Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigate ubiquitylation of RPS27A/eS31 by the E3 ligase RNF25 in response to translational stress. Previous studies have identified RPS27A/eS31 ubiquitylation at Lys113 under conditions where translation factors are trapped in the ribosomal A-site. Here, the authors extend this work by testing whether additional translational stress conditions, including amino acid deprivation, induce RPS27A/eS31 ubiquitylation. They further show that GCN1 is required and explore a possible competition between RNF25 and GCN2 for GCN1.

Strengths:

This study expands on the range of stress conditions leading to RPS27A/eS31 ubiquitylation, reporting that it occurs in a variety of conditions associated with ribosome stalling, including amino acid deprivation. These observations are useful because they suggest that the RNF25 pathway may not require translation factors trapped in the ribosomal A-site, but may instead respond more broadly to translational perturbations associated with ribosome collisions.

We wish to point out that our study in fact suggests that the RNF25 pathway is activated by translation factors in the A-site, in agreement with what has been previously proposed, and in addition by stalling conditions that are assumed to not trap translation factors in the A-site. We do not exclude that these conditions might be sampled by A-site binding quality control factors before recognition by RNF25.

Weaknesses:

The evidence supporting several of the major claims is incomplete, and additional controls and orthogonal approaches would greatly strengthen the evidence presented.

We appreciate adding more controls to further substantiate our novel findings. In the course of the revisions we will focus our work on those experiments that do not merely reproduce established facts in the field.

In particular:

(1) It is unclear whether the different conditions used to induce translational stress lead to ribosome stalling or collisions. The model presented by the authors seems to rely on ribosomal collisions, but this is not shown. In addition, further investigating amino acid deprivation beyond the removal of Arg or Lys would strengthen the paper.

We thank the reviewer for the comment. It is correct that we don’t formally show collisions.

However, the conditions we use have been previously established in the field to induce ribosome stalls and/or collisions, which we may not have pointed out clearly enough. In the revised version, we will include all relevant citations, i.e. for ternatin (Oltion et al., 2023): collisions, anisomycin (Juszkiewicz et al., 2018, Sinha et al., 2020): collisions, emetine (Sinha et al., 2020): collisions, didemnin B (Juszkiewicz et al., 2018, Stoneley et al., 2022): accumulation of ubi-eS10 and changes in polysome profiles indicative of collisions, MMS (Stoneley et al., 2022): changes in polysome profiles indicative of stalls or collisions, starvation -Arg/-Lys (Darnell et al., 2018, Stoneley et al., 2022): accumulation of collided ribosomes only upon GCN2 inhibition, indicative of collisions.

Secondly, we do not claim to induce collisions when describing the inhibition data (Figure 1 and Figure S1) and were careful to say that we use ‘conditions that cause ribosome stalling’.

Thirdly, we conclude on collisions when interpreting the data on amino acid starvation (and in our model (Figure 6)), based on our data demonstrating that RNF25 activity in RPS27A/eS31 ubiquitylation is dependent on GCN1 (Figure 3), an established sensor of collided disomes (Pochopien et al., 2021). This conclusion is thus based on the current knowledge in the field.

We will carefully screen the text for potential points of overinterpretation or confusion between stalling and collisions.

To address the request of further investigating amino acid deprivation beyond the removal of Arg or Lys, we will include an additional experiment in which we will deplete another amino acid.

(2) Ubiquitylation of RPS27A/eS31 by RNF25 is used throughout the paper as a readout of RNF25 activity and is assumed to be on Lys113 based on previous work, but is not formally shown here.

It is established that Lys113 is the main target of RNF25, not only by our work (Montellese et al., 2020), but also by recent work of other groups to which we had referred in our manuscript (Gurzeler et al., 2023, Oltion et al., 2023, Zhao et al., 2026).

To experimentally address this point, we will add an experiment testing ubiquitylation of RPS27A/eS31 in cells carrying the K113R mutation.

(3) Rescue experiments of the different mutants used in this study with wild-type and different domain deletions (i.e., ΔRWD for RNF25, ΔRWD-binding for GCN1) would help confirm specificity and strengthen the mechanistic claims.

Minimally, we will include rescue experiments for RNF25 (using WT, DRWD and enzymatically dead mutant) and, if possible, also for GCN1, which might be more challenging due to its large size and anticipated problems with cloning, cell line generation and protein expression.

(4) The conclusion that RPS27A/eS31 ubiquitylation supports translation (Figure 4) is based entirely on polysome/monosome ratios, which are difficult to interpret without additional assays of translation output, elongation, or collision.

It is correct that we base our conclusion on polysome profiles and agree that these are an indirect measure of translation output. However, this assay is well established in the field to show dysregulation of polysome/monosome ratio upon ribosome stalling (Garzia et al., 2017), (Wu et al., 2020), (Chatterjee et al., 2024), (Gurzeler et al., 2023).

Elongation defects would be expected to lead to stalls and/or collisions (which we conclude on). However, we cannot exclude that there is more initiation when RPS27A/eS31 carries the K113R mutation, although this is hard to rationalize mechanistically and experimentally challenging to exclude. Therefore, to address the point, we will add a sentence that we cannot exclude indirect effects on initiation but consider these unlikely.

(5) The idea that RNF25 competes with GCN2 for GCN1 binding is interesting, and related models have recently been proposed in RNA damage. The effect of GCN2 KO on RNF25dependent ubiquitylation appears modest, and the data would be strengthened by rescue experiments with wild-type GCN2 and GCN2 mutants defective in GCN1 binding. The authors propose: "that the RNF25 pathway acts as a first line of defence to resolve ribosome collisions, outcompeted by GCN2 binding to GCN1 under acute stress." This model would suggest a further increase in RPS27A/eS31 ubiquitylation upon Arg/Lys deprivation in GCN2 KO cells, since this is the condition in which GCN2 is expected to be activated and engaged with GCN1 (i.e., when it would be competing with RNF25), but no further increase in RPS27A ubiquitylation is observed. It is therefore not clear that these data support the proposed model. Contributing to this may be the fact that many of these assays are performed in a USP16 KO background, which may make it difficult to assess changes in RPS27A/eS31 ubiquitylation.

We thank the reviewer for the comment. We measure on average a 50% increase in the level of ubiquitinated RPS27A/eS31 in GCN2 KO cells. Considering the large number of ribosomes in a cell (~10⁷ per HeLa cell), this 50% increase (from 12.5 to 25% ubiquitinated RPS27A/eS31) amounts to an estimated number of 1,25 x 10⁶ of RPS27A/eS31 molecules that get additionally modified, which is clearly a substantial difference, especially compared to the naturally very low levels of RNF25 (in the range of 23’000 molecules (Itzhak et al., 2016)).

We respectfully disagree that performing experiments in USP16 KO background makes it difficult to assess RPS27A/eS31 ubiquitination. On the contrary. The natural levels of RPS27A/eS31 ubiquitination in WT cells are very low, making quantification sensitive to background fluctuations (see Figure S1). Therefore, in our experience, the usage of USP16 KO makes the quantitative analysis of RPS27A/eS31 ubiquitination robust, allowing us to analyse both increase and decrease in the levels of ubiquitination. We agree that with increasing collisions, the level of ubiquitinated RPS27A/eS31 reaches a plateau in USP16 KO, which may limit the observable increase. Therefore, the substantial 50% increase might indeed underestimate the effect as compared to WT cells. Still, the measurable increase is substantial and robust.

To experimentally address the point of the reviewer, we will try generating GCN2 KO cells in a WT background, i.e. in absence of USP16 KO, to strengthen our model.

(6) Given that several RWD domain proteins can interact with GCN1, and that DRG2 KO appears to affect RPS27A/eS31 ubiquitylation (Figure S5), the data do not support the GCN2specific title. The results are more consistent with a broader, incompletely characterized network of GCN1-associated RWD domain-containing proteins that seems to affect RNF25-dependent ubiquitylation rather than with a demonstrated RNF25-GCN2 competition mechanism. Further characterization of GCN2-dependent ISR activation (p-eIF2a and ATF4 WB) in the absence of RNF25 in Arg/Lys starvation will help shed light on the RNF25-GCN2 competition. The authors use K113R, but this is not shown to prevent RNF25 engagement with GCN1, so a RNF25 KO should be used.

While we fully agree that our data point at a broader network of competition on GCN1, we wished to avoid an overstatement on other pathways than GCN2, since our experimental evidence on DRG2 is limited at the moment. As it stands, changing the title of the manuscript to a more general message, would indeed fuel the view that our claims are incomplete. But we are glad to reconsider this suggestion if further supporting evidence can be obtained in the course of the revision work.

The reviewer suggests experiments on competition of RNF25 with GCN2. In contrast to the expectation of the reviewer, we do not expect KO of RNF25 to manifest in defects in ISR activation due to the low expression levels of RNF25. In the revised manuscript, we will make clearer that our model refers to competition in the other direction, i.e., of GCN2 with RNF25, which our data supports. The reverse competition of RNF25 with GCN2 is expected to be inefficient to enable a robust activation of the ISR by GCN1 when needed. In addition, other pathways (such as DRG2) might also contribute to the resolution of collisions in the absence of RNF25, affecting the level of ISR activation.

We feel that further working out these competitive relationships will be interesting to perform in future work. Currently, it is also not clear whether all involved RWD-containing factors bind GCN1 with the same affinity, which is important to consider for the effectiveness of a mutual competition model as suggested by the reviewer.

Reviewer #2 (Public review):

Summary:

The authors show that deprivation of Arginine and Lysine induces a ~50% increase in the ratio of ubi-RPS27A to RPS27A, and this induction requires E3 ubiquitin ligase RNF25. The authors show ZAKalpha and EDF1 are not required for steady state or ribosome stalling-induced ubiRPS27A, while GCN1 is required. The ratio of polysomes to monosomes is increased in RNF25 knockdown cells or when translation is activated by ISRIB in a RPS27A K113R mutant cell line. GCN2 KO cells indicate elevated levels of ubi-RPS27A, and overexpression of the GCN2 RWD domain reduces levels of ubi-RPS27A.

Strengths:

(1) The authors identified a novel pathway to sense amino acid deprivation, indicated by ubiRPS27A, previously implicated in ribosome stalling.

(2) The authors find antagonism between two proteins known to act downstream of GCN1, giving insight into how signaling occurs from an upstream sensor of ribosome stalling to multiple downstream pathways.

Weaknesses:

(1) The authors suggest that, based on increased Polysome/Monosome ratios, there is more disome stalling in RNF25 KD cells and RPS27A K113R cells treated with ISRIB, but this readout is very indirect and could be driven by other changes in the cell other than ribosome stalling.

We thank the reviewer for this important comment. We intentionally used ISRIB in Figure 4F, G to avoid possible effects on initiation, and the results are consistent with our model. While we agree that ISRIB itself might have indirect consequences, these should be the same for the control (WT cells) and the assay condition (K113R cells). We also show the data without ISRIB, which show a similar trend but are less robust (Figure 4D, E). It is very hard to exclude other possible effects which would selectively affect K113R cells in presence of ISRIB.

(2) While the authors propose that GCN2 and RNF25 compete for binding to GCN1, no evidence was shown that RNF25 binds to GCN1 in cells, nor that the interaction increases when GCN2 is absent.

The idea of RNF25 binding to GCN1 is based on a previously published work (Oltion et al., 2023, Seidel et al., 2026, Zhao et al., 2026). We will design additional experiments to potentially confirm the interaction between RNF25 and GCN1.

(3) The use of USP16 to enhance the detection of ubi-RPS27A in many experiments brings the question of whether USP16 KO may alter the protein levels of any known regulators of ribosome collisions? (i.e. ZNF598, GCN1, EDF1, ZAKalpha, etc.) If USP16 KO causes changes in other important regulators of collisions, the authors could be identifying genetic interactions with USP16 in their experiments throughout the paper.

Indeed, we can’t exclude the effect of USP16 KO on the expression levels of other collision sensors. We will experimentally confirm the levels of other ribosome collision sensors in USP16 KO cells.

(4) In Figure 5E, the expression level of the GCN2 3K RWD domain looks to be lower than the WT RWD domain; perhaps this could be what is driving the smaller decrease of ubi-RPS27A seen with GCN2 3K vs WT.

We thank the reviewer for pointing at this issue, which we will experimentally address in the revised version.

Reviewer #3 (Public review):

Summary:

This study examines the role of RNF25 in translational quality control. Previous work indicated that RNF25 is activated by ribosomes stalled with defective elongation or termination factors bound in the A-site. Here, the authors provide evidence that RNF25 is activated by other treatments that evoke ribosome stalling, including amino acid starvation, where the A-site may be empty, leading to ubiquitination of RPS27A in a manner requiring the ISR collision sensor Gcn1, but not EDF1 and ZAKα, involved in the RQC and RSR surveillance pathways. They present some evidence from polysome profiling that RNF25 and its ubiquitination of RPS7A help resolve ribosome collisions and support translation elongation in basal conditions. They further show that KO of Gcn2 increases RPS27A ubiquitination in basal conditions, but not in amino acid-starved cells, and that RPS27A ubiquitination was reduced on overexpressing the WT RWD domain of Gcn2 but not a variant harboring substitutions of residues predicted to bind Gcn1. Based on these findings, they propose a model that, in response to ribosome stalling induced by various stresses, Gcn1 recruits RNF25 via the latter's RWD domain to ubiquitinate RPS27A and thereby resolve ribosome stalling and promote continued elongation. If collisions increase even further, GCN1 recruits GCN2 instead of RNF25 to elicit the ISR.

Strengths:

The data is convincing that a variety of triggers leading to diverse stalled ribosomal states, including amino acid limitation, can activate RNF25, suggesting that activation of this pathway does not require the presence of trapped protein factors in the ribosomal A-site but is a more general response to ribosome collisions. It is also convincing that Gcn1 is required for RNF25 activation under all of these conditions, which is consistent with previous findings that Gcn1 is required for RNF25 function in the presence of trapped elongation or termination factors. The finding that EDF1 and ZAK are not needed for RNF25 activation in amino acid starvation conditions is of interest for EDF1, given the recent claim that it is required for full ISR activation.

Weaknesses:

(1) The evidence presented from polysome profiling that RNF25 helps resolve naturally occurring ribosome collisions in basal conditions is not compelling, as eliminating RNF25 could be increasing the rate of initiation rather than increasing stalled ribosomes as the means of increasing the P/M ratio. The Rps27A-K113R mutation could have the same effect of increasing initiation, which could have been obscured by inhibiting the ISR with ISRIB.

Our results indicate that P/M ratio increases upon ISRIB treatment of K113R cells compared to WT cells, aligning with the idea that ISRIB enhances initiation, causing increased loading of ribosomes on mRNA and consequent increased frequency of collisions. As outlined above, we agree that this experiment is indirect and results might be affected by secondary effects. However, we cannot rationalize how inhibition of the ISR by ISRIB would specifically obscure the effect for the K113R mutation but not the WT.

(2) The evidence that RNF25 competes with Gcn2 for Gcn1 binding is also not compelling. While it's convincing that Rps27A-Ubi is elevated in basal conditions on eliminating Gcn2, loss of GCN2 would be expected to increase ribosome loading on mRNAs, potentially elevating the frequency of collisions and thereby stimulating RNF25 activity indirectly.

We have not made sufficiently clear that we did not intend to claim that RNF25 efficiently competes with GCN2 (see also response to reviewer 1), which we do not expect due to the low levels of RNF25. Our manuscript is focussed on competition in the reverse direction, i.e. of GCN2 with RNF25.

We agree that loss of GCN2 may increase ribosome loading on mRNA similar to ISRIB treatment, which could lead to more collisions by enhanced translation and hence increased Rps27A-Ubi. At the same time, however, this does not exclude that loss of GCN2 contributes more directly at the level of RNF25 recruitment. Therefore, the experiment also supports the competition model, and both effects together may contribute to the observed increase in ubiquitylated RPS27A/eS31. Without other evidence, the experiment would remain inconclusive.

Therefore, to directly test the competition model, we had overexpressed the GCN1-binding RWD domain of GCN2, which leads to decreased levels of ubiquitinated RPS27A/eS31, lending direct support to the competition model of GCN2 with RNF25, which is consistent with similar models recently proposed by two other manuscripts (Seidel et al., 2026, Zhao et al., 2026).

(3) It's also quite puzzling and left unexplained why they observed no further increase in Rps27AUbi on -Arg/-Lys starvation in the cells lacking Gcn2. Why wouldn't -Arg/-Lys starvation lead to further stalling and RNF25 activation in the absence of Gcn2? (Since Gcn2 KO increases Rps27A-Ubi in the presence +Arg/+Lys conditions, it can't be that Gcn2 is required for RNF25 function.) The same puzzling and unresolved observation was made in the cells lacking DRG2. One possible explanation for this conundrum is that low-level RNF25 abundance limits further activation.

Over all of our experiments, we have observed that RPS27A-Ubi reaches a plateau of about 30% to 35% of total RPS27A in the USP16 KO background (GCN2 deletion or amino acid starvation). This plateau indeed limits seeing further increases. We do not know the underlying reason but note that under these conditions about one third of 40S subunits carry ubiquitin on RPS27A/eS31. As the reviewer suggests, RNF25 is expressed at low levels (in the range of 23’000 molecules, (Itzhak et al., 2016); see point 5 of reviewer 1), likely rendering it the limiting factor for further ubiquitination events.

To circumvent the plateau issue, we will attempt to generate GCN2 KO cell lines in the WT background for the starvation experiments (see also response to reviewer 1, point 5).

(4) The quantitative effects of overexpressing the Gcn2 RWD domain on Rps27A-Ubi, constituting their other evidence presented to support the competition model, are quite small in magnitude.

We respectfully disagree with the reviewers’ comment concerning the magnitude of the effect. There is a ~27% decrease in ubiquitination, which is substantial considering the number of 40S ribosomal subunits and possible consequences of such change. It should also be noted that this is a transient transfection experiment not hitting all cells of the population. We will repeat the experiment, optimizing the expression of the negative control construct.

Cited literature:

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Darnell AM, Subramaniam AR, O'Shea EK (2018) Translational Control through Differential Ribosome Pausing during Amino Acid Limitation in Mammalian Cells. Mol Cell 71: 229-243 e11

Garzia A, Jafarnejad SM, Meyer C, Chapat C, Gogakos T, Morozov P, Amiri M, Shapiro M, Molina H, Tuschl T et al. (2017) The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs. Nat Commun 8: 16056

Gurzeler LA, Link M, Ibig Y, Schmidt I, Galuba O, Schoenbett J, Gasser-Didierlaurant C, Parker CN, Mao X, Bitsch F et al. (2023) Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control. Cell Rep 42: 113056

Itzhak DN, Tyanova S, Cox J, Borner GH (2016) Global, quantitative and dynamic mapping of protein subcellular localization. Elife 5

Juszkiewicz S, Chandrasekaran V, Lin Z, Kraatz S, Ramakrishnan V, Hegde RS (2018) ZNF598 Is a Quality Control Sensor of Collided Ribosomes. Mol Cell 72: 469-481 e7

Montellese C, van den Heuvel J, Ashiono C, Dorner K, Melnik A, Jonas S, Zemp I, Picotti P, Gillet LC, Kutay U (2020) USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit. Elife 9

Oltion K, Carelli JD, Yang T, See SK, Wang HY, Kampmann M, Taunton J (2023) An E3 ligase network engages GCN1 to promote the degradation of translation factors on stalled ribosomes. Cell 186: 346-362 e17

Pochopien AA, Beckert B, Kasvandik S, Berninghausen O, Beckmann R, Tenson T, Wilson DN (2021) Structure of Gcn1 bound to stalled and colliding 80S ribosomes. Proc Natl Acad Sci U S A 118

Seidel AS, Nemcekova L, Grønbæk-Thygesen M, Shi X, Ramalho S, Mordente KC, Bekker-Jensen S, Haahr P (2026) RNF25 restrains GCN2 hyperactivation to sustain protein synthesis and cell proliferation in response to RNA damage. bioRxiv

Sinha NK, Ordureau A, Best K, Saba JA, Zinshteyn B, Sundaramoorthy E, Fulzele A, Garshott DM, Denk T, Thoms M et al. (2020) EDF1 coordinates cellular responses to ribosome collisions. Elife 9

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Figures and data

Ubiquitination of RPS27A/eS31 is induced by conditions that cause ribosome stalling including amino acid starvation.

Ubiquitination of RPS27A/eS31 depends on RNF25.

Ubiquitination of RPS27A/eS31 depends on GCN1 but not on EDF1 or ZAKα.

Ubiquitination of RPS27A/eS31 is needed to support translation.

Level of ubiquitinated RPS27A/eS31 increases in the absence of GCN2

Model for the activation of the RNF25 pathway.