Clu1/Clu form mitochondria-associated granules upon metabolic transitions and regulate mitochondrial protein translation via ribosome interactions

  1. Leonor Miller-Fleming
  2. Wing Hei Au
  3. Laura Raik
  4. Pedro Rebelo-Guiomar
  5. Jasper Schmitz
  6. Ha Yoon Cho
  7. Aron Czako
  8. Alexander J. Whitworth

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Abstract

Mitochondria perform essential metabolic functions and respond rapidly to changes in metabolic and stress conditions. As the majority of mitochondrial proteins are nuclear-encoded, intricate post-transcriptional regulation is crucial to enable mitochondria to adapt to changing cellular demands. The eukaryotic Clustered mitochondria protein family has emerged as an important regulator of mitochondrial function during metabolic shifts. Here, we show that the Drosophila melanogaster and Saccharomyces cerevisiae Clu/Clu1 proteins form dynamic, membraneless, mRNA-containing granules adjacent to mitochondria in response to metabolic changes. Yeast Clu1 regulates the translation of a subset of nuclear-encoded mitochondrial proteins by interacting with their mRNAs while these are engaged in translation. We further show that Clu1 regulates translation by interacting with polysomes, independently of whether it is in a diffuse or granular state. Our results demonstrate remarkable functional conservation with other members of the Clustered mitochondria protein family and suggest that Clu/Clu1 granules isolate and concentrate ribosomes engaged in translating their mRNA targets, thus, integrating metabolic signals with the regulation of mitochondrial protein synthesis.

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  1. Version published to 10.1371/journal.pgen.1011773
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    Reply to the reviewers

    Reply to the Reviewers

    We thank the reviewers for their evaluation of our previous submission and have responded to each point in detail below. Overall, we have revised the manuscript with the addition of several new data and corresponding figure panels that strengthen our previous conclusions and add new insights allowing us to extend the conclusions of the study. Important additions include new data showing the impact of loss of CLU on adapting to additional stressors during metabolic transitions that supports a mechanistic understanding of our omics results; by poly(dT) FISH we show that fly Clu granules indeed contain mRNAs; FRAP microscopy analysis supports that Clu1 granules have dynamic content similar to other LLPS membraneless organelles; and we have re-analysed our data to demonstrate more clearly the impact of Clu1 on translation efficiency and also the relative binding of mRNAs during translation. In addition, we provide some extra control analyses for completeness.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary:

    In this manuscript the authors study the Clustered mitochondrial proteins Clu of Drosophila melanogaster and Clu1 of Saccharomyces cerevisiae, two homologues of the mammalian protein CLUH. They show in compelling microscopy analysis that both proteins form granules. This was the case for flies fed on yeast paste after starvation and in yeast in post-diauxic phase, in respiratory media or during mitochondrial stress. They show that these granules are found in proximity to mitochondria and that they behave like liquid-liquid-phase separated condensates. They show by co-staining for P-bodies and stress granules that Clu1-granules are distinct from these RNA granules. Furthermore, they found that the formation required active translation. In the second part, they show that Clu1 interacts with ribosomal and mitochondrial proteins by BioID. The deletion of Clu1 leads to slightly impaired growth on media containing Ethanol as a carbon source. They find that nascent polypeptides of some mitochondrial precursor proteins are decreased in the deletion of Clu1 and conclude that Clu1 regulates translation of these proteins. Using RNA immunoprecipitation of Clu1-GFP in presence of cycloheximid, EDTA and puromycin. The mRNAs of nuclear-encoded mitochondrial proteins found to be interacting with Clu1 were purified in conditions when the ribosomes are intact and the RNAs showed no interaction when ribosomes were disassembled. They show in sucrose gradients that Clu1 co-migrates with polysomes independent of its distribution state or carbon source. However, when cells are grown in conditions of granule formation, then polysomes and Clu1 run less deeply into the gradient. Form these data, the authors conclude that Clu/Clu1 regulates the translation of nuclear-encoded mitochondrial proteins.

    Major comments:

    -The authors state that Clu1 is regulating translation during metabolic shifts. However, it is not clear what the real impact on mitochondrial function is. They show that there is a minor growth defect on ethanol media when CLU1 is deleted. However, if Clu1 is necessary mainly for adaptation, the phenotype will be strongest observed in conditions where cells switch carbon sources. Growth curves would be suitable in which the lag-phase of yeast cells precultured either in glucose or glycerol switched to media of different carbon sources (glucose to glycerol or glycerol to glucose) are measured. One would expect that the deletion mutant shows a longer lag-phase compared to the wild type when shifted from glucose to glycerol media.

    We agree that this is an important question, and, duly, we previously attempted to address this exactly as the reviewer described. Surprisingly, we were not able to observe any substantial differences in the duration of the lag phase between the wild-type and CLU1 knockout strains under these conditions. However, we did note that CLU1 knockout cells consistently reached stationary phase with a lower optical density when switched to ethanol media, consistent with these cells having a different metabolic efficiency during growth on ethanol media.

    To further explore the role of Clu1, we noted that several of the Clu1 mRNA interactors were mitochondrial heat shock proteins (HSPs), which are crucial for mitochondrial protein folding and import during the transition from fermentation to respiration. Hence, we hypothesised that the absence of Clu1 might lead to increased sensitivity to heat shock during the metabolic shift.

    To test this, we subjected both wild-type and CLU1 knockout cells to heat shock under three different conditions: (1) during growth on glucose-containing media (fermentation), (2) after shifting cells to media containing ethanol during the lag phase, when cells are adapting to respiration, and (3) after cells had fully adapted to ethanol and resumed growth. Interestingly, CLU1 knockout cells were more sensitive to heat shock selectively during the adaptation to respiration, which involves the translation of an extensive number of mitochondrial proteins. We think that the small difference in translation of mitochondrial HSPs becomes evident only upon additional heat shock, likely due to a deficient mitochondrial protein folding and import. These findings support our hypothesis that Clu1 is essential for optimal mitochondrial function during metabolic shifts.

    These results have been added to the manuscript and shown in Fig. S6 and described on page 9.

    -In line with this, how different is the mitochondrial proteome of the WT and the mutant? Do hits of the BioID, RIP and Punch-P experiments change at steady state or during metabolic shifts? Either proteomics of isolated mitochondria or western blots of whole cells or isolated mitochondria of WT and the deletion mutant grown in conditions of Clu1-granule formation or no granules for the hits could answer this question.

    We also considered this question during the course of the work. However, in exploratory analyses we saw no obvious differences in overall mitochondrial proteomics at steady-state which is what prompted us to look at more subtle effects on translation. Considering this further, changes in steady-state levels can be complex to interpret as they represent the combined effects of protein production and degradation. Small changes arising from altered production could be masked by compensatory changes in turnover rate. In light of this, we believe that the translational regulation differences identified in our study remain central to understanding the role of Clu1, and any downstream proteomic changes would not alter our primary conclusions.

    -The authors analyze RNAs bound in polysomes to assess translation efficiency. Translation efficiency is usually calculated by the fraction of RNA bound by ribosomes to the total RNA amount of an RNA species. Thus, doing RT-qPCR from whole cells would be necessary to assess if the occupancy of ribosomes on the transcripts is due to changes in RNA abundance or other regulatory pathways and would help to further assess what causes the observed changes.

    Thanks for this recommendation. To address this and expand our analysis to other proteins differentially translated in *clu1Δ *cells, we measured the mRNA steady-state levels by performing RNAseq on WT and *clu1Δ *strains grown under the same conditions as used for Punch-P. We then calculated the translation efficiency by dividing the nascent protein levels (Punch-P) by steady-state mRNA levels (RNAseq), as previously described for Punch-P data (PMID: 26824027). The translation efficiency for the majority of proteins with reduced translation in the *clu1Δ *cells by Punch-P analysis was lower. Similarly, the majority of proteins with increased translation had higher translation efficiency.

    The mRNA quantification in polysomes we originally presented in the manuscript, further showed that the decrease in translation efficiency is not caused by a simple decrease of mRNA engaged in translation and that Clu1 is regulating protein translation at the ribosome level. In contrast, for higher translated proteins, we detected an increase in mRNAs engaged in polysomes, likely underlying the increased translation. These results further support our conclusions regarding the regulatory effects of Clu1 on translation.

    These results have been added to the manuscript and shown in Fig. 7E and described on page 9.

    OPTIONAL:

    -The authors show a co-localization of Clu/Clu1 with mitochondrial fission factors and conclude that the granules appear likely near fission sites. Indeed, CLUH has been implied in the past to play a role in mitochondrial fission (Yang, H., Sibilla, C., Liu, R. et al. Clueless/CLUH regulates mitochondrial fission by promoting recruitment of Drp1 to mitochondria. Nat Commun 13, 1582 (2022). https://doi.org/10.1038/s41467-022-29071-4). Thus, are fission sites required for Clu-granule localizations? What is the role of the mitochondrial network integrity for the granule distribution? Expressing Clu-GFP/Clu1-GFP in cells depleted for the fission factors would provide information on that.

    Thanks for this suggestion. We agree that it would be interesting to know whether Clu1 granules still appear when mitochondrial fission is blocked. We tried to address this question but encountered some technical limitations. First, overexpression of Clu1-GFP via a plasmid did not replicate the endogenous Clu1 behaviour, making it necessary to delete the fission factors in the Clu1-GFP background. While crossing the Clu1-GFP strain with already available knockout strains would be straightforward, we would need access to a tetrad dissecting microscope, which unfortunately was not available to us. We also attempted PCR-based gene deletion but the sequence homology between the GFP-tagging cassette and the deletion cassettes made this very challenging. Given these limitations, and as the lab's yeast expert had already left, we were not able to pursue this experiment further and have removed these observations from our manuscript. We hope that future studies will explore this question in more detail.

    -The author assess convincingly that Clu1 interacts with ribosomes and runs with polysomal fractions. However, how it actually regulates translation is not clear. To answer this question, selective ribosomal profiling would be necessary. The authors have established conditions which would be suitable for the experiment. They could use crosslinking and sucrose cushions to IP ribosomes with Clu1-GFP bound to be used for ribosomal profiling. However, this experiment is quite time-intensive (3-4 months) and expensive, thus, an optional suggestion.

    We thank the reviewer for this suggestion. We agree that ribosome profiling could provide novel insights into the function of Clu1/Clu. While we recognise the potential of this approach, as the reviewer points out, this experiment would indeed be time- and resource-intensive. Based on our initial tests, where we included cross-linked samples (UV and formaldehyde) we anticipate that it could even take longer than the estimated 3-4 months, as the IP using cross-linked lysates was not as successful as the IP using non-cross-linked samples: we were not able to immunoprepitate Clu1 so efficiently likely to the epitope being poorly exposed to the antibody. Although we have optimised working conditions for co-immunoprecipitating Clu1 with ribosomes, performing ribosome profiling using our setup within the timeframe and resources of this study is unfortunately not currently feasible.

    Minor comments:

    Fig1: B, C, please add scale bars into the zoom ins.

    These have been added.

    Fig 2 would profit from inlets of zoom ins to visualize the distribution better.

    These have been added.

    Fig.3: Panel C does not really add much information. I would rather remove it or put it into supplements and therefore show a zoom of Panel E with a line plot showing the rings. It is not clear from the represented images where the rings are formed.

    We think some confusion has arisen from the text description. It seems that the reviewer was under the impression that Fig. 3C and 3E were intended to be showing the Clu1 rings around the mitochondria, but this was shown only in Fig. S3A. We have re-written these sentences for better clarity. To be clear, Fig. 3C is a 3D rendering of the left-hand cell in 3B (3D is a line plot of part of the right-hand cell) and 3E is a different experiment showing the formation of Clu1 granules under a different respiratory stress (galactose plus CCCP). We have also added a line plot showing Clu1-GFP and mito-mCherry fluorescence intensity to highlight the Clu1 rings around the mitochondria in Fig. S3A.

    Fig.3 panel F: Max projections are not appropriate to show colocalization as they can lead to false-positive overlaps. Just remove the max projections.

    We tried a number of different approaches to improve this analysis but, ultimately, we were not able to generate sufficiently robust data to be convincing so we decided to remove this from the manuscript. The coincidence of Clu1 granules with mitochondrial fission factors was an adjunct observation and not a major part of the story and has been discussed by others relating to fly Clu (PMID: 35332133), so removal from the current manuscript does not impact the key conclusions of the study.

    References 21 and 22 are the same.

    Thanks. This has been fixed.

    Reviewer #1 (Significance (Required)):

    This manuscript shows in a convincing way that Clu and Clu1 form RNA granules and that Clu1 interacts with ribosomes. It is written in a clear way and the figures support the conclusions drawn in the text. The finding that Clu/Clu1 is important for metabolic adaptation has not been shown in fly or yeast to my knowledge. It is in line with findings for the mammalian homologue CLUH. Thus, the findings are supported by earlier work. This study is of value for a broader audience of the basic research field, especially of the mitochondrial and RNA granule field, as it supports the idea of post-transcriptional regulation of nuclear-encoded mitochondrial protein gene expression for dynamic adaptation of mitochondrial function. The conditions when Clu granules form is studied in detail, followed up by identification of target RNAs and interaction partners. Though the interaction of Clu1 with ribosomes is shown in a compelling way, a detailed mechanism of the function of Clu/Clu1 is missing and would require more experiments. Thus, even though a detailed mechanism is missing, the study does expand on our understanding of Clu/Clu1 in regulating mitochondrial biogenesis and is therefore of high interest of the mitochondrial field.

    Expertise: mitochondria, yeast, RNA granules, mitochondrial biogenesis, next-generation sequencing, fluorescence microscopy

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary:

    In this manuscript the authors use D. melanogaster and S. cerevisiae to study the role of CLUH in the translation of nuclear-encoded mitochondrial proteins. During conditions requiring aerobic respiration, CLUH forms RNA-dependent granules that localise in the proximity to mitochondria. Furthermore, the authors demonstrate that CLUH interacts with translating ribosomes to facilitate the translation of specific target mRNAs. For this, the authors use a combination of GFP-tagged CLUH models. BioID, polysome translating proteomics, RNA-IP. The authors' main conclusions are that (i) CLUH forms dynamic, membrane-less, RNA-dependent granules under conditions that demand aerobic respiration, (ii) CLUH interacts with specific mRNAs encoding metabolic factors, and (iii) CLUH interacts with the translating ribosome. The manuscript is well written and the conclusions stand in proportion to the experimental output and the results. The main concern is with regards to lack of advancement in relationship to published data.

    We appreciate the reviewer's feedback and specific comments which we respond to individually below. However, we would like to first address the point regarding "lack of advancement" and the use of the "CLUH" terminology which the reviewer uses throughout their critique. We would like to reiterate, as the reviewer states, our work focussed exclusively on yeast Clu1 and Drosophila Clu. None of our data relates to mammalian CLUH. While these proteins share substantial sequence homology, it is imprudent and scientifically unsound to assume cross-species equivalence without directly testing. Indeed, one of the central aims of our study was to characterise the molecular function of yeast Clu1, which remains almost entirely unstudied.

    We acknowledge that some of the observations contained within our study have been described by others and we have appropriately noted and cited these in context. Nevertheless, (a) independent replication is always valuable but easily criticised as lacking novelty, and (b) the majority of the work was analysing the molecular dynamics and function of yeast Clu1 which is almost completely unstudied and may help provide hypotheses for others to test for conservation in mammalian CLUH. Hence, we consider that summarising the work as 'lacking advancement' is misplaced.

    Comments:

    To this reviewer it is not clear how CLUH can regulate the translation of specific mRNAs while being bound to ribosomes, regardless of being in a diffuse or granular state. The authors suggest that under metabolically active conditions, CLUH might aggregate translating ribosomes, forming the granular structures. How CLUH though can both be bound to translating ribosomes and recruit specific mRNAs at the same time is not explained.

    It was indeed surprising to us that the data indicate that Clu1 can bind both mRNAs and ribosomes to affect translation, and we share the reviewer's curiosity about the precise mechanism of how this occurs. While we have provided novel insights into this situation, dissecting the precise molecular mechanisms is beyond the scope of the current study.

    The authors might want to discuss how changes in metabolic demands signal the aggregation of CLUH, and how CLUH can recognise its target mRNAs.

    We appreciate the reviewer's point here but as this would be pure speculation we have made only brief comments on this at the end of the Discussion.

    What was the rationale to perform the RIP or the PUNCH-P experiments only under non-challenged conditions, but not under conditions demanding aerobic respiration?

    We appreciate the reviewer's question. In fact, the Punch-P analysis was carried out on cells that had been transferred to ethanol to induce respiration. This was stated in the Methods, but we appreciate that this may have been missed so we have now clarified this in the main text (p9).

    Regarding the RIP, our initial tests showed that mRNAs encoding proteins found to interact with Clu1 by BioID were interacting with Clu1 in both fermenting and respiring conditions. Due to this consistency, it did not seem necessary to perform the RIP experiments under both metabolic conditions, so we chose to conduct the experiment under the simpler growth condition.

    If CLUH is ubiquitously bound to ribosomes, has CLUH been seen in any structural representation of the cytosolic ribosome?

    This is a good question, and we wondered the same. To our knowledge, Clu1/Clu/CLUH has not been observed in any structural studies of the ribosome, and no formal structure of any Clu family proteins has been resolved.

    Nevertheless, we would like to clarify that we do not think, or suggest in the manuscript, that Clu/Clu1 is ubiquitously bound to ribosomes. First, current evidence supports that Clu/Clu1 only regulates a specific subset of mRNAs. Second, our work, particularly the sucrose gradient experiments, shows that Clu1 interacts transiently with ribosomes, as cross-linking was required to capture the full extent of this interaction. This transient and selective interaction of Clu/Clu1 with the ribosome, together with the fact that transient interactors are often lost during ribosome purification, makes Clu/Clu1 detection in structural studies unlikely. Due to the transient interaction and dynamic localisation of Clu/Clu1, capturing Clu/Clu1 in ribosomal structures will require significant work in the future.

    Reviewer #2 (Significance (Required)):

    CLUH has been studied in various publications, showing data very similar to that presented in this manuscirpt. However, the authors provide a comprehensive analysis on both yeast and fly CLUH. The strength of the manuscript is the combination of several elegant methods and genetically modified model systems in two species to elucidate the role of CLUH during the translation of specific mRNA. In my view through, the advancement of understanding the function of CLUH is limited.

    Although the authors work in yeast and DM, the results seem applicable to other species, including humans, and thus, the presented results will be of interest in a range of researchers working in the field of metabolic regulation and gene expression.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary: This study from Miller-Fleming et al. employs yeast and Drosophila as model systems to explore the function of the RNA-binding protein Clu1, which is involved in mitochondrial biogenesis. The first part of the manuscript characterizes so called "Clu1 granules", and their dependance from metabolic transitions. In particular, using yeast, they find a relocalisation of Clu1 upon starvation and several mitochondrial stress conditions. These granules are not stress granules, and are dissolved by RNAse and puromycin treatment. The second part of the study aims to understand the molecular function of the protein and its link to translation. The results confirm an evolutionary conserved role of Clu1 in binding mRNAs for mitochondrial proteins and in interacting with mitochondrial proteins, ribosomal components and polysomes. In addition, the authors claim that binding of Clu1 to RNA is enhanced when mRNAs are trapped in polysomes by treatment with cycloheximide (CHX), leading to the proposal that Clu1 binds mRNAs during active translation.

    Major comments:

    -The claim of Clu1 granule localization next to mitochondria (Figure 3) would be more convincing if any of the experiment would be quantified. Especially in the case of panel 3G in Drosophila egg chambers where there are a lot of mitochondria, one wonders whether the closeness to mitochondria is just random. Furthermore, mdv1-signal does not look very convincing, being blurry and not dotty as expected. Thus, the conclusion that Clu1 granules partially colocalization with site of fission appears premature.

    The claim that Clu/Clu1 granules are often found in close proximity to mitochondria was inferred from observations from multiple analyses from yeast (we looked at hundreds of cells in several different conditions) and flies, where it had already been demonstrated (Cox and Spradling, 2009). We agree that observations of the fly egg chambers are challenging due to the very high density of mitochondria (and other cellular components - see the new analysis of poly(A) mRNAs) in these highly active cells. These considerations motivated us to take the CLEM approach (in addition to investigating the membraneless nature), to gain a much higher resolution view of the localisation of the granules. This analysis unequivocally showed that the Clu granules were exactly juxtaposed to several mitochondria. It is noteworthy that even in the TEM images shown, there is ample cytoplasm in which the Clu granule could be located if the association with mitochondria was coincidental and all granules had mitochondria in close proximity.

    Regarding the possible coincidence of Clu1 with mitochondrial fission factors, as mentioned above for Reviewer 1, we tried a number of different approaches to improve this analysis but, ultimately, we were not able to generate sufficiently robust data to be convincing so have decided to remove this from the manuscript. Since this was an adjunct observation and not a major part of the story and has been discussed by others relating to fly Clu (PMID: 35332133), removal from the current manuscript does not impact the key conclusions of the study.

    Based on the ability of 1,6-hexanediol to dissolve the granules (Figure 4), the authors conclude that: "Clu1 foci have membraneless nature". As they correctly state in the discussion, treatment with 1,6-hexanediol can have other effects. I suggest to be more cautious with the conclusions or add additional experiments. Are the granules dynamics if using FRAP? Do they fuse?

    The inference that the Clu1 granules are membraneless organelles was not solely based on the observation that they disassemble upon 1,6-hexanediol treatment but was made in conjunction with the CLEM analysis that showed unambiguously that Clu granules are not associated with any detectable membrane, which is strong evidence that these granules are membraneless in nature. Indeed, as the reviewer mentioned, we are cautious in concluding they have been formed by liquid-liquid phase separation (LLPS) and we do acknowledge that 1,6-hexanediol can have other effects in cells. Nevertheless, following the reviewer's suggestion we have analysed Clu1 granule dynamics using FRAP, even though we are aware that FRAP is also not a definitive proof that a structure is formed by LLPS. The FRAP analysis, shown in new Figure 4C, D, revealed approximately 50% recovery over 10 min imaging timeframe. As discussed on page 13, this indicates a dynamic nature of these granules, but this dynamism can vary widely between different types of granules and even different proteins within the same granule. Further work is warranted to fully investigate the dynamic nature of Clu/Clu1 granule components.

    The experiment in which the granules are dissolved by treatment with RNAse is very interesting. However, per se this does not directly demonstrate that the granules contain mRNA. To state this the author should perform FISH experiments for example using a probe to detect poly-A.

    We thank the reviewer for this suggestion. We have performed poly(dT) FISH in egg chambers. Initial analysis showed that the fluorescence was diffuse and widely distributed, as expected for these highly active cells, but with no specific accumulation in Clu granules. Interestingly, we observed that treatment with RNase A, which we initially used to demonstrate probe specificity, revealed an enrichment of poly(A) RNAs in Clu granules. So, while treating the live egg chambers with RNase revealed that granules depend on RNA for their stability, treating fixed egg chambers revealed more directly the presence of RNAs in granules.

    These results have been added to the manuscript and shown in Fig. 5 and described on page 7.

    The authors show that puromycin prevents the granule formation before insulin addition in the fly. Are these results (upon RNAse treatment and puromycin treatment) recapitulated in the yeast system? The authors conclude that Clu1 formation depends on mRNAs being engaged in translation, but never show that the granules are site of active translation. More experiments in this direction (for example using puro-PLA of specific mRNAs) are missing and would clearly improve the manuscript.

    Thanks for this very interesting consideration. We agree that we have not formally shown that the Clu1 granules are sites of active translation. A major limitation to addressing this is that puromycin is not able to penetrate the yeast cell wall, so cannot be used for analysis of intact cells as would be needed in this case. We agree that this would be a welcome addition but is beyond the scope of the current study.

    The interactome of Clu1-neighbouring proteins (Figure 6) is interesting and a valuable addition to data in other organisms. I am wondering why the authors have not used as a control a cytosolic BirA-GFP, which would have been the right control for this experiment, especially since GFP tends to form aggregates.

    We thank the reviewer for this comment. With hindsight, we agree that a cytosolic BirA-GFP would have been a better control. However, we are confident in our results for the following reasons:

    1. The levels of GFP obtained from Clu1-GFP expression are low, and under these conditions, we observed no evidence of GFP aggregation. Even in experiments where GFP is overexpressed from a high-copy 2µ plasmid under a strong promoter, we do not detect aggregation. Aggregation is not a concern in our experimental setup.
    2. Our conclusions are not solely based on the interactome analysis (BioID) but are supported by complementary findings. Specifically, several proteins identified in the proximity to Clu1 in the BioID analysis showed reduced translation in Clu1 knockout cells, and their corresponding mRNAs were found to interact with Clu1 during translation. These complementary results from independent techniques provide strong evidence for Clu1's role and validate the findings of the interactome analysis. Given this robust and complementary dataset, having BirA as a control strain was sufficient to validate our conclusions.

    Figure 7B: The log 2 FC for the changed proteins are in many cases small, implying that the difference in translation for these proteins is not so large. For this reason, it is relevant to know how was the statistical significance calculated for these MS measurements. In the supplementary Tables and in Fig 7B, a p value is indicated and it is not clear if this is a simple p value or an adjusted p value (FDR or q value). If not shown, I recommend showing the adjusted p value, so that one can have an idea of the solidity of the data and the claim. Again, this is an important piece of evidence, since the authors base on this experiment the conclusion that Clu1 controls translation of these mRNAs.

    Thanks for this comment. We have now included the q-value in the supplementary table.

    Minor comments:

    -Figure 1: The change in Clu1 localisation in post-diauxic phase or upon changing of the medium is evident from the images shown. However, it seems that the experiment has been performed only once (the same for Figure 2). Is this the case? An important information would be to show the expression levels of Clu1-GFP in the different conditions. Does recruitment of CLU1 to granules associate to increased expression levels?

    The experiments shown in figures 1 and 2 were performed independently at least three times, as stated in the figure legends. The numbers shown are indicative values from one of the replicate experiments. This has now been added to the figure legends.

    We agree that providing the information regarding the expression levels of Clu1-GFP is important to address whether the recruitment of Clu1 to granules is associated with changes in its abundance. To this end, we have performed an additional experiment to quantify Clu1-GFP levels under the conditions where Clu1 is diffuse (log growth phase in glucose-containing media) and when Clu1 is in granules (sodium azide treatment).

    These results have been added to the manuscript and shown in Fig. S2 and described on page 4.

    Figure 2 A-B. The authors claim that the only stressor capable of inducing Clu1 granules formation alone is inhibition of complex IV activity via sodium azide treatment. Other mitochondrial stresses like CCCP treatment or OA treatment are efficient only when combined to starvation. It should be mentioned that sodium azide treatment is not only capable of inhibiting complex IV but has also uncoupling function.

    Thanks for this comment. We have now mentioned this (p4).

    Figure 2 D-E: investigation of colocalization with Bre5 would help to understand how similar the yeast Clu1 granules are compared to the mammalian CLUH granules (Pla-Martin et al., 2020).

    This is an interesting suggestion and one that we also considered, but with limited time and resources we were not able to pursue this line of inquiry as well.

    Figure 8. This figure summarizes one of the most novel pieces of data about Clu1, the interaction with mRNAs via the ribosome. The way how panel A-C are represented is however a bit misleading. The Y axis in Figure B and C has the same amplitude as the one in A. Therefore, potential differences in Clu1-RNA pull-down in presence of EDTA or puromycin cannot be assessed. It is true that in presence of CHX there is much more pulled down RNA, but one cannot judge from these panels if there is any difference between Clu1 targets and controls also in the other conditions. The graphs should be modified and statistics added.

    We appreciate the reviewer's feedback regarding the presentation of the RIP-qPCR data in Fig. 8. Based on the comments, we have revised how the results are represented, improved the normalisation of the data and added statistical analysis.

    First, it is worth clarifying that the presentation of the original charts was done specifically to highlight the huge differences between RNA-pulldown in CHX versus disrupted ribosomes. It is also important to note that these RIP experiments were performed simultaneously under identical experimental conditions, so any differences lie in the treatments applied. To improve cross-comparison between treatments we have now incorporated an additional normalisation step. We normalised the enrichment levels of each mRNA tested against the non-specific binding observed with the negative control housekeeping genes (UBC6 and TAF10). This ensures that differences in bead loss or other technical variations are accounted for.

    We now show the comparison of the six positive hits and two negative controls normalised as described above, on the same scale (Fig. 8A). We now also present the relative effects of the three conditions (CHX, EDTA, and puromycin) within the same graph for each mRNA tested (Fig. 8B). This format enables direct comparison of Clu1 target mRNA enrichment and two negative controls across treatments, which is the relevant comparison for testing the hypothesis of ribosome-dependent interactions. We have adjusted the Y-axis scaling for each mRNA, as requested by the reviewer, and added statistical comparisons. For clarity, the data shown in Fig. 8A are also represented in the panels of Fig. 8B (CHX). We have amended the text appropriately and hope that these changes improve the comparisons between treatments and more readily demonstrate that Clu1 target enrichment is lost upon ribosome disassembly, either by EDTA or by puromycin.

    In addition, RNAse treatment in panel L does not seem to have really worked.

    These samples were cross-linked prior to treatment to preserve the transient interaction of Clu1 with the ribosome, hence, the normal dramatic effect of RNase to collapse the polysomes is much less pronounced. Nevertheless, the purpose of this experiment was to monitor whether Clu1 co-migrated with ribosomes, which it does.

    The authors should cite Vornlocher et al. (PMID: 10358023), who were the first to implicate Clu1 (Tif31) with translation.

    Thank you for this prompt. We have now added a comment on this in the Discussion (page 13).

    References 21 and 22 are the same.

    Thanks. This has been fixed.

    Reviewer #3 (Significance (Required)):

    The data reported in this manuscript are valuable, because they confirm an evolutionary conserved role of Clu1 in binding mRNAs for mitochondrial proteins and regulating their translation. It is also interesting that in yeast, similar to Drosophila and mammalian cells, Clu1 can form granular structures upon metabolic rewiring. A limitation of the study is that direct experiments to support the claim that Clu1 concentrates ribosomes engaged in translation are not provided. Furthermore, it is not clear what is the functional role of the Clu1 granules, since the proximity interactome and the binding of Clu1 to the polysomes is not affected by treatments that dissolve or stimulate granule formation.

    The study is of interest to a general cell biology audience.

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    Referee #3

    Evidence, reproducibility and clarity

    This study from Miller-Fleming et al. employs yeast and Drosophila as model systems to explore the function of the RNA-binding protein Clu1, which is involved in mitochondrial biogenesis. The first part of the manuscript characterizes so called "Clu1 granules", and their dependance from metabolic transitions. In particular, using yeast, they find a relocalisation of Clu1 upon starvation and several mitochondrial stress conditions. These granules are not stress granules, and are dissolved by RNAse and puromycin treatment. The second part of the study aims to understand the molecular function of the protein and its link to translation. The results confirm an evolutionary conserved role of Clu1 in binding mRNAs for mitochondrial proteins and in interacting with mitochondrial proteins, ribosomal components and polysomes. In addition, the authors claim that binding of Clu1 to RNA is enhanced when mRNAs are trapped in polysomes by treatment with cycloheximide (CHX), leading to the proposal that Clu1 binds mRNAs during active translation.

    Major comments:

    • The claim of Clu1 granule localization next to mitochondria (Figure 3) would be more convincing if any of the experiment would be quantified. Especially in the case of panel 3G in Drosophila egg chambers where there are a lot of mitochondria, one wonders whether the closeness to mitochondria is just random. Furthermore, mdv1-signal does not look very convincing, being blurry and not dotty as expected. Thus, the conclusion that Clu1 granules partially colocalization with site of fission appears premature.
    • Based on the ability of 1,6-hexanediol to dissolve the granules (Figure 4), the authors conclude that: "Clu1 foci have membraneless nature". As they correctly state in the discussion, treatment with 1,6-hexanediol can have other effects. I suggest to be more cautious with the conclusions or add additional experiments. Are the granules dynamics if using FRAP? Do they fuse?
    • The experiment in which the granules are dissolved by treatment with RNAse is very interesting. However, per se this does not directly demonstrate that the granules contain mRNA. To state this the author should perform FISH experiments for example using a probe to detect poly-A.
    • The authors show that puromycin prevents the granule formation before insulin addition in the fly. Are these results (upon RNAse treatment and puromycin treatment) recapitulated in the yeast system? The authors conclude that Clu1 formation depends on mRNAs being engaged in translation, but never show that the granules are site of active translation. More experiments in this direction (for example using puro-PLA of specific mRNAs) are missing and would clearly improve the manuscript.
    • The interactome of Clu1-neighbouring proteins (Figure 6) is interesting and a valuable addition to data in other organisms. I am wondering why the authors have not used as a control a cytosolic BirA-GFP, which would have been the right control for this experiment, especially since GFP tends to form aggregates.
    • Figure 7B: The log 2 FC for the changed proteins are in many cases small, implying that the difference in translation for these proteins is not so large. For this reason, it is relevant to know how was the statistical significance calculated for these MS measurements. In the supplementary Tables and in Fig 7B, a p value is indicated and it is not clear if this is a simple p value or an adjusted p value (FDR or q value). If not shown, I recommend showing the adjusted p value, so that one can have an idea of the solidity of the data and the claim. Again, this is an important piece of evidence, since the authors base on this experiment the conclusion that Clu1 controls translation of these mRNAs.

    Minor comments:

    • Figure 1: The change in Clu1 localisation in post-diauxic phase or upon changing of the medium is evident from the images shown. However, it seems that the experiment has been performed only once (the same for Figure 2). Is this the case? An important information would be to show the expression levels of Clu1-GFP in the different conditions. Does recruitment of CLU1 to granules associate to increased expression levels?
    • Figure 2 A-B. The authors claim that the only stressor capable of inducing Clu1 granules formation alone is inhibition of complex IV activity via sodium azide treatment. Other mitochondrial stresses like CCCP treatment or OA treatment are efficient only when combined to starvation. It should be mentioned that sodium azide treatment is not only capable of inhibiting complex IV but has also uncoupling function.
    • Figure 2 D-E: investigation of colocalization with Bre5 would help to understand how similar the yeast Clu1 granules are compared to the mammalian CLUH granules (Pla-Martin et al., 2020).
    • Figure 8. This figure summarizes one of the most novel pieces of data about Clu1, the interaction with mRNAs via the ribosome. The way how panel A-C are represented is however a bit misleading. The Y axis in Figure B and C has the same amplitude as the one in A. Therefore, potential differences in Clu1-RNA pull-down in presence of EDTA or puromycin cannot be assessed. It is true that in presence of CHX there is much more pulled down RNA, but one cannot judge from these panels if there is any difference between Clu1 targets and controls also in the other conditions. The graphs should be modified and statistics added. In addition, RNAse treatment in panel L does not seem to have really worked.
    • The authors should cite Vornlocher et al.. ( PMID: 10358023), who were the first to implicate Clu1 (Tif31) with translation.
    • References 21 and 22 are the same.

    Significance

    The data reported in this manuscript are valuable, because they confirm an evolutionary conserved role of Clu1 in binding mRNAs for mitochondrial proteins and regulating their translation. It is also interesting that in yeast, similar to Drosophila and mammalian cells, Clu1 can form granular structures upon metabolic rewiring. A limitation of the study is that direct experiments to support the claim that Clu1 concentrates ribosomes engaged in translation are not provided. Furthermore, it is not clear what is the functional role of the Clu1 granules, since the proximity interactome and the binding of Clu1 to the polysomes is not affected by treatments that dissolve or stimulate granule formation. The study is of interest to a general cell biology audience.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript the authors use D. melanogaster and S. cerevisiae to study the role of CLUH in the translation of nuclear-encoded mitochondrial proteins. During conditions requiring aerobic respiration, CLUH forms RNA-dependent granules that localise in the proximity to mitochondria. Furthermore, the authors demonstrate that CLUH interacts with translating ribosomes to facilitate the translation of specific target mRNAs. For this, the authors use a combination of GFP-tagged CLUH models. BioID, polysome translating proteomics, RNA-IP. The authors' main conclusions are that (i) CLUH forms dynamic, membrane-less, RNA-dependent granules under conditions that demand aerobic respiration, (ii) CLUH interacts with specific mRNAs encoding metabolic factors, and (iii) CLUH interacts with the translating ribosome. The manuscript is well written and the conclusions stand in proportion to the experimental output and the results. The main concern is with regards to lack of advancement in relationship to published data.

    Comments:

    • To this reviewer it is not clear how CLUH can regulate the translation of specific mRNAs while being bound to ribosomes, regardless of being in a diffuse or granular state. The authors suggest that under metabolically active conditions, CLUH might aggregate translating ribosomes, forming the granular structures. How CLUH though can both be bound to translating ribosomes and recruit specific mRNAs at the same time is not explained.
    • The authors might want to discuss how changes in metabolic demands signal the aggregation of CLUH, and how CLUH can recognise its target mRNAs.
    • What was the rationale to perform the RIP or the PUNCH-P experiments only under non-challenged conditions, but not under conditions demanding aerobic respiration?
    • If CLUH is ubiquitously bound to ribosomes, has CLUH been seen in any structural representation of the cytosolic ribosome?

    Significance

    CLUH has been studied in various publications, showing data very similar to that presented in this manuscirpt. However, the authors provide a comprehensive analysis on both yeast and fly CLUH. The strength of the manuscript is the combination of several elegant methods and genetically modified model systems in two species to elucidate the role of CLUH during the translation of specific mRNA. In my view through, the advancement of understanding the function of CLUH is limited.

    Although the authors work in yeast and DM, the results seem applicable to other species, including humans, and thus, the presented results will be of interest in a range of researchers working in the field of metabolic regulation and gene expression.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript the authors study the Clustered mitochondrial proteins Clu of Drosophila melanogaster and Clu1 of Saccharomyces cerevisiae, two homologues of the mammalian protein CLUH. They show in compelling microscopy analysis that both proteins form granules. This was the case for flies fed on yeast paste after starvation and in yeast in post-diauxic phase, in respiratory media or during mitochondrial stress. They show that these granules are found in proximity to mitochondria and that they behave like liquid-liquid-phase separated condensates. They show by co-staining for P-bodies and stress granules that Clu1-granules are distinct from these RNA granules. Furthermore, they found that the formation required active translation. In the second part, they show that Clu1 interacts with ribosomal and mitochondrial proteins by BioID. The deletion of Clu1 leads to slightly impaired growth on media containing Ethanol as a carbon source. They find that nascent polypeptides of some mitochondrial precursor proteins are decreased in the deletion of Clu1 and conclude that Clu1 regulates translation of these proteins. Using RNA immunoprecipitation of Clu1-GFP in presence of cycloheximid, EDTA and puromycin. The mRNAs of nuclear-encoded mitochondrial proteins found to be interacting with Clu1 were purified in conditions when the ribosomes are intact and the RNAs showed no interaction when ribosomes were disassembled. They show in sucrose gradients that Clu1 co-migrates with polysomes independent of its distribution state or carbon source. However, when cells are grown in conditions of granule formation, then polysomes and Clu1 run less deeply into the gradient. Form these data, the authors conclude that Clu/Clu1 regulates the translation of nuclear-encoded mitochondrial proteins.

    Major comments:

    • The authors state that Clu1 is regulating translation during metabolic shifts. However, it is not clear what the real impact on mitochondrial function is. They show that there is a minor growth defect on ethanol media when CLU1 is deleted. However, if Clu1 is necessary mainly for adaptation, the phenotype will be strongest observed in conditions where cells switch carbon sources. Growth curves would be suitable in which the lag-phase of yeast cells precultured either in glucose or glycerol switched to media of different carbon sources (glucose to glycerol or glycerol to glucose) are measured. One would expect that the deletion mutant shows a longer lag-phase compared to the wild type when shifted from glucose to glycerol media. In line with this, how different is the mitochondrial proteome of the WT and the mutant? Do hits of the BioID, RIP and Punch-P experiments change at steady state or during metabolic shifts? Either proteomics of isolated mitochondria or western blots of whole cells or isolated mitochondria of WT and the deletion mutant grown in conditions of Clu1-granule formation or no granules for the hits could answer this question.
    • The authors analyze RNAs bound in polysomes to assess translation efficiency. Translation efficiency is usually calculated by the fraction of RNA bound by ribosomes to the total RNA amount of an RNA species. Thus, doing RT-qPCR from whole cells would be necessary to assess if the occupancy of ribosomes on the transcripts is due to changes in RNA abundance or other regulatory pathways and would help to further assess what causes the observed changes.

    Optional:

    • The authors show a co-localization of Clu/Clu1 with mitochondrial fission factors and conclude that the granules appear likely near fission sites. Indeed, CLUH has been implied in the past to play a role in mitochondrial fission (Yang, H., Sibilla, C., Liu, R. et al. Clueless/CLUH regulates mitochondrial fission by promoting recruitment of Drp1 to mitochondria. Nat Commun 13, 1582 (2022). https://doi.org/10.1038/s41467-022-29071-4). Thus, are fission sites required for Clu-granule localizations? What is the role of the mitochondrial network integrity for the granule distribution? Expressing Clu-GFP/Clu1-GFP in cells depleted for the fission factors would provide information on that.
    • The author assess convincingly that Clu1 interacts with ribosomes and runs with polysomal fractions. However, how it actually regulates translation is not clear. To answer this question, selective ribosomal profiling would be necessary. The authors have established conditions which would be suitable for the experiment. They could use crosslinking and sucrose cushions to IP ribosomes with Clu1-GFP bound to be used for ribosomal profiling. However, this experiment is quite time-intensive (3-4 months) and expensive, thus, an optional suggestion.

    Minor comments:

    Fig1: B, C, please add scale bars into the zoom ins.

    Fig 2 would profit from inlets of zoom ins to visualize the distribution better.

    Fig.3: Panel C does not really add much information. I would rather remove it or put it into supplements and therefore show a zoom of Panel E with a line plot showing the rings. It is not clear from the represented images where the rings are formed.

    Fig.3 panel F: Max projections are not appropriate to show colocalization as they can lead to false-positive overlaps. Just remove the max projections.

    References 21 and 22 are the same.

    Significance

    This manuscript shows in a convincing way that Clu and Clu1 form RNA granules and that Clu1 interacts with ribosomes. It is written in a clear way and the figures support the conclusions drawn in the text. The finding that Clu/Clu1 is important for metabolic adaptation has not been shown in fly or yeast to my knowledge. It is in line with findings for the mammalian homologue CLUH. Thus, the findings are supported by earlier work. This study is of value for a broader audience of the basic research field, especially of the mitochondrial and RNA granule field, as it supports the idea of post-transcriptional regulation of nuclear-encoded mitochondrial protein gene expression for dynamic adaptation of mitochondrial function. The conditions when Clu granules form is studied in detail, followed up by identification of target RNAs and interaction partners. Though the interaction of Clu1 with ribosomes is shown in a compelling way, a detailed mechanism of the function of Clu/Clu1 is missing and would require more experiments. Thus, even though a detailed mechanism is missing, the study does expand on our understanding of Clu/Clu1 in regulating mitochondrial biogenesis and is therefore of high interest of the mitochondrial field.

    Expertise: mitochondria, yeast, RNA granules, mitochondrial biogenesis, next-generation sequencing, fluorescence microscopy

  6. Version published to 10.1101/2024.08.17.608283 on bioRxiv