Perturbations in eIF3 subunit stoichiometry alter expression of ribosomal proteins and key components of the MAPK signaling pathway

Reviewer #1 (Public Review):

Summary:

In this manuscript, Herrmannova et al explore changes in translation upon individual depletion of three subunits of the eIF3 complex (d, e, and f) in mammalian cells. The authors provide a detailed analysis of regulated transcripts, followed by validation by RT-qPCR and/or Western blot of targets of interest, as well as GO and KKEG pathway analysis. The authors confirm prior observations that eIF3, despite being a general translation initiation factor, functions in mRNA-specific regulation, and that eIF3 is important for translation re-initiation. They show that the global effects of eIF3e and eIF3d depletion on translation and cell growth are concordant. Their results support and extend previous reports suggesting that both factors control the translation of 5'TOP mRNAs. Interestingly, they identify MAPK pathway components as a group of targets coordinately regulated by eIF3 d/e. The authors also discuss discrepancies with other reports analyzing eIF3e function.

Strengths:

Altogether, a solid analysis of eIF3 d/e/h-mediated translation regulation of specific transcripts. The data will be useful for scientists working in the Translation field.

Weaknesses:

The authors could have explored in more detail some of their novel observations, as well as their impact on cell behavior.

Reviewer #2 (Public Review):

Summary:

mRNA translation regulation permits cells to rapidly adapt to diverse stimuli by fine-tuning gene expression. Specifically, the 13-subunit eukaryotic initiation factor 3 (eIF3) complex is critical for translation initiation as it aids in 48S PIC assembly to allow for ribosome scanning. In addition, eIF3 has been shown to drive transcript-specific translation by binding mRNA 5' cap structures through the eIF3d subunit. Dysregulation of eIF3 has been implicated in oncogenesis, however the precise eIF3 subunit contributions are unclear. Here, Herrmannová et al. aim to investigate how eIF3 subcomplexes, generated by knockdown (KD) of either eIF3e, eIF3d, or eIF3h, affect the global translatome. Using Ribo-seq and RNA-seq, the authors identified a large number of genes that exhibit altered translation efficiency upon eIF3d/e KD, while translation defects upon eIF3h KD were mild. eIF3d/e KD share multiple dysregulated transcripts, perhaps due to both subcomplexes lacking eIF3d. Both eIF3d/e KD increase the translation efficiency (TE) of transcripts encoding lysosomal, ER, and ribosomal proteins. This suggests a role of eIF3 in ribosome biogenesis and protein quality control. Many transcripts encoding ribosomal proteins harbor a TOP motif, and eIF3d KD and eIF3e KD cells exhibit a striking induction of these TOP-modified transcripts. On the other hand, eIF3d KD and eIF3e KD lead to a reduction of MAPK/ERK pathway proteins. Despite this downregulation, eIF3d KD and eIF3e KD activate MAPK/ERK signaling as ERK1/2 and c-Jun phosphorylation were induced. Finally, in all three knockdowns, MDM2 and ATF4 protein levels are reduced. This is notable because MDM2 and ATF4 both contain short uORFs upstream of the start codon, and further support a role of eIF3 in reinitiation. Altogether, Herrmannová et al. have gained key insights into precise eIF3-mediated translational control as it relates to key signaling pathways implicated in cancer.

Strengths:

The authors have provided a comprehensive set of data to analyze RNA and ribosome footprinting upon perturbation of eIF3d, eIF3e, and eIF3h. As described above in the summary, these data present many interesting starting points for understanding additional roles of the eIF3 complex and specific subunits in translational control.

Weaknesses:

- The differences between eIF3e and eIF3d knockdown are difficult to reconcile, especially since eIF3e knockdown leads to a reduction in eIF3d levels.

- The paper would be strengthened by experiments directly testing what RNA determinants allow for transcript-specific translation regulation by the eIF3 complex. This would allow the paper to be less descriptive.

- The paper would have more biological relevance if eIF3 subunits were perturbed to mimic naturally occurring situations where eIF3 is dysregulated. For example, eIF3e is aberrantly upregulated in certain cancers, and therefore an overexpression and profiling experiment would have been more relevant than a knockdown experiment.

Reviewer #3 (Public Review):

Summary:

In this article, Hermannova et al catalog the changes in ribosome association with mRNAs when the eukaryotic translation initiation factor 3 is disrupted by knocking down subunits of the multisubunit protein. They find that RNAs relying on TOP motifs for translation, such as ribosomal protein RNAs, and RNAs encoding proteins that modify other proteins in the ER or components of the lysosome are upregulated. In contrast, proteins encoding components of MAP kinase cascades are downregulated when subunits of eIF3 are knocked down.

Strengths:

The authors use ribosome profiling of well-characterized mutants lacking subunits of eIF3 and assess the changes in translation that take place. They supplement the ribosome association studies with western blotting to determine protein level changes of affected transcripts. They analyze what is being encoded by the transcripts undergoing translation changes, which is important for understanding more broadly how translation initiation factor levels affect cancer cell translatomes.

Weaknesses:

(1) The data are presented as a catalog of effects, and the paper would be strengthened if there were a clear model tying the various effects together or linking individual subunit knockdown to cancerous phenotypes. It is unclear what the hypothesis is for cells having more MAPK activity with less of the MAPK proteins being translated, so the main findings of the paper become observational without context.

(2) The conclusions drawn are presented as very generalized other than in the last paragraph, but the experiments were only done in Hela cells. Since conclusions are being made about how translation changes affect MAP kinase signaling and there is mention in the abstract that dysregulation of these subunits is observed in cancer, at least one other cell line would need to be analyzed to provide evidence that the effects of subunit knockdown aren't cell-line specific.

(3) It is also unclear how replicates were performed and how many replicates were performed for several experiments. Biological replicates are mentioned, but what the authors did for biological replicates isn't defined and the description of the collection of cells for polysome/ribosome footprint/RNA seq samples makes it unclear whether the "biological replicates" are samples from separate transfections (true biological replicates) or different aliquots or wells from a single transfection (technical replicates) being run over a separate gradient. If using technical replicates, the data comparing the effects of knocking down D vs E vs H subunits are substantially weakened because subunit-specific differences could be the result of non-specific events that occurred in a transfection. It's also notable that while the pooled siRNAs will increase the potency of knockdown, it is possible that one or more of the siRNAs could have off-target effects, and analyzing individual siRNAs would be better for ensuring effects are specific.

(4) Many of the changes in protein levels reported by Western are subtle. Data from all western blots making claims of quantitative differences should really be quantified relative to nontreated over-loading control or total protein quantified from the gel, and presented with a degree of error from biological replicates to make conclusions about differences in protein levels between samples.

Author Response

eLife assessment

This study demonstrates mRNA-specific regulation of translation by subunits of the eukaryotic initiation factor complex 3 (eIF3) using convincing methods, data, and analyses. The investigations have generated important information that will be of interest to biologists studying translation regulation. However, the physiological significance of the gene expression changes that were observed is not clear.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, Herrmannova et al explore changes in translation upon individual depletion of three subunits of the eIF3 complex (d, e, and f) in mammalian cells. The authors provide a detailed analysis of regulated transcripts, followed by validation by RT-qPCR and/or Western blot of targets of interest, as well as GO and KKEG pathway analysis. The authors confirm prior observations that eIF3, despite being a general translation initiation factor, functions in mRNA-specific regulation, and that eIF3 is important for translation re-initiation. They show that the global effects of eIF3e and eIF3d depletion on translation and cell growth are concordant. Their results support and extend previous reports suggesting that both factors control the translation of 5'TOP mRNAs. Interestingly, they identify MAPK pathway components as a group of targets coordinately regulated by eIF3 d/e. The authors also discuss discrepancies with other reports analyzing eIF3e function.

We would like to note that the first sentence contains a typo; the correct expression is: “…of three subunits of the eIF3 complex (d, e, and h) in mammalian cells”.

Strengths:

Altogether, a solid analysis of eIF3 d/e/h-mediated translation regulation of specific transcripts. The data will be useful for scientists working in the Translation field.

Weaknesses:

The authors could have explored in more detail some of their novel observations, as well as their impact on cell behavior.

Many experiments are on-going in this direction. The original plan was to map all the effects in general and in as much detail as possible to select a few of them for future long-term projects.

Reviewer #2 (Public Review):

Summary:

mRNA translation regulation permits cells to rapidly adapt to diverse stimuli by fine-tuning gene expression. Specifically, the 13-subunit eukaryotic initiation factor 3 (eIF3) complex is critical for translation initiation as it aids in 48S PIC assembly to allow for ribosome scanning. In addition, eIF3 has been shown to drive transcript-specific translation by binding mRNA 5' cap structures through the eIF3d subunit. Dysregulation of eIF3 has been implicated in oncogenesis, however the precise eIF3 subunit contributions are unclear. Here, Herrmannová et al. aim to investigate how eIF3 subcomplexes, generated by knockdown (KD) of either eIF3e, eIF3d, or eIF3h, affect the global translatome. Using Ribo-seq and RNA-seq, the authors identified a large number of genes that exhibit altered translation efficiency upon eIF3d/e KD, while translation defects upon eIF3h KD were mild. eIF3d/e KD share multiple dysregulated transcripts, perhaps due to both subcomplexes lacking eIF3d. Both eIF3d/e KD increase the translation efficiency (TE) of transcripts encoding lysosomal, ER, and ribosomal proteins. This suggests a role of eIF3 in ribosome biogenesis and protein quality control. Many transcripts encoding ribosomal proteins harbor a TOP motif, and eIF3d KD and eIF3e KD cells exhibit a striking induction of these TOP-modified transcripts. On the other hand, eIF3d KD and eIF3e KD lead to a reduction of MAPK/ERK pathway proteins. Despite this downregulation, eIF3d KD and eIF3e KD activate MAPK/ERK signaling as ERK1/2 and c-Jun phosphorylation were induced. Finally, in all three knockdowns, MDM2 and ATF4 protein levels are reduced. This is notable because MDM2 and ATF4 both contain short uORFs upstream of the start codon, and further support a role of eIF3 in reinitiation. Altogether, Herrmannová et al. have gained key insights into precise eIF3-mediated translational control as it relates to key signaling pathways implicated in cancer.

Strengths:

The authors have provided a comprehensive set of data to analyze RNA and ribosome footprinting upon perturbation of eIF3d, eIF3e, and eIF3h. As described above in the summary, these data present many interesting starting points for understanding additional roles of the eIF3 complex and specific subunits in translational control.

Weaknesses:

• The differences between eIF3e and eIF3d knockdown are difficult to reconcile, especially since eIF3e knockdown leads to a reduction in eIF3d levels.

We agree and discuss this problem thoroughly in the corresponding section of our study.

• The paper would be strengthened by experiments directly testing what RNA determinants allow for transcript-specific translation regulation by the eIF3 complex. This would allow the paper to be less descriptive.

We carried out bioinformatic analysis dealing with specific RNA determinants that is presented as the last chapter of our study. A detailed, transcript-specific analysis of these determinants is underway, however, we consider them beyond the scope for this article.

• The paper would have more biological relevance if eIF3 subunits were perturbed to mimic naturally occurring situations where eIF3 is dysregulated. For example, eIF3e is aberrantly upregulated in certain cancers, and therefore an overexpression and profiling experiment would have been more relevant than a knockdown experiment.

This is indeed true and so far we have generated several stable cell lines individually overexpressing selected eIF3 subunits implicated in the observed cancer phenotypes. However, this is a completely different project of one of our PhD students, which will be published as a comprehensive study when completed.

Reviewer #3 (Public Review):

Summary:

In this article, Hermannova et al catalog the changes in ribosome association with mRNAs when the eukaryotic translation initiation factor 3 is disrupted by knocking down subunits of the multisubunit protein. They find that RNAs relying on TOP motifs for translation, such as ribosomal protein RNAs, and RNAs encoding proteins that modify other proteins in the ER or components of the lysosome are upregulated. In contrast, proteins encoding components of MAP kinase cascades are downregulated when subunits of eIF3 are knocked down.

Strengths:

The authors use ribosome profiling of well-characterized mutants lacking subunits of eIF3 and assess the changes in translation that take place. They supplement the ribosome association studies with western blotting to determine protein level changes of affected transcripts. They analyze what is being encoded by the transcripts undergoing translation changes, which is important for understanding more broadly how translation initiation factor levels affect cancer cell translatomes.

Weaknesses:

(1) The data are presented as a catalog of effects, and the paper would be strengthened if there were a clear model tying the various effects together or linking individual subunit knockdown to cancerous phenotypes. It is unclear what the hypothesis is for cells having more MAPK activity with less of the MAPK proteins being translated, so the main findings of the paper become observational without context.

As the signaling pathways are very complex and there is a frequent crosstalk among them (c-Jun can be activated by the ERK pathway as well as the JNK pathway, activated ERKs can phosphorylate many different transcription factors, etc.), we opted not to investigate the reported results any further in this study. As mentioned above, we have several ongoing, long-term projects aiming to elucidate the consequences of the observed changes in protein levels as well as in the phosphorylation status of the MAPK pathway constituents. The take home message of the present study is that eIF3 subunits (d and e) have control over the expression of many proteins involved in the MAPK/ERK pathway and that there is an independent effect (already present in the downregulation of eIF3h, which does not affect the MAPK protein expression) that leads to activation of the ERK pathway, which may be a direct consequence of compromised eIF3 function in general.

(2) The conclusions drawn are presented as very generalized other than in the last paragraph, but the experiments were only done in Hela cells. Since conclusions are being made about how translation changes affect MAP kinase signaling and there is mention in the abstract that dysregulation of these subunits is observed in cancer, at least one other cell line would need to be analyzed to provide evidence that the effects of subunit knockdown aren't cell-line specific.

There are several notes emphasizing that the data presented in this study were obtained only in HeLa cells. We agree that further research in other cell lines will be needed to confirm that what we observed is a general phenomenon. Nonetheless, as noted in the discussion, other reports have already been published strongly indicating that this phenomenon is not unique to HeLa cells (Li et al., 2021, PMID:34520790, HTR-8/SVneo cells). We will review our conclusions and further clarify that our results so far only apply to Hela cells.

(3) It is also unclear how replicates were performed and how many replicates were performed for several experiments. Biological replicates are mentioned, but what the authors did for biological replicates isn't defined and the description of the collection of cells for polysome/ribosome footprint/RNA seq samples makes it unclear whether the "biological replicates" are samples from separate transfections (true biological replicates) or different aliquots or wells from a single transfection (technical replicates) being run over a separate gradient. If using technical replicates, the data comparing the effects of knocking down D vs E vs H subunits are substantially weakened because subunit-specific differences could be the result of non-specific events that occurred in a transfection. It's also notable that while the pooled siRNAs will increase the potency of knockdown, it is possible that one or more of the siRNAs could have off-target effects, and analyzing individual siRNAs would be better for ensuring effects are specific.

We can reassure this reviewer that our Ribo-seq and RNA-Seq libraries were prepared from true biological replicates, grown, and transfected at different times. In fact, for each biological replicate, we used a new aliquot of cells from cryostock from the same batch and transfected the cells with the same passage number only. Multiple biological replicates were grown and all underwent a series of control experiments (polysomes, qPCR, western blot) as described in the article. Based on the results, 3 samples were selected for Ribo-Seq library preparation and 4 for RNA-Seq. We decided to add a fourth replicate for RNA-Seq to increase the data robustness, because RNA-Seq is used to normalize FPs to calculate TE, which was our main metric analyzed in this article.

As for the usage of the siRNA pool from Dharmacon/Horizon – our current article builds on our previous studies (Wagner et al. 2014 PMID: 24912683; Wagner et al. 2016 PMID: 27924037 and Herrmannová et al. 2020 PMID: 31863585), where we thoroughly characterized the effects of downregulation of individual eIF3 subunits on the growth, translation, composition and stability of eIF3 complex and on the 43S preinitiation complex assembly and subsequent mRNA recruitment. In all of these studies, we used the same siRNAs pools, the same cells and the same transfection protocol; therefore, we are convinced that our results are as coherent and reproducible as can possibly be. We have never noticed any off-target effects. Moreover, the ON-TARGETplus siRNA technology we employed uses a patented modification pattern that reduces the incidence of off-targets by up to 90% compared to unmodified siRNA (see the supplier's website for more information).

(4) Many of the changes in protein levels reported by Western are subtle. Data from all western blots making claims of quantitative differences should really be quantified relative to nontreated over-loading control or total protein quantified from the gel, and presented with a degree of error from biological replicates to make conclusions about differences in protein levels between samples.

Generally speaking, we agree with the reviewer’s opinion. In the original version of our study, we felt that it was not necessary to perform a quantification analysis to support our conclusions as it was not important whether a given protein was downregulated to, for example, 60% or 70%, as long as its amount was visibly reduced. The main message resided in the general trend, i.e. that the whole pathway is affected in a similar way. Nevertheless, in order to properly address this criticism, we will provide quantifications in the revised paper.