Nucleus translocation of tRNA synthetase mediates late integrated stress response

  1. Na Wei
  2. Haissi Cui
  3. Yi Shi
  4. Guangsen Fu
  5. Navin Rauniyar
  6. John R. Yates
  7. Xiang-Lei Yang

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Various stress conditions are signaled through phosphorylation of translation initiation factor eIF2α to inhibit global translation while selectively activating transcription factor ATF4 to aid cell survival and recovery. However, this integrated stress response is acute and cannot resolve lasting stress. Here we report that TyrRS, a member of the aminoacyl-tRNA synthetase family capable of responding to diverse stress factors through cytosol-nucleus translocation and activating stress-response genes, also inhibits global translation, however at a later stage than eIF2α/ATF4 and mTOR responses. Excluding TyrRS from the nucleus over-activates protein synthesis and increases apoptosis in cells under prolonged oxidative stress. Nuclear TyrRS transcriptionally represses translation genes by recruiting TRIM28 and/or NuRD complex. We propose TyrRS, possibly along with other family members, can sense a variety of stress signals through intrinsic properties of this enzyme and its strategically located nuclear localization signal and integrate them by nucleus-translocation to effect protective responses against prolonged stress.

Article activity feed

  1. Version published to 10.1101/2020.06.07.138792v3 on bioRxiv
  2. eLife

    ##Author Response

    We would like to thank all three reviewers for their great effort and their helpful and detailed comments on our manuscript. The reviewers noted the significance of the novel concept we present here, however, major weaknesses of the manuscript were cited in the comments from each reviewer. The criticisms can be summarized into three major categories: 1) missing key controls and analyses in the HEK293 cell models we used; 2) the HEK293 cell models being the only system used for this study; and 3) some evidences that support the mechanistic conclusion are based on correlations and lack direct demonstration for causality. We have addressed some of their concerns in the updated version of the manuscript and believe that it improved our manuscript. We would like to also briefly respond to the comments here:

    First of all, we apologize for not including some key controls and analyses in our manuscript. We have now revised Figure 1 and added 5 additional Supplementary Figures to provide those controls and analyses. The mistake was caused in part by our lack of perception from an audience point of view. Our HEK293 cell system has been rigorously validated for studying TyrRS nuclear deficiency at endogenous level of expression. Those evidence were published (Wei et al., 2014, Molecular Cell, PMID: 25284223) and cited in this manuscript. But this clearly was not enough; each new experiment needs to have its independent controls and analyses, which we did preform and confirm but failed to include in the original manuscript. This mistake caused major confusion and a lack of confidence in our conclusions. Now those controls and analyses have been included in the revised manuscript as listed below:

    Supplementary Figure S1 shows that 1) the ΔY/YARS and ΔY/YARS-NLSMut HEK293 cells we generated express TyrRS (WT or NLS mutant) at a level similar to endogenous TyrRS expression in the original, unmodified HEK293 cells; 2) H2O2 treatment stimulates the nuclear translocation of TyrRS; and 3) ΔY/YARS-NLSMut cells are deficient in TyrRS nuclear localization with or without H2O2 treatment.

    Figure 1A is expanded to include nuclear fractionation and Western blot results as controls to show that 1) overall and cytosolic levels of TyrRS (WT or NLS mutant) do not change obviously during H2O2 treatment; and 2) ΔY/YARS-NLSMut cells are deficient in TyrRS nuclear localization with or without H2O2 treatment.

    Supplementary Figure S2 shows equal expression of different transgenes in our experiments (Figure 1C and Figure 2D).

    Supplementary Figure S5 is added to strengthen the evidence that co-factors are required for TyrRS to regulate target gene expression. Because HDAC1 is a shared co-factor for both TRIM28 and the NuRD complex, we used an HDAC1 inhibitor Trichostatin A (TSA) to test if it can affect the transcriptional repressor activity of TyrRS. Indeed, TSA treatment blocks the inhibition effect of overexpressed TyrRS on its target gene transcription.

    Supplementary Figure S6 shows equal expression of WT and E196K TyrRS and the gain-of-function effect of the E196K mutation in suppressing target gene expression and protein synthesis.

    Supplementary Figure S7 shows the quantification analysis of caspase-3 cleavage as detected by Western blot analysis in Figure 5B.

    For the second major criticism which is the sole use of the engineered HEK293 cell models in the study, we agree that the main conclusions of this paper need to be confirmed in an additional cell system and ideally with the endogenous TyrRS. In fact, we have generated TyrRS nuclear deficient mice by mutating the NLS of the endogenous YARS gene and, by using the mouse fibroblasts, we have confirmed that protein synthesis is overactivated in TyrRS nuclear deficient cells. Because the study of the mouse model has not been completed and it is a separate in vivo study of nuclear TyrRS with its own objectives, we prefer not to add the mouse fibroblasts data to this manuscript but will share these data with the reviewers. However, we would like to point out that the ΔY/YARS and ΔY/YARS-NLSMut HEK293 cell lines are not stable cell lines derived from single clones but instead transient transfections that were selected for in bulk. Therefore, they originated from the same starting cell line and diverged only 1-2 passages before the experiments were performed. Genetic diversion between the NLSMut and the control cell line should therefore be limited. We apologize if that was not clear from the Material and Method section.

    For the last major criticism, we acknowledge that some mechanistic aspects of nuclear TyrRS have not been unequivocally demonstrated. For example, whether the direct binding of TyrRS to its target genes and the interactions of TyrRS with TRIM28 and/or NuRD complex are responsible for the endogenous TyrRS to regulate target gene expression in cells, and whether the level of transcriptional regulation on protein synthesis genes by nuclear TyrRS is sufficient and responsible for the observed suppression in cellular protein synthesis activity. While this issue is partially addressed by the new Supplementary Figure S5 (Treatment with an inhibitor of HDAC1, the shared co-factor of TRIM28 and the NuRD complex), we acknowledge that these weaknesses are in part due to the use of ectopically expressed TyrRS in the current system and can be addressed in the future by using the mouse fibroblasts mentioned above.

  3. eLife

    ###Reviewer #3:

    Many of the genes whose expression is induced by the integrated stress response (ISR) encode aminoacyl tRNA synthetases. Why is expression of so many synthetases enhanced in the ISR and what is the functional significance of this induction are important unresolved questions. This manuscript focuses on the tyrosyl tRNA synthetase, which is induced by the ISR in response to different stress conditions. The study suggests that induced expression of TyrRS in response to oxidative stress leads to nuclear localization of the enzyme where it then binds to DNA targets and recruits key transcription factors that control selected gene expression that ultimately controls protein synthesis levels late in the ISR. The TyrRS dampening of translation late in the ISR apparently occurs independent of the levels of eIF2 phosphorylation.

    These ideas are a potentially interesting mechanistic feature of the ISR that builds on prior reports from this lab. However, there are major reviewer concerns about the manuscript. The manuscript uses different HEK cell models do not appear to be comparable in key ways. Hence one cannot readily integrate the results between the different models and there are important gaps in each. Additionally, key controls and assays are missing from each of the studied models. Because of these major concerns, the stated conclusions are not sufficiently supported from the experimental results. A portion of these concerns are highlighted below. These concerns diminished enthusiasm for the manuscript.

    Reviewer concerns:

    1. Figure 1: A major concern with the manuscript is that key controls and measurements are missing in experiments. The manuscript implies that prior publications have some of these measurements but this is problematic in many ways. In Figure 1A should also measure TyrRS levels and compare these to endogenous TyrRS induced in by oxidative stress. Determine the timing and duration of the anticipated induction of TyrRS expression for endogenous translation. Are the levels comparable with the rescued expression system (shown in this study) and is there induced expression of the engineered TyrRS by stress? If not, is this problematic with the proposed ISR induction model? Does this proposed translation dampening (Fig. 1B) involve continued reduction of translation initiation or elongation? Does the TyrRS +/- nuclear localization reduce global translation in the absence of eIF2 phosphorylation function?

    The H2O2 treatment involves an initial insult and presumably the H2O2 is quickly dissipated. Therefore, one is likely not measuring the length of H2O2 exposure but rather the time after a short duration of stress. Other stress treatment regimens, including those involving oxidative damage, can be continuous. In Fig. 1C and other measures the synthetases, especially TyrRS, to show the level of overexpression.

    1. Figure 2 and supplement: The ChIP analyses appears to feature overexpression of TyrRS (tagged versions different than those used in Fig. 1?). Are immunoblot measurements of the versions of TyrRS in Fig 1A applicable to those in Fig 2? A key feature of this pathway is that TyrRS expression late in the ISR directs the nuclear localization of the enzyme. Test this model with versions of TyrRS whose expression levels and regulation are appropriate in the ISR. Does the mRNA measurements in Fig. 2B involve +/- oxidative stress? This is critical to the proposed model.

    2. Figure 3: Explain more clearly the mini-TyrRS and its utility. This point is also germane to earlier figures.

    3. Figure 4: Be clear about the expression levels of the tagged TyrRS for the MS studies. Be sure to provide statistical information and support documentation in the methods and supplemental tables. Would be helpful to include the nuclear exclusion mutant with the co-IP. The analysis of the E196K mutant of TyrRS needs fuller development (e.g. with the stress condition) and clarity.

    4. Figure 5: Regarding biological implications and cell survival, one finds it difficult to separate altered TyrRS charing of tRNA(Tyr) in this equation. Show the different mutants and arrangements do not alter aminoacylation of tRNA(Tyr).

  4. eLife

    ###Reviewer #2:

    This paper presents a very compelling story: TyrRS has an important moonlighting function in the nucleus involving regulated gene expression via the recruitment of transcriptional co-regulators that is subordinate to TyrRS' ability to sense changes in the cellular environment. If proven correct this notion stands to influence our thinking about cellular stress responses. Therefore, the task of the reviewers is simply to critically evaluate the evidence; the significance of the claims is not in question.

    According to the authors, by a mysterious process, that is not expanded on here, under oxidative stress conditions (200 µM H2O2-treatement of HEK293 cells for extended periods) a small fraction of TyrRS finds its way to the nucleus, where it selectively represses genes involved in the ability of cells to synthesize new proteins. The consequence of this selective transcriptional repression is a sustained oxidative stress-induced repression of protein synthesis that is entirely dependent on this nuclear translocation event.

    The formative experiment supporting this chain of events is a comparison of cells in which the endogenous TyrRS has been inactivated by RNAi and rescued in trans, either by a wildtype TyrRS (i.e. one subject to this regulated nuclear translocation event) or a TyrRS bearing mutations in its nuclear localization signal (242KKKLKK247 to NNKLNK. Figure 1A shows that rescue with the NLS mutant TyrRS leads to superbasal (> complete) recovery of protein synthesis, whereas rescue with the wildtype TyrRS is associated with sustained stress-dependent decrease in protein synthesis.

    This foundational experiment is not described in any detail, nor are its key tenets confirmed experimentally, instead the reader is referred to two previous papers, Fu 2012 describing the NLS mutations and Wei 2014 describing the implementation of this allele swap). Neither the extent of the inactivation of the wildtype allele nor the extent of the rescue are presented. Nor, for that matter, is there evidence that in the cells tested in Figure 1A the NLS mutation indeed abolishes the stress-dependent nuclear import of TyrRS. The WT-rescued cells are not even compared to the parental cells. These weaknesses are compounded by the inherent unreliability of any comparison of two clades of cells, as near as one can tell the authors have compared here two preparations of cells to which they attribute diverse properties.

    Given how much is hanging off the phenotypic comparison of the WT and NLS mut TyrRS, it seems reasonable to impose a much higher standard on the experimental system. In 2020, this amounts to an allele replacement of the endogenous TyrRS with a silently-marked wildtype and NLS (and other) mutant coding sequences. Given the essentiality of TyrRS this should be a simple matter, using CRISPR/Cas9 to target the endogenous locus and offering a repair template to bring in the new alleles. Once implemented this method will produce numerous independent stable clones with the desired genotypes that can then serve in a comprehensive phenotypic analysis that traverses the problem of random clonal variation and phenotypic drift in clades of puro-resistant cells (that plagues the interpretation of the experiments shown here) It is uncertain if the above would be enough. The NLS of TyrRS is also involved in tRNA binding and potentially in other aspects of the charging reaction. Thus, mutations in that sequence rather than purely interfering with the putative nuclear functions of TyrRS, may also compromise the protein's more conventional function, with important and unanticipated phenotypic consequences. Fu et al. 2012, have made an effort to address this issue by comparing the affinity of WT TyrRS and the NLS mutants for tyr-tRNA (Table 1 therein) and by measuring tRNA acylation (Figure 2B, therein). The upshot of these measurements is that mutations in NLS severely compromise tRNA binding and acylation and even the weakest mutation, used here, has a measurable defect. These findings call into question the sweeping conclusions regarding the functionality of the NLS mutation. Therefore, to convince the sceptic the authors need to provide parallel evidence that selectively compromising nuclear transport of TyrRS is at the heart of the phenotypes observed.

    In this vein it is notable that whereas in Wei 2014, study of the phenotypic consequences of the NLS mutation (on the cells' response to DNA damage) was buttressed by manipulation of angiogenin, an agent putatively implicated in the signal that sends TyrRS to the nucleus in stressed cells, no such attempt is made here; is angiogenin no longer believed to play a role? If not, it is incumbent on the authors to discover such trans-acting factors, and study the effect of their manipulation on the phenotype. This may be challenging, but the important claims for discovery made here must be matched by equally convincing experiments.

    And then there is the surprising fact that in Wei 2014 and here the same cells exposed to the same stress seem to have very different consequences to gene expression programmes - where was the nuclear TyrRS-induced downregulation of 'translation' genes in 2014? Were none included in the 718 genes on the SmartChip Real-Time PCR System (WaferGen Biosystems)? Furthermore in 2014 Wei et al were concerned about the confounding effects of the different TyrRS alleles on protein synthesis, as the basis for the effects on DNA damage response (in their words: 'Considering that a simple knockdown of TyrRS may affect global transcription through a general effect on translation...'), yet dismissed this concern only to return now with a new version of reality whereby translational effects are all important. These issues need to be discussed and accounted for.

    In summary, this is a paper presenting a very interesting but inadequately supported idea.

  5. eLife

    ###Reviewer #1:

    Previous work has shown that the nuclear import of TyrRS is stimulated under stress and that nucleus-localized TyrRS functions through the transcriptional machinery to promote the expression of DNA damage response genes for cell protection. In this work, evidence is presented that nuclear TyrRS also inhibits bulk translation in a manner correlated with its association with several AARS-encoding genes and that for elongation factor eEF1A, and recruitment to these genes of HDACs. Mutation of the TyrRS NLS, whose function in nuclear localization provides for coupling between low tRNATyr binding and nuclear localization, was found to derepress bulk translation after prolonged oxidative stress by H2O2, without altering eIF2 phosphorylation levels or mTOR activation, and overexpression (o/e) of TyrRS can reduce protein synthesis, in a manner enhanced by the E196K mutation associated with Charcot-Marie-Tooth disease (CMT), shown previously to enhance TyrRS association with transcriptional co-repressors. ChIP-Seq of overexpressed V5-tagged TyrRS showed binding to only 17 sites, of which 15 are within gene coding sequences, among which four encode TyrRS, TrpRS, SerRS and GlyRS, and a fifth encodes elongation factor eEF1A. These results were confirmed by ChIP analysis of endogenous TyrRS, using the HisRS gene as negative control; and the occupancies were shown to increase on H2O2 treatment. The expression of these AARS/eEF1A gene transcripts was shown to be reduced by o/e of TyrRS, in a manner enhanced for at least some of them by the E196K CMT mutation; and the repression was shown to be eliminated by the NLS_mut for YARS expressed at native levels. Reductions in AARS/eEF1A protein expression were also observed on WT TyrRS o/e. Sequence analysis of the genes showing TyrRS binding by ChIP-seq led to identification of a motif that was shown to be required for binding to TyrRS in vitro in EMSA assays with either purified TyrRS or in extracts from cells overexpressing it, in a manner requiring the full-length TyrRS and not only the catalytic core of the enzyme. It was not shown however that eliminating this motif from any of the target genes attenuated their repression by nuclear-localized TyrRS. Mass spec analysis of affinity-purified, overexpressed TyrRS identified interacting proteins, and several of which were shown to be coimmunoprecipitated with endogenous TyrRS in non-stressed cells, including the transcription cofactors Trim28, HDAC1, and subunits of the NURD co-repressor/histone deacetylase complex. ChIP assays showed that overexpression of TyrRS lead to decreased levels of H3K27Ac, a histone mark of active transcription, and elevated occupancies HDAC1, TRIM28, or NURD subunit CHD4 in non-stressed cells at the AARS/eEF1A genes, with either TRIM28/HDAC1 or CHD4 being observed for all of the genes except the TyrRS gene that shows all three cofactors present. Based on these results, the authors conclude that increased nuclear localization of TyrRS on oxidative stress leads to increased binding of TyrRS to the AARS/eEF1A genes with attendant direct recruitment of either TRM28/HDAC1 or NURD, leading to transcriptional repression of these genes, which is responsible for the reduction in bulk protein synthesis observed after prolonged H2O2 treatment. They go on to provide evidence that cell survival in H2O2 is enhanced by nuclear association of TyrRS (dependent on the NLS), and that in its absence, conferred by the NLS_mut, apoptosis is increased. They also show that ROS increases by preventing TyrRS nuclear localization by the NLS_mut, and that this effect as well as decreased cell survival for this mutant in H2O2 can be rescued by the translation elongation inhibitor harringtonine.

    The results presented in this report provide some support for the main conclusions of the paper and the overall model presented in Fig. 4F. However, as detailed below, many of the main conclusions of the paper are based on correlations and lack direct experimental support, and a number of the experiments are not comprehensive enough with sufficient conditions and controls to establish that the effects observed can be attributed to enhanced nuclear localization of TyrRS in response to H2O2. Considering the statements in the abstract, the evidence is reasonably strong that nuclear localization of TyrRS leads to inhibition of global translation at a stage later than that of eIF2α/ATF4 and mTOR responses, and that excluding TyrRS from the nucleus increases apoptosis under prolonged oxidative stress (although even this last point requires better documentation). However, the evidence is inadequate in several respects to claim that TyrRS directly represses the transcription of translation-related genes by recruiting TRIM28 or NURD complex, and as claimed on p. 13 of the Discussion, that the repression of the four AARS genes and the gene for eEF1A accounts for the reduction in bulk protein synthesis on H2O2 treatment.

    Major issues:

    -Evidence is lacking that the binding of TyrRS to the AARS/eEF1A genes is functionally important for the repression of any of the 6 putative target genes upon increased nuclear localization of TyrRS conferred by the NLS_mut or in response to H2O2. This would require ChIP analysis of TyrRS binding to the target genes for WT vs. NLS_mut TyrRS in H2O2-treated cells; and CRISPR mutagenesis of the putative TryRS binding site in the genome and analysis of transcription in the presence and absence of H2O2 for at least one of the putative TyrRS target genes.

    -Evidence from ChIP analysis is lacking that TRIM28, HDAC1, or the NURD complex are recruited to the AARS/eEF1A genes at native levels of TyrRS in a manner dependent on the NLS and stimulated by H2O2, as the ChIP experiments involved only overexpressed WT TyrRS in non-stressed cells. It is also unclear whether H3K27Ac levels at the putative target genes decline at endogenous levels of TyrRS on treatment with H2O2. Similarly, evidence is lacking that the physical association of TyrRS with these co-repressors is dependent on the NLS and stimulated by H2O2, as the co-IP analysis was limited to endogenous WT TyrRS in non-stressed cells.

    -Evidence is lacking that the cofactors TRIM28, HDAC1, or CHD4 are required for the down-regulation of target gene transcription on H2O2 treatment, which would require knock-down or elimination of these factors by CRISPR accompanied by analysis of target gene transcription +/- H2O2.

    -Direct evidence is lacking from ChIP analysis of RNA Pol II that the transcription of the AARS/eEF1A genes is reduced on H2O2.

    -Evidence is lacking that the repression of bulk protein synthesis is actually mediated by the reduced expression of the 4 AARSs and eEF1A. The fact that the TyrRS-E196K mutation enhances repression of bulk translation and also repression of 3 of the 5 target genes does support the idea that the repression of the target genes is instrumental in reducing protein synthesis, but again, this is still a correlation. There is no evidence that the reduced expression of the AARSs is sufficient to reduce charging of the cognate tRNAs, or that the reduced expression of eEF1A decreases the rate of translation elongation in cells or cell extracts.

    -There is an important lack of information provided needed to evaluate the quality and significance of the ChIP-seq analysis of TyrRS binding to DNA. No details are provided concerning the ChIP-seq analysis of V5-tagged TyrRS to indicate how the TyrRS occupancy peaks were identified and distinguished above background signal from the cells expressing V5 tag alone, whether replicates were examined to provide statistical significance for the identified occupancy peaks, and the sequencing library depths. No genome browser views were provided to show the signals from the cells expressing V5-TyrRS vs V5 alone to demonstrate the quality and reproducibility of data from replicates. The supplementary table S1 describing these data was even omitted from the submission, and it's unclear whether these data are being deposited in GEO.

    -There is an important lack of information provided needed to evaluate the quality and significance of the mass-spec analysis of TyrRS interacting proteins. No details are provided about the statistical significance of the protein interactions identified by mass-spec analysis of the affinity-purified TyrRS; and a negative control for non-specific association seems not to have been included in the analysis. The supplementary table describing these data was even omitted from the submission.

    -It's unclear whether the motif described in Fig. 3A was found under the peaks of TyrRS occupancy in the various genes showing TyrRS binding in the ChIP-seq experiments, nor whether its occurrence is statistically significant. It was not indicated that the motif coincides with the peak ChIP-seq occupancies for TyrRS, and if not, how this could be explained.

    -Evidence is lacking that harringtonine treatment reduced bulk protein synthesis under the conditions where it suppressed the effects of the TryRS NLS mutation in elevating ROS and decreasing cell survival.

    -In general, the figure legends are poorly written in lacking important details about the nature of the TyrRS being examined in the experiment (tagged vs endogenous; overexpressed vs. native levels), and also whether oxidative stress was imposed in the experiment, and if so, the exact conditions for the treatment. Figure legends should contain all of the critical details needed to understand and evaluate the significance of the experimental results without having to search elsewhere in the paper for them.

    -It needs to be clarified whether the mini-TyrRS construct lacks the NLS, and the significance of its behavior as a negative control for the effects of overexpressing WT TyrRS.

    -For the experiment in Fig. 5B, quantification of the fraction of caspase-3 or PARP cleaved from biological replicates is required.

    -The experiment in Supp. Fig. S4 lacks the results from cells untreated with H2O2 to ensure that these proteins were being induced by H2O2 in their hands.

  6. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript. Alan G Hinnebusch (Eunice Kennedy Shriver National Institute of Child Health and Human Development) served as the Reviewing Editor.

  7. Version published to 10.1101/2020.06.07.138792v2 on bioRxiv
  8. Version published to 10.1101/2020.06.07.138792v1 on bioRxiv