonucleotide substraction using Dynabeads MyOne Streptavidin C1 (Invitrogen, #65001) and DNA oligos that are the reverse complement of ribosomal RNAs (listed in Table S2). The samples were then amplified by PCR using Phusion DNA Polymerase (ThermoFisher Scientific, #F530L) and resulting product were resolved in hand-poured 8% native TBE gel. The libraries were extracted using DNA gel extraction buffer (0.3 M NaCl, 1 mM EDTA, 10 mM Tris pH 8) with gentle agitation (700 rpm) overnight at 20 C. The next day, DNA was precipitated and the pellet was resuspended
Briefly, fastq files of sequencing samples were provided by NIDDK Genomics Core and NHLBI DNA Sequencing and Genomics Core (NIH). We used Cutadapt (Martin, 2011) to remove linkers and demultiplex for retrieving individual samples from pooled data. For RNA-seq samples, we trimmed all the reads to 50 nt by using following parameters: -j 0l 57 --discard-untrimmed. To remove rRNA and tRNA reads, we then aligned the files to an index of noncoding RNAs with Bowtie version 1.1.2 (Langmead et al., 2009) by using following parameters: -v 2 -y -S -p 12. We removed PCR duplicates by using a custom python script. We then aligned the deduplicated files to coding regions and splice junctions of R64-1-1 S288C reference genome assembly (SacCer3, Saccharomyces Genome Database Project) by using the following parameters: -v 1 -y -a -m 1 --best -strata -S -p 4. The number of reads that were obtained after each of these steps are outlined in Table 3.
Custom python scripts are used for the data analysis by using biopython version 1.72 and python 2.7.18. For Ribo-seq and Disome-seq experiments, only the reads between 25-34 and 57-63 nt were analyzed, respectively. Ribo-seq and Disome-seq reads were aligned by their 3 ends. For RNA-seq experiments, 50 nt reads were analyzed and coverage of reads was used instead of 3 alignment. All reads were normalized in units of rpm (reads per million mapped reads), which was computed by dividing the read count at each nt position by the total number of mapped reads and then multiplying the result with 10 6 .
Quantitation of Ribo-seq and RNA-seq data was performed by summing the total number of normalized reads mapping to each coding sequence or UTR regions obtained from published studies (Ng et al., 2020;Pelechano et al., 2014). These total number of reads per gene was normalized by the gene s length (in kilobases) to obtain rpkm values.
Ribo-seq reads were shifted 15 nt from their 3 end to align the P-site to the beginning of each gene. Data from 15 nt of either end of the ORFs was eliminated to reduce the effects of initiation and termination on ribosome occupancy. For differential expression analysis by DESeq2 (Love et al., 2014), raw counts were first generated for each gene. The gene expression profiles were compared by running DESeq2 on Rstudio and Padj values were obtained. We used Padj < 0.05 for significance cut-off, log2FoldChange value > 0.8 for upregulated and < -0.8 for the downregulated genes. Volcano plots were generated by using ggplot2 in Rstudio (Wickham, 2016). Gene ontology analysis was performed by using PANTHER Classification System (http://www.pantherdb.org/) (Mi et al., 2013;Thomas et al., 2022) with following parameters: PANTHER version 17.0

Overrepresentation Test, FISHER test with FDR correction, PANTHER GO-Slim
Biological Process with Saccharomyces Cerevisiae -REFLIST (6050) as a reference gene list. Gcn4-target mRNAs were obtained from a published ChIP-seq dataset (Rawal et al., 2018). From this dataset, the first 250 genes that had > 2 fold increase in Rbp3 (RNA polymerase B) occupancy in starved cells with reproducible induction by Gcn4 in other datasets (Natarajan et al., 2001;Qiu et al., 2016;Saint et al., 2014) were defined as Gcn4 targets.
Metagene plots were generated by averaging rpm around the start and stop codons normalized by the total number of reads in a given window for each gene (100 nt upstream of the ORF and 300 nt into the ORF for start codon metagene; 300 nt of the ORF and 100 nt downstream of the ORF for stop codon metagene). ORFs that were unidirectionally overlapping with other ORFs, the genes with features smaller than the window size, and the genes without any mapped reads were excluded from the analysis.
Average reads plots of XIP motifs were generated by first creating a list of occurrences of XIP motifs in the yeast transcriptome and then averaging normalized monosome or disome occupancy from a region of interest (50 nt upstream and 50 nt downstream of XIP motif). Normalization was done by dividing the rpm at each position in the region of interest by the average rpm of the gene.
Pause scores were computed by dividing the rpm of a motif by the average rpm in a region of interest (±50 nt of each motif). Pause scores for sites that are smaller than the ±50 nt window were eliminated from the analysis. Average pause scores were generated by averaging the individual pause scores for each tri-amino acid motif. We excluded the motifs that were represented in the genome less than 100 times to reduce noise, which resulted in 6267 motifs that were compared across datasets. Individual pause scores for XIP motifs were visualized in a box plot to show the distribution and significance of XIP pause scores. The significance of differences in the median of these individual pause scores were computed by independent 2-group Mann Whitney U Test in Rstudio.

Plasmids Building
RLuc-P2A-X-P2A-Fluc plasmids (where X represents a variable sequence) were assembled using NEB Builder HiFi DNA Assembly Cloning Kit (New England Biolabs, #E2621S) by combining the original plasmid digested with HindIII (New England Biolabs, #R3104S) and NotI (New England Biolabs, #R3189S) and the gene fragments and oligo listed in Table S2.

Luminescence activity measurement
Yeast strains transformed with the plasmids described above in log phase grown in SD-Ura medium were pelleted down and transferred to SD-Ura-Met to induce plasmid expression for 90 minutes. For cells treated with indicated H2O2 concentrations, plasmid expression was induced for 60 minutes and then H2O2 was added to the medium and

Northern blotting
The tRNA-Pro coding sequence was ordered as gBlock (listed in Table S2) and was assembled with digested YCplac33 backbone using NEBuilder HiFi DNA Assembly Cloning Kit (NEB, #E5520). The tRNA-Pro probe sequence was amplified from this plasmid using the primers in Table S2 and in vitro transcribed by using Digoxigenin-11-UTP included in DIG Northern Starter Kit (Sigma, #12039672910). 25 µg total RNA each from WT and rad6 cells was resolved on 15% TBE-Urea polyacrylamide gel (Bio-Rad, #3450091). The RNAs were then transferred onto positively charged nylone membrane (Sigma, #11209299001) in 20x SSC buffer for 3 hours by using Nytran SuPerCharge turboblotter system (Cytiva, #10416302) following manufacturer s instructions. The RNA was UV-crosslinked to the membrane by using VWR UV crosslinker (VWR, at 120,000 microjoules per cm 2 and 100 ng/mL of the probe diluted in DIG Easy Hyb Granules Working Buffer was hybridized overnight at 42 C with gentle agitation (Sigma, #11796895001). Next day, the membranes were washed with low stringency wash buffer (2X SSC, 0.1% SDS) and then with high stringency wash buffer (1X SSC, 0.1% SDS), twice for 5 minutes at room temperature for each. The membrane was washed and subjected to Anti-DIG-AP antibody by using DIG Wash and Block Buffer Set (Sigma, #11585762001) and immonological detection of the membrane was conducted by CDP-Star chemiluminescent substrate included in the northern blotting kit.

Translation rate assays
Indicated yeast strains in logarithmic phase grown into SD medium were back-diluted to

Gene fragments and oligo used for generating p416-Met25-RLuc-P2A-X-P2A FLuc plasmids
No-Stall (dsDNA)    as reference list and test type as FISHER with FDR correction.

TAATACGACTCACTATAGGGGAGCCGGGACTCGAACCCGG
(D) Schematics for calculation of pause score, which is computed by dividing reads at a motif by the average reads in a local window around the motif of interest ( 50 nt). We calculated pause score at tri-amino acid motifs that map within to the ribosomal E (penultimate position of the nascent peptide), P, A sites.   (C) Disome detection by Gcn1/2 induces eIF2 phosphorylation and ISR activation.
Western blotting demonstrates that eIF2 is phosphorylated upon oxidative stress, reaching a maximum at 0.6 mM peroxide, suggestive of increased ribosome stalling events. Peroxide-induced eIF2 -P is reduced in the absence of Rad6, consistent with less ribosome stalling. GAPDH is used as a loading control.
(D) Western blotting shows that the rad6 cells complemented with WT Rad6 (Rad6-HA) restores peroxide-induced eIF2 phosphorylation. Actin is used as a loading control.
(E) Western blotting shows that the rad6 cells complemented with WT Rad6 (Rad6-HA) restores peroxide-induced eIF2 phosphorylation but the catalytically dead mutant (Rad6 C88S -HA) cannot, even at longer incubation times with 0. 6 mM peroxide. GAPDH is used as a loading control.    These data show the responses observed in the SUB280 strain are consistent in the S288C strain.
(C) Quantification of HPG incorporation during peroxide treatment is shown as a normalized rate for HPG incorporation in treated vs untreated S288C cells. The translation rates were calculated by fitting the mean fluorescence values to a linear regression as a function of time. Significance is determined by two-tailed unpaired t-test.
These data show the responses observed in the SUB280 strain are consistent in the S288C strain. replicates. The genes that are differentially expressed (DESeq2, padj < 0.05) are indicated with an asterisk (*).
(C) RNA-seq data of ribosomal protein-encoding genes (n=148, gene names obtained from SGD) in WT vs rad6 cells peroxide show that mRNAs that encode ribosomal proteins are lower in rad6 cells, which is a hallmark of TOR pathway inactivation.
(D) Translation efficiency (Ribo-seq reads normalized by RNA-seq reads) of 21 mRNAs that encode redox enzymes shows translational upregulation of these genes in rad6 vs WT cells in the presence of peroxide. Significance is calculated by one-way Anova test. ns = not significant.
(E) Western blotting demonstrates that the phosphorylation of eIF2 due to peroxide is minimal in rad6 cells in the SUB280 background but not in the S288C background, suggesting that other inputs regulate eIF2 phosphorylation in the S288C background.
Note that total eIF2 levels do not change with peroxide and in the different strains.
(F) Bar graphs of the translation efficiency (left) and RNA levels (right) of GCN1, GCN2, GCN20 genes show that the abundance and translation level of the mRNAs encoding these proteins are not affected by loss of Rad6. (B) RNA-seq (top) and translation efficiency (bottom) data corresponding to mRNAs that encode "Gcd" genes indicate that the expression of these genes does not change in the cells lacking Rad6 peroxide.
(C) GCN4-lacZ assay performed in the presence of peroxide. The data show that Gcn4 translation is higher in rad6 cells compared to WT, with and without peroxide.