DHX30 coordinates cytoplasmic translation and mitochondrial function contributing to cancer cell survival
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Abstract
DHX30 was recently implicated in the translation control of mRNAs involved in p53-dependent apoptosis. Here we show that DHX30 exhibits a more general function by integrating the activities of its cytoplasmic isoform and of the more abundant mitochondrial one. The depletion of both DHX30 isoforms in HCT116 cells leads to constitutive changes in polysome-associated mRNAs, enhancing the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing the translational efficiency of the nuclear-encoded mitoribosome mRNAs. Furthermore, depletion of both DHX30 isoforms exhibits higher global translation but slower proliferation, and reduced mitochondrial energy metabolism. Isoform-specific silencing established a role for cytoplasmic DHX30 in modulating global translation. The impact on global translation and proliferation were confirmed in U2OS and MCF7 cells, although the effect of DHX30 depletion on mitochondrial gene expression was observed only in MCF7 cells. Exploiting RIP, eCLIP, and gene expression data, we identified a gene signature comprising DHX30 and fourteen mitoribosome transcripts that we candidate as direct targets: this signature shows prognostic value in several TCGA cancer types, with higher expression associated with reduced overall survival. We propose that DHX30 contributes to cell homeostasis by coordinating ribosome biogenesis, global translation, and mitochondrial metabolism. Targeting DHX30 could, thus, expose a vulnerability in cancer cells.
Author summary
Translation occurs in the cell both through cytoplasmic and mitochondrial ribosomes, respectively translating mRNAs encoded by the nuclear and the mitochondrial genome. Here we found that DHX30, an RNA-binding protein implicated in p53-dependent apoptosis, enhances the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing that of the mitoribosome mRNAs when silenced. This coordination of the cytoplasmic and mitochondrial translation machineries affected both cell proliferation and energy metabolism, suggesting an important role for this mechanism in determining the fitness of cancer cells. Indeed, the analysis of publicly available cancer datasets led us to define a 15-genes signature that is able to affect the prognosis of a subset of cancer types. In this subset, we found that higher expression of the genes composing the signature is associated with a worse prognosis. We thus propose DHX30 as a potential vulnerability in cancer cells, that could be targeted to develop novel therapeutic strategies.
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###Reviewer #2:
The manuscript addresses a very interesting topic, namely the possibility that DHX30 protein exists in two alternatively transcribed variants that have a role, respectively, in the cytoplasm and in the mitochondria. The first of the two functions is relatively new and barely addressed in the literature. The mitochondrial localization has already been described in previous works where, among others, has been shown to be important for mitochondrial function, possibly acting at the transcriptional level. The experimental approach is largely based on the "specific" depletion of either one of the two isoforms, and a downstream analysis (RNAseq, a few biochemical endpoints). The phenotypic results are relatively few and the authors conclude that DHX30 may have a role in "...coordinating ribosome biogenesis, global translation …
###Reviewer #2:
The manuscript addresses a very interesting topic, namely the possibility that DHX30 protein exists in two alternatively transcribed variants that have a role, respectively, in the cytoplasm and in the mitochondria. The first of the two functions is relatively new and barely addressed in the literature. The mitochondrial localization has already been described in previous works where, among others, has been shown to be important for mitochondrial function, possibly acting at the transcriptional level. The experimental approach is largely based on the "specific" depletion of either one of the two isoforms, and a downstream analysis (RNAseq, a few biochemical endpoints). The phenotypic results are relatively few and the authors conclude that DHX30 may have a role in "...coordinating ribosome biogenesis, global translation and mitochondrial metabolism...".
The main criticism that I have of this work is that...although this term is often abused by editor's polite answers, it is rather preliminary. There are a consistent number of shortcuts that, in my mind, when taken all together, cast some doubts on the correct message. I will describe these limits by going systematically through the data.
In Figure 1, the authors describe the effects of shDHX30 on several endpoints: 1. The authors employ here a single shRNA which is really not sufficient given the very well known problem of off-target effects; 2. With the exception of a few confirmatory experiments the whole analysis is based on a single cell line; 3. In 1B there is a plot indicating the relative translation efficiency of ribosomal protein mRNAs. However the Supplementary Table 1 is not properly annotated and not all ribosomal mRNAs seem equally regulated; 4. The polysomal profiles have very low polysomes and very high 80S, raising some questions on the actual relevance of the regulation of Pol/Sub peak described in Fig. 1g (seen with a single shRNA); 5. The statement of increased ribosome biogenesis is not solid. The authors mention quantitation of 18S rRNA and nucleolar intensity of 18S staining. However, the state of the art must be pulse-chase analysis followed by autoradiography and/or Northern blotting of rRNA precursor, possibly with two shRNAs and perhaps even with a couple of cell lines; 6. The logic by which an increase in rRNA is co-regulated with an increase of translation of ribosomal protein mRNA is obscure and has no explanations: is signalling involved? Is it indirect? 7. The authors claim an effect on translation. The correct interpretation of the polysomal profile is a reduction in initiation of translation (which in itself brings back to the question of 6. what happens to mTOR signalling?). 8. The authors show a very clear increase in AHA. How does this increase in incorporation fit with the data of Fig. 2/3 showing a reduction in mitochondrial fitness? In short this Figure assembles several data without building a strong case. All these points are touched upon but not developed properly in the following tables.
In Figure 2, the authors show the effects of shDHX30 on mitochondrial proteins. In general, this set of data is relatively convincing. What is not totally convincing is the existence of a cytosolic form of DHX30 (Fig. 2f, for instance). I believe that the existence of a cytosolic form of DHX30 is a potentially very cool finding. But a) the levels of this cytosolic form seem minimal, b) the effects of its specific downregulation with a (single) specific shRNA are absent or a bit contradictory (Fig. 2g, MRPS22 versus MRPL11), and c) none of the assays of Fig. 1 (global DHX30 downregulation) has been reproduced by the interesting experiment, here, of the specific downregulation of either a cytosolic or a mitochondrial form of DHX30.
Finally, in Figure 3, the authors explore the effects of downregulation of DHX30 on mitochondrial functionality. Overall, the biological effects are very convincing (in short, a reduction in the oxygen consumption rate), although the mitochondrial analysis is really rudimentary (EM? ATP? ). What strikes me is that the authors started with the point of translation of mitochondrial mRNAs and then, here, look at data on mRNA levels of the OxPhos machinery. I fail to see the mechanistic connection.
The manuscript is written in an approximate way with some confused statements. Example, methods "rRNA biogenesis was performed" (??), fluorescence is low quality with bad resolution, I failed to find Supplementary Table 2 and 3 (perhaps it is my browser, but they seem empty). If the authors would be able to clearly define a) the effects of downregulating DHX30, b) convince about the presence of a cytosolic isoform and c) its role, this paper is really interesting.
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###Reviewer #1:
In this manuscript, Bosco et al. propose that DHX30 coordinates cytoplasmic translation and mitochondrial function to impact on cancer cell survival. They deplete DHX30 and report that this causes an enhancement of translation including those of mRNAs encoding for cytoplasmic ribosomal proteins, while paradoxically reducing the translation of mitoribosome protein mRNAs. There are cytoplasmic and mitochondrial isoforms of DHX30 and the authors assess the long-term consequences of knockdown of the cytoplasmic versus mitochondrial + cytoplasmic proteins. Some of the novelty of this paper has been preempted by a previous publication by Antonicka and Shoubridge showing that loss of DHX30 results in impaired mitochondrial ribosome assembly, impaired mitochondria OXPHOS assembly, impaired mitochondrial mRNA precursor …
###Reviewer #1:
In this manuscript, Bosco et al. propose that DHX30 coordinates cytoplasmic translation and mitochondrial function to impact on cancer cell survival. They deplete DHX30 and report that this causes an enhancement of translation including those of mRNAs encoding for cytoplasmic ribosomal proteins, while paradoxically reducing the translation of mitoribosome protein mRNAs. There are cytoplasmic and mitochondrial isoforms of DHX30 and the authors assess the long-term consequences of knockdown of the cytoplasmic versus mitochondrial + cytoplasmic proteins. Some of the novelty of this paper has been preempted by a previous publication by Antonicka and Shoubridge showing that loss of DHX30 results in impaired mitochondrial ribosome assembly, impaired mitochondria OXPHOS assembly, impaired mitochondrial mRNA precursor processing, and a very severe decrease in mitochondrial translation. I think the work, while interesting, is preliminary and should aim to provide mechanistic insight for the phenotype associated with DHX30 knockdown.
As far as I can see, none of the targets obtained from the polysome profiling are validated in this study. This is concerning since polysome profiling was previously reported in a Cell Report 2020 publication by the authors (GSE 95024; available at the GEO database), but the origin of the RNA-seq data in the current paper is not clear (GSE 154065; not available at the GEO database). We do not know if the RNA-seq data was generated from the same samples as the polysome profiling samples previously reported or completely independent of these (this information is lacking). Regardless, validation of any putative translation responsive genes predicted from polysome profiling data would appear to be a reasonable expectation these days.
The authors claim that depletion of DHX30 leads to increased global translation (Figs 1f, g). They also provide evidence that translation of mRNAs encoding cytoplasmic ribosomal proteins is increased, while the translation of mRNAs encoding mitoribosome ribosomal proteins is decreased (Fig 1b). DHX30 is associated with ribosomal subunits, 80S monosone and low-molecular weight polysomes, and it also interacts with a CG-rich motif for p53-dependent death (CGPD) in 3' UTRs of mRNAs. What is lacking is a mechanism to explain these observations (if the data validates)? To this reviewer the lack of mechanistic insight is a serious shortcoming of the current submission. What is responsible for the general translational increase (including cytoplasmic rps encoding mRNAs), yet mitochondrial rp mRNA translation decrease, upon DHX30 knockdown? Many rp mRNAs have TOP motifs at their 5' ends, is this pathway affected?
The authors previously identified DHX30 as a CGPD-motif interactor. They published this as a specific DHX30 binding motif, yet this motif is not enriched in the new data set established by the authors. I don't understand the statement put forth by the authors on line 286 that " While we cannot exclude that the CGPD motif can be implicated, only a subset of RP transcripts harbors instances of it". Either it is significantly enriched or it is not. In any event, there appears to be an inconsistency with previously published data.
The ENCODE eCLIP data suggests that DHX30 can bind to 67 cytoplasmic ribosomal and 23 mitochondrial protein transcripts. Yet in their eCLIP validation experiments using RIP, the authors probe for the potential of DHX30 to bind to only MRPL11 and MRPS22 (Fig 2a). They write "These findings suggest that DHX30 directly promotes the stability and/or translation of mitoribosome transcripts." What about the cytoplasmic ribosome protein mRNAs, which according to the ENCODE data can also bind DHX30, yet their response to DHX30 depletion is the opposite of that of the mitoribosome protein mRNAs. I think it may be premature to correlate DHX30 with mitoribosome protein regulation.
The comparison of the efficiency of knockdown using siRNAs targeting the cytoplasmic form versus the mitochondrial + cytoplasmic forms versus shRNA knockdown efficiency is confusing and, in my humble opinion doesn't add insight into mechanism of action. "Transient silencing of DHX30" (ie, using siRNAs) achieves ~50% mRNA reduction in HCT and U2OS cells 48-96s following transfection. On the other hand, silencing of DHX30 mRNA using shRNA achieved better levels of reduction (60-75% decrease) in U2OS and MCF7 cells (Fig S2e). The authors use these differences in knockdown efficiencies to correlate differences in expression response of several mitochondrial encoded genes. The authors need to show the extent to which DHX30 protein levels are reduced in the siRNA treated cells (only changes in mRNA levels are presented). As well, there should be a genetic rescue experiment to show that siRNA or shRNA resistant DHX30 cDNA can overcome this effect. Lane 3 of Fig 2h appears underloaded as assessed by the actin intensity. MRPL11 protein levels appear greater in lane 2 (siDHX30-C) compared to lane 1, why is that?
Please provide details on the siRNA and shRNAs used. It appears that only one shDHX30 was used to target cytoplasmic DHX30 and one shRNA to target cytoplasmic + mito DHX30. I couldn't find information on this.
If mutations in DHX30 are known to trigger stress granules formation, does knockdown of DHX30 do the same. Is eIF2 alpha phosphorylated upon HDX30 knockdown?
There appears to be several DHX30 mRNAs made through alternative splicing (see https://www.ncbi.nlm.nih.gov/gene/22907). In this study, when the authors refer to cytoplasmic DHX30, is the equivalent function being attributed to these different potential isoforms?
The pictures in Figs 1e, 2d, and S3g are quite difficult to appreciate and should be provided at higher magnification.
Fig 2f. Why is there so much tubulin in the mitochondrial protein extract lane?
Suppression of DHX30 mRNA leads to lowered proliferation rates in HCT116 cells. This however was not due to significant alterations in the cell cycle (Fig 4e). Apoptotic rates do not appear to be affected (compare HCT_shNT to HCT_shDHX30 in the DMSO samples of Fig 4g). Can the authors please provide an understanding into what is leading to the lowered proliferation rates if cell cycle progression and cell death are unaffected. Confusingly, "transient" silencing of DHX30 mRNA (protein levels were not assessed) in U2OS cells did not impact proliferation while in MCF7 cells it did. Although the authors attribute this difference in response to better depletion of DHX30 mRNA in MCF7 cells, they do not actually measure DHX30 protein levels and the use of different cell lines complicates the interpretation.
Line 267 "none of the DHX30 closer homologs showed strong evidence of such localized translation". What homologs are being referred to here?
Line 269. "Although our experiments did not enable us to confirm this in HCT116, a previous report also showed evidence for DHX30 interaction with mitochondrial transcripts in human fibroblasts by RIP-seq (Antonicka and Shoubridge, 2015). Our data instead point to a direct interaction with mitoribosome transcripts and their positive modulation as another means by which DHX30 can indirectly affect mitochondrial translation." DHX30 thus interacts with many different mRNAs and in my view it becomes difficult to ascribe a particular biological response to DHX30 to a particular set of transcripts based on interaction data.
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##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 1 of the manuscript.
###Summary:
The major weaknesses of the paper are: 1. The work is preliminary as there is very little mechanistic insight to explain the major findings. 2. Some of the conclusions are not substantiated by the data. 3. Targets from the ribosome profiling were not validated.
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