Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression

Editorial comment:

When you submit the revised version of your manuscript, we would also like you to revise the title to make it more broadly understandable and more readable. The presence of three acronyms in the present title, combined with its length, reduce the readability of the title so, if possible, we would be grateful if you could reduce the number of acronyms and/or length of the title.

We revised the title of the manuscript to a shorter variant and reduced the number of acronyms. The new title is: “Translation of leaders is pervasive in genes resistant to eIF2 repression”.

Two of the reviewers noted that a mechanistic explanation of how uORFs provide translational resistance to stress is lacking, however.

In addition, the work does not break new ground in our understanding of translational control during stress and eIF2 phosphorylation. No new mechanisms of translational control were described, but rather the study provides a broader picture supporting the idea that certain uORFs are central to mechanisms facilitating translation during stress.

Because the results are novel and important we are prepared to consider a revised version that will address some important questions raised by the reviewers as follows:

1) Given the small number of eIF2-alpha phosphorylation resistant transcripts, it is important to validate them by RT-qPCR/polysome profiling. In addition, it is rather surprising that the list of stress-resistant mRNAs does not include mRNAs that were reported to possess IRESs. This should be discussed.

We agree that validation of the predictions using an alternative method is important. However, we have concerns whether RT-qPCR of polysome fractions would be applicable to our case. For most stress-resistant mRNAs identified in our study, the translational response is accompanied with a significant reduction of ribosome occupancy at their uORFs. Therefore, these mRNAs may shift to lighter polysome fractions even if translation of the main coding region is increased. While ribosome profiling provides information on local changes of ribosome density within the same mRNA, polysome profiling cannot distinguish translation of the main coding region from translation of a uORF.

Therefore, in order to validate ions we chose to test the ability of 5’ leaders to provide resistance using mRNA constructs with reporter genes. We created three new reporters and demonstrated that in addition to PPP1R15B, and IFRD1 (tested earlier) leaders of UCP2, SLC35A4 and PTP4A1 are sufficient for stress-resistant translation (Figure 4–figure supplement 2).

The phenomenon of internal ribosome entry site (IRES) initiation has been known for decades since its discovery in EMCV and Poliovirus. However, only a few examples of IRESes were reported to be independent of eIF2 inactivation and to be able to operate in eIF2 independent mode. We extracted the list of human mRNAs (reported to contain IRES) from IRESite and address the question whether these mRNAs are resistant to arsenite treatment. We did not find any of these mRNAs to be resistant to arsenite treatment. However, not all of these mRNAs are expressed at a level sufficient for detecting resistance. We discuss this now in the manuscript, we also made a new table (Table 1) that lists mRNAs that were reported to have IRES.

2) The manuscript should be more precise in explaining the calculation that was used to classify gene transcripts that are resistant to arsenite stress. Classification of resistant mRNAs could be based on either the change in RNAseq reads or the Z score. This clarification is important because mRNAs that have a positive Z score may still experience a reduction in translation during arsenite treatment, in which case the Z score may not be an the best measure of mRNA resistance. Explain and justify which calculation was used to classify mRNAs as being resistant and provide a full description of what delineates translation resistance from repression during arsenite stress.

Z-score transformation is widely used for differential gene expression analysis, because it provides a simple method for comparing changes of gene expression across genes expressed at different levels. This cannot be done with fold changes because the same fold change may be statistically significant for a highly expressed gene, but not for a low expressed gene. This is because measurements for low expressed genes are expected to have higher variability and lower accuracy due to poorer sequencing coverage. This is clearly seen in scatter plots (Figure 1–figure supplement 1A) where the distributions of read counts have higher variability for genes with low read count.

Nonetheless, we agree that the information on absolute changes of expression is also important and therefore we provide both. However, our ranking of resistance is based on the Z-score and we now describe this in greater detail in the manuscript. We also provide a new reference to a seminal review that describes the topic in further detail: Quackenbush, 2002, Nat Genet, 2002, 32:496-501 PMID: 12454644.

It is true that genes experiencing a reduction of translation may have a high positive Z-score. However, a high Z-score would indicate that this reduction is exceptionally low in comparison with the reduction experienced by the rest of genes. This indicates resistance. On the contrary a high fold change does not necessarily indicate the resistance. If the Z-score magnitude is low, it means that such change (no matter how high) may occur between two replicas produced under the same conditions and therefore such a change may not have biological meaning. A low Z-score magnitude does not necessarily mean that the corresponding mRNA is not resistant. It means that we cannot detect it, either because the mRNA is not resistant or because the level of its expression is insufficient for resistance detection.

3) The manuscript should delineate preferential translation from translation resistance (sometimes called tolerance). In preferential translation there is poor mRNA translation during non-stressed conditions and high levels of translation in response to stress and eIF2 phosphorylation. The literature indicates that ATF4 is an example of preferential translation in response to eIF2 phosphorylation as supported by Figure 1A. Curiously in this manuscript, arsenite stress did not induce the expression of the luciferase reporter with the 5'-leader of the ATF4 transcript (Figure 4A) and there was perhaps only a modest enhancement of ribosome occupancy in the mRNA CDS during arsenite stress as judged by ribosome profiling. Perhaps arsenite differs from other stress conditions that were reported to induce preferential translation of ATF4 mRNA (e.g. pharmacological inducers of ER stress).

In order to delineate “preferential translation” from “tolerance” in a high-throughput study we need to establish a quantitative definition of “preferential translation” and “tolerance”. If we define “preferential translation” as an increase of translation and “resistance” as a weak decrease in translation, then genes with a high Z-score and a fold change >1 should be classified as “preferentially translated” and those with high a Z-score and a fold change <1 as “tolerant”.

Based on this definition, ATF4 is preferentially translated in our conditions. Western blotting (Figure 1A) shows increase of ATF4 protein levels. The actual level of increase is likely to be an overestimate because under normal conditions ATF4 is hydroxylated and rapidly degrades (see Koditz et al., 2007, Blood 110:3610-7). Stabilization of ATF4 under stress conditions may lead to its accumulation irrespective of a change in translation. Ribosome profiling (Figures 1G and 2 as well as Supplementary Table) also indicates increase of ATF4 mRNA translation by ∼30%. The reporter assay in HEK293 cells (Figure 4A) shows almost no change. The reporter assay in Huh7 cells (Supplementary Figure S3B) shows about 25% increase of translation. Given the technical limits of the accuracy of the methods (note the error bar for the reporter assays), the data are in agreement. Importantly, we report normalized absolute Fluc values rather than Fluc/Rluc values (which is more common practice). In our case upon arsenite treatment Fluc/Rluc values significantly increase due to strong inhibition of Rluc translation.

Therefore, while it is possible and even likely that the stress response to arsenite treatment differs from the response to other stresses inducing ATF4 translation, we cannot draw such a conclusion solely based on ATF4 behavior. Relevant to this, in experiments not described in the manuscript we also observed variation in ATF4 translation response depending on the severity of the stress (concentration of arsenite and duration of treatment). The translation of a particular ORF under stress condition is modified by the global reduction of translation and an individual mechanism of translation activation. It can be expressed as TE(stress)=[TE(normal)Ka]/Ki, where Ka represents mRNA-specific activation under given condition and Ki represents global inhibition. When Ka>Ki, TE(stress)>TE(normal) and the mRNA is preferentially translated. When Ka<Ki, TE(stress)<TE(normal) and the mRNA is stress tolerant. Ka/Ki is unlikely to be a constant, thus the same mRNA could be tolerant or preferentially translated depending on conditions. To illustrate, consider the extreme points: under very low concentrations of eIF2 phosphorylation no change is likely; under very high concentrations, all eIF2 dependent translation will cease, including for resistant genes. Between these points there may be specific conditions under which translation of a specific mRNA is higher than that under normal conditions (preferentially translated), but it certainly would not be so at the entire range of phosphorylated eIF2 concentrations.

We decided to introduce an alteration to our reporter assay in comparison with the original to make it more relevant to address the effect on ongoing translation. In the original work mRNAs were transfected into the cells simultaneously with arsenite treatment. In the modified assay, we treated cells with either arsenite or cycloheximide an hour after transfection. While cycloheximide blocks all translation, arsenite treatment permits translation of reporter mRNAs with leaders from resistant mRNAs. Interestingly, for SLC35A4 mRNA, arsenite treatment results in the production of 15 times more luciferase than that for cycloheximide treatment.

4) SLC35A4 appears to be the only gene that showed strong enhanced translation in response to arsenite translation (i.e. preferential translation). Does the 5'-leader of the SLC35A4 gene transcript confer enhanced expression in a reporter assay during arsenite stress?

For the revised version of the manuscript we designed a reporter construct with SLC35A4 5’ leader. The reporter assay demonstrated only moderate upregulation of the main ORF translation (Figure 4–figure supplements 1 and 2). However, SLC35A4 resistance was still the strongest in comparison with other tested 5’ leaders. Although the gene was found to have an apparently large translational increase detected with ribosome profiling it was associated with a large degree of uncertainty due to its very low expression as explained in our answer to the comment 2. Thus the results are not conflicting.

5) Do the uORFs in the resistant gene transcripts display any differences from those whose translation was repressed during arsenite stress?

The organization of uORFs (in terms of their numbers and length) varies considerably among 5’ leaders of all resistant mRNAs as can be seen in Figure 2. However, the presence of at least one comparatively long uORF (>20 codons) is a common feature (see Figure 3E).

To compare 5’ leaders of resistant and repressed mRNAs we analyzed the nucleotide context of uORFs initiation starts (Kozak) and secondary structure potential within 5’ leaders. We have not found significant difference in the distribution of Kozak contexts (Figure 3–figure supplement 1). Also see our response to the comment 7 below.

We found that the resistant mRNAs have lower secondary structure potential (based on free energy estimates of putative RNA structures) within the first 240 nt of the leaders in comparison with an average for non-resistant mRNAs. However, many non-resistant mRNAs have even lower RNA secondary structure potential (Figure 3–figure supplement 1).

6) The manuscript refers to eIF2 phosphorylation and translational control, as highlighted in the title, but there were no experiments establishing cause/effect between eIF2 phosphorylation and the translation expression of a gene. Rather, it was only inferred based on translational control mechanisms previously described in the literature for ATF4 and the related ISR genes.

We believe that the comment can be formulated as the following. There are no experiments demonstrating that the translation control observed in response to the arsenite treatment is mediated via eIF2 phosphorylation.

To demonstrate that translational control in response to arsenite treatment is mediated through eIF2 phosphorylation, it is necessary to show that the treatment results in eIF2 phosphorylation and that it does not affect translation by other means. The first part, the causative link between arsenite treatment and eIF2 phosphorylation has been established before (see McEwan et al., 2005, JBC, 280: 16925-33, PMID: 15684421. To address the second part we used two positive controls, HCV and ATF4. HCV can operate in eIF2 independent mode. Its translation was not inhibited by arsenite treatment suggesting that translation elongation was not affected (Figure 4). As noted by the referee ATF4 was also a control whose translation control mechanism was established previously.

To rule out other pathways affecting cap-dependent initiation we tested whether 5’ leaders of arsenite resistant mRNAs also resistant to mTOR inhibition and tested the reports in the presence of torin-1. No differential suppression was observed (Figure 4–figure supplement 3). Finally, in order to eliminate a possibility of a translational response to arsenite treatment instigated by means other than eIF2 phosphorylation we analyzed expression of the reporters in the presence of DTT, another well-known inducer of eIF2 phosphorylation (see Prostko et al., 1993, MCB, 127-128:255-65, PMID: 7935356). The results were similar to those observed under the arsenite treatment (Figure 4–figure supplement 1). It is, of course, unlikely that arsenite treatment effects eIF2 exclusively. However, based on our data, we believe that the major effect on translation is due to eIF2 phosphorylation.

7) Could you include analysis of mRNAs either for the context around the AUG in the uORF or whether non-AUG codons might be used (if in a good context)? This would require additional data mining but no additional experiments. This might also allow for an explanation of the 11 AUGs in the SLC35A4 mRNA that do not seem to serve as initiation codons.

Thank you for this suggestion. We have included such analysis in the revised version of the manuscript (Figure 3–figure supplement 1). Surprisingly not all of the translated uORFs contain initiation codons in good context.

We also checked how the Kozak context of uORF in 5’ leader of IFRD1 affects the resistance. The wild type uAUG in its leader is in suboptimal Kozak context (-3A but +4U). The +4U/G substitution slightly inhibited the translation of the reporter, most likely due to increased translation of the uORF, but it did not alter the reporter response to the stress (Figure 4–figure supplement 3).

8) A major concern is the sampling time. From Figure 1A it appears that the hyper-phosphorylation of 4E–BP1 disappears at 1 hour (although the gel is not of high enough quality to tell; there is the possibility of smiling of the gel bands). The GAPDH loading control is overexposed and, therefore, inadequate. The phosphorylation of eIF2alpha is up at 0.5 hours but maximal at 2 hours. The concern is the possible crossover of 4E–BP activation (loss of phosphorylation) and the onset of eIF2alpha phosphorylation. The authors should more rigorously exclude indirect targeting of mTORC1–4EBP and mTORC1–S6K pathways of protein synthesis regulation by arsenite treatment. The 4EBP1 blot on Figure 1A should be less exposed to make the bands more discernible. The blots showing no change in p-4EBP1 and p-S6K levels under their conditions of arsenite treatment should also be shown. Do the authors have an independent control for determining the level of cap-dependent translation vs. the level of eIF2-dependent translation?

Thank you for pointing this out. We substituted the western blot in Figure 1A with one of a better quality and a lower exposure. Further we added an additional line with the same control lysate (last line) to ensure the absence of gel artifacts. We did not detect any significant changes in 4E–BP1 dephosphorylation after 30 min of 40 µM arsenite treatment (compare lanes 1 and 2). We did not detect changes in the levels of phosphorylated p70 S6k and phosphorylated rpS6 during such treatment. We added the western gel to Figure 1A panel.

We also addressed this question by analyzing how mTOR sensitive mRNAs responded to the stress using our ribosome profiling dataset. We mapped mRNAs which were shown to be downregulated upon treatment with mTOR inhibitor PP242 (Hsieh et al., 2012, Nature, 485:55-61 PMID: 22367541). Their translational efficiency decrease upon arsenite treatment was ∼25% greater than the average (6.54-fold v 5.2-fold), see (Figure 1–figure supplement 2D). Thus, while it is possible that arsenite treatment effects mTOR pathway, its impact would be subsidiary to the eIF2 pathway. Interestingly, there are several outliers like PABPC1 and RPL12 (Figure 1–figure supplement 2D, left bottom corner) which behaves differently from the rest of mTOR sensitive mRNAs and which are exceptionally sensitive to the arsenite treatment. Perhaps, these mRNAs are sensitive to both types of stress pathways.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

There are two remaining major concerns:

The first concern was the bioinformatics and reluctance to incorporate fold-change into the core statistics of the manuscript. This is a shortcoming and confuses interpretation of the identification of genes whose translation is induced upon arsenite stress. This is the potential novelty of the manuscript.

We feel that this concern, at least in part, is a result of misunderstanding. The fold-change statistics was incorporated in both, the original and the revised version of the manuscript. Fold changes of RNAseq, riboseq and TE signals are given for every gene in the source data file 1. They are provided for selected genes in Figure 1F and are shown in axis y in scatter plots in Figure 1E. Perhaps, the confusion came from the rebuttal letter where we overzealously explained a need to estimate statistical significance of change folds using Z-score. We apologise for the confusion caused.

The other major concern was the experimental validation of the ribosome profiling. The reporter assays do not fully support the profiling data, and the controls, e.g. ATF4, did not follow the expected induced translation with increased eIF2 phosphorylation. In the rebuttal letter (the Methods of the manuscript were not detailed on this point), the reporter mRNA was stated to be transfected in cultured cells for 1 hour prior to arsenite treatment for an additional 2 hours (the Methods section stated the later treatment time). There is a concern that the non-stressed cells are not truly non-stressed as there is likely to be insufficient recovery from the transfection using lipofectamin 2000. This could explain why there were virtually no genes that were preferentially translated in the reporter assay, just different degrees of repression. The non-stressed conditions were not without the underlying membrane stress of the transfection agent. Also 40 micromolar is a large amount of arsenite to stress the cells.

We considered the possibility that transfection, as any other manipulation with cultured cells, may stress the cells inducing eIF2 phosphorylation. We conducted a new experiment that we now describe in the manuscript (also see response further below). In this experiment we overexpressed ppp1r15a encoding GADD34 for one day prior to stress induction. GADD34 should dephosphorylate eIF2 if it occurs due to the membrane stress. However, we did not observe significant increases in reporter assays in comparison with cells where GADD34 was not overexpressed, see Figure 4–figure supplement 4. Therefore the stress induced by transfection, if occur, is unlikely to be significant.

In addition, it was demonstrated earlier by Anderson and coauthors that, unlike nucleofection, lipofection does not induce eIF2 phosphorylation (see Figure 2 in BR Anderson et al., 2013, Gene Therapy, PMID: 22301437).

We agree that it is possible that 40 uM concentration of arsenite is too high for obtaining the conditions for maximum translation of ATF4 mRNA. We optimized the arsenite concentration and duration of the stress to generate conditions under which global translation would be substantially but not completely inhibited as can be judged from the polysome profiles. We believe that such conditions would be the most informative for the ribosome profiling experiment.

Below is the description of the concerns in more detail:

The first is centered on reviewer concern number 3 that indicated that there should be clear delineation between the levels of translational control, e.g. preferential translation and translation resistance (or tolerance). In the first paragraph of the response to reviewer concern 3 the authors provide a definition of preferential translation as an increase of translation with a high Z-score and a fold-change >1 and tolerant with a high Z-score and fold change as <1. This was a straightforward conceptual framework involving Z-score and fold-change that should be incorporated into the manuscript analysis and ranking.

We agree that such a classification could be useful and incorporated it in the new version of the manuscript (see the beginning of the subsection “Efficient translation of uORFs combined with inefficient translation of CDS is a predictor of stress resistant mRNAs”).

The second concern is how the results from ribosomal profiling analysis compare to other approaches previously used in the literature. This is the heart of the manuscript. In Reviewer concern 1, the response stated that qPCR and polysome analysis would not be appropriate for this analysis despite its utility in the literature. As a consequence, the manuscript elected to rely on reporter assays involving transfections of mRNAs featuring 5'-leaders of targeted gene transcripts upstream of the firefly luciferase coding sequence. ATF4, a well-characterized preferentially translated gene transcript was used as a positive control, and the artificial gene transcript pGL3 was an example of a gene that is repressed by eIF2 phosphorylation and stress. Curiously, ATF4 translation was not preferentially translated, but displayed a partial resistance to arsenite stress. The authors’ argument for why ATF4 ribosome profiling data and reporter data are in agreement (even though the ATF4 reporter is not induced) is based on “technical limits of methods accuracy”. The details of the reporter assay were not fully clear in the original or present manuscript, but the response letter provided additional details in the response to reviewer concern number 3. The mRNAs were transfected into HEK293T cells and 1 hour after transfection the cells were treated with arsenite for 2 hours prior to harvesting and analysis. There are some concerns about the timing in this protocol, such as would 1 hour be sufficient time for the resolution of the membrane stress triggered by the transfection protocol? Furthermore, it is suggested that even though the SLC35A4 reporter was not inducible during arsenite stress, that this is not in disagreement with the ribosome profiling dataset because SLC35A4 mRNA levels are low in the profiling dataset. As the purpose of using the Z-score was to eliminate variability and error in the assessment of the ribosome profiling dataset, is this sufficient justification for why the two assay results do not appear to be consistent? In a related point in concern number 6, it was suggested that the manuscript should focus on “establishing cause/effect between eIF2 phosphorylation and translation expression of a gene.” This could be directly addressed in the reporter assay, and the explanation involving an alternative stress (DTT) and mTOR did not appear to adequately address this basic concern.

Thank you for the useful comments and suggestions.

1) We now addressed the “cause/effect” concern by conducting the following experiment to address the impact of eIF2 phosphorylation on arsenite treatment in our reporter assay:

One day prior to reporter mRNA transfection, we transfected the cells with the plasmid for overexpression of FLAG-tagged full length human ppp1r15a (GADD34) phosphatase subunit. This almost completely abrogates eIF2 phosphorylation and the inhibitory effect of arsenite on translation of reporters (see new panel D in Figure 4). Upon arsenite treatment, control Rluc mRNA translation was downregulated 2 fold. Compare this with more than 6 fold when GADD34 was overexpressed. Translation of IFRD1 reporter was not affected at all. From this experiment, we can conclude that inhibitory effect of arsenite is mostly accounted for by eIF2 phosphorylation. This experiment is described now in the penultimate paragraph of the subsection “5’ leaders of several newly identified mRNAs are sufficient to provide resistance to the translation inhibition”.

2) Regarding small disagreement between ribosome profiling experiments and reporter assays:

We do not expect full concordance between these two assays. First, the signals provided by these two approaches are quantitatively different. Ribosome profiling provides a snapshot of translation after 0.5 hours of stress. The reporter assay relies on the amount of luciferase produced during 2 hours post transfection. In other words, if we think of translation as a function, ribosome profiling provides a signal at single time point, while the reporter assay corresponds to the integral of the signal after a long time period. Hence the only scenario when both approaches are expected to fully converge would be when upon very rapid phosphorylation of eIF2 its levels would not change over time. It is not the case with arsenite treatment when continuous increase of eIF2 phosphorylation is evident. Second, reporter assays do not depend on natural gene expression levels, while ribosome profiling does. Because of that discordance is particularly likely for low expressed genes. As noted by the referee, SLC35A4 is a good example of this. The change fold observed in ribosome profiling data was high enough to surpass the threshold for statistical significance (Z>4), yet the absolute level is unreliable due to high signal variability for such a lowly expressed gene. Subsequently reporter assays confirmed SLC35A4 as the most resistant (even induced) mRNA, but did not confirm the absolute levels.

3) Regarding the low levels of ATF4 mRNA preferential translation:

Assuming that all mRNAs identified in this study operate in eIF2-dependent manner, their translation efficiency is likely to change during progression of the stress for the reasons we outlined above. It is conceivable that some “preferentially translated” mRNAs could become “resistant” when the level of active eIF2 decreases further. Indeed, we observed increases in absolute levels of ATF4 reporter when a lower concentration of arsenite was used. Thus it is likely that the concentration of arsenite is too high for the optimal levels of ATF4 translation. As we pointed out earlier we did not aim to create conditions for the optimal ATF4 translation.

Other comments:

1) The English is still a bit rough, but not to the point where the reader cannot understand what is intended.

We have made several minor alterations to the draft and have shown it to other colleagues who are native English speakers.

2) Although in the position of being a “break out” paper, it would be nice if there was some effort expended at a possible explanation although it would be obviously speculative (see below). To just leave the paper to end on “there a black box that allows for this resistance to eIF2 repression” is unsatisfying (although accurate).

Thank you. We conducted an experiment which may shed some light on the features required for preferential/resistant translation. We observed earlier that all stress resistant mRNAs possess efficiently translated uORFs. So, we hypothesized that some features of 5’ leaders upstream of uORFs may be important for the regulation. We decided to address this issue with reporter constructs. To this end, we created two additional reporters based on control pGL3 and IFRD1, where we added 5’ terminal stem loop of intermediate stability. As expected, addition of this stem loop resulted in a 3-4 fold decrease of both reporters yield under normal conditions. Interestingly, when arsenite stress was induced, SL+IFRD1 (IFRD1 with stem loop) construct was no longer resistant, while translation of SL+pGL3 and pGL3 constructs was similarly reduced. From this, we can propose that high loading of preinitiator complexes to uORF is necessary for stress resistance. Next, we addressed the question of whether the specific sequence upstream of IFRD1 uORF is required for the regulation. To this end, we substituted it with an artificial single stranded (CAA)6 leader of the same length. This modification did not affect stress resistance. Thus, we hypothesize that for resistant translation, the uORF have to be situated under the control of a leader that allows high initiation rates at uORF. These experiments are now described in a new subsection “Unstructured leader sequence upstream of IFRD1 uORF is necessary for stress resistance”.

3) The authors stress the resistance to eIF2 repression, but do not stress as well that most of the proteins are poorly expressed. Using the number of reads scale in Figure 2 where ATF4 is 43, the other proteins fall mostly into the 0 to 2-8 range. This inefficiency is also reflected in the reporter assays in Figure 4 where pGL3 is 100%, the remaining normal configuration for most constructs yields expression levels in the 5 to 10% of pGL3 range.

Thank you. It is indeed so, as can be seen in Figure 3C. We now mention this in the Discussion.

4) That said, the low level of expression may be entirely appropriate for phosphatases, kinases, transcription factors, etc. that are not required in large amounts.

We agree and thank you for providing a thought for the Discussion, which we have now expanded to accommodate this suggestion.