FAM210A mediates an inter-organelle crosstalk essential for protein synthesis and muscle growth in mouse

  1. Jingjuan Chen
  2. Feng Yue
  3. Kun Ho Kim
  4. Peipei Zhu
  5. Jiamin Qiu
  6. W. Andy Tao
  7. Shihuan Kuang

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Abstract

Mitochondria are not only essential for energy production in eukaryocytes but also a key regulator of intracellular signaling. Here, we report an unappreciated role of mitochondria in regulating cytosolic protein translation in skeletal muscle cells (myofibers). We show that the expression of mitochondrial protein FAM210A (Family With Sequence Similarity 210 Member A) is positively associated with muscle mass in mice and humans. Muscle-specific Myl1 Cre -driven Fam210a knockout ( Fam210a MKO ) in mice reduces mitochondrial density and function, leading to progressive muscle atrophy and premature death. Metabolomic and biochemical analyses reveal that Fam210a MKO reverses the oxidative TCA cycle towards the reductive direction, resulting in acetyl-CoA accumulation and hyperacetylation of cytosolic proteins. Specifically, hyperacetylation of several ribosomal proteins leads to disassembly of ribosomes and translational defects. Transplantation of Fam210a MKO mitochondria into wildtype myoblasts is sufficient to elevate protein acetylation in recipient cells. These findings reveal a novel crosstalk between the mitochondrion and ribosome mediated by FAM210A.

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    Reply to the reviewers

    The authors sincerely appreciate the editors’ and the reviewers’ dedication in providing constructive and insightful comments aimed at enhancing the quality of the manuscript. In response to the valuable feedback received, we have implemented significant revisions to the manuscript, including the addition of key experiments, reorganization of the figures as well as providing detailed point-to-point responses to address the reviewers’ concerns. With these changes, we are confident that we have effectively addressed the comments raised by all three reviewers and have strengthened the overall quality of the manuscript.

    Below are the major improvements we have made in the revised manuscript:

    1. Figure 4  new figure with polysome profiling assay to strengthen the link between translational regulation and mitochondrial defects.
    2. Figure 7  added confocal images showing the transfer of mitochondria into recipient cells.
    3. Figure S2  added RER data further supporting a shift of metabolism to favor fatty acid oxidation as shown by proteomics data.
    4. Figure S4  added WB data showing that protein degradation was not affected, strengthening a protein synthesis defect due to Fam210a KO.
    5. Figure S5B, S6C  added quantification to the staining and blots.

    1. Point-by-point description of the revisions

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

    In the manuscript entitled "FAM210A mediates an inter-organelle crosstalk essential for protein synthesis and muscle growth in mouse", Chen et al, found that knocking out of FAM210A specifically in muscle using Myl Cre resulted in abnormal mitochondria, hyperacetylation of cytosolic proteins, and translation defects. The manuscript uncovered the new functions of FAM210A in regulating metabolism and translation. I have the following the concerns about the manuscript.

    Comments

    One of the major phenotypes of FAM210A is the decrease of muscle mass after 6 weeks after birth. Is this phenotype caused by the accumulation of progressive loss of muscle mass from birth? Are the body weight and muscle mass reduced in FAM210A knocking out new-born mice? Is the muscle mass growth curve the same in FAM210A and WT mice from birth to 6 weeks after birth? These results will reveal more mechanism of FAM210A mediated muscle mass control. Answer: Indeed, the phenotype of the Fam210aMKO was caused by the progressive loss of muscle mass. The body weight of the mice was not different before 3-weeks of age (Figure 2B). We reasoned that myonuclei accretion occurred before Myl1Cre induced knockout of Fam210a, accounting for the relative normal muscle development and nuclei accretion prior to 21 days after birth (refer to Response Figure 2). However, due to the small muscle mass, it is hard to accurately evaluate whether the muscle mass in very young mice. Regardless, we believe that body weight and muscle weight closely mimic each other and exhibit similar slopes in WT and KO mice (Response Figure 1).

    Beyond 21 days, muscle growth is mainly attributed to hypertrophy of myofibers, a process that relies on protein synthesis. Yet the Fam210aMKO myofibers has defects in protein synthesis, explaining why the muscles cannot gain weight after 3 weeks and started to lose weight. We have shown that at 4 weeks the TA muscle weight was 13 mg in Fam210aMKO compared to 25 mg in WT control. At 6-weeks, the TA weight in the Fam210aMKO mouse was 10 mg compared to 28 mg in the WT control. Furthermore, the TA weight of the Fam210aMKO mouse was 8.7 mg compared to 36mg in the WT control. These results provide compelling evidence that the Fam210aMKO muscles are progressively wasted.

    Response Figure 1. Changes of body weights and TA muscle weights during postnatal growth. The muscle weights increased (in wildtype mice) or decreased (in KO mice) with body weights at similar trends.

    Does the muscle mass continue to decrease after 8 weeks?

    Answer: Based on the trend (see Response Figure 1), we believe the answer is “yes”. However, we were not allowed to monitor the Fam210aMKO mice after 8 weeks of age, as they were severely lethargic and can barely move, reaching the humane endpoint determined by the IACUC guidelines.

    FAM210A knockout mice displayed high lethal rate. Is there any potential mechanism for the high lethality?

    Answer: We performed extensive necropsy and could not identify a direct cause. The potential cause for the lethality could be the difficulty of breathing as the diaphragm muscle was very thin in the Fam210aMKO mouse compared to the WT control. Besides, the diminished muscle contraction force (Figure 3) might have prohibited normal activities (including eating), leading to exhaustive death.

    In Figure 2, the muscle mass decreased significantly, while the fat mass only decreased slightly in FAM210A knockout mice. However, the ratio of the lean mass and fat mass to body mass did not change in FAM210A knockout mice compared to WT mice. How do the authors reconcile this?

    Answer: Just to clarify, Figure 2D-E shows that fat mass was significantly reduced at 4-week old but not reduced at 6-week old. We interpret the significant reduction of the mass but not the ratio (to body weight) as the result of the concomitant reduction of the body weight in the Fam210aMKO mice.

    Are there changes of the number of nuclei per myotube? Is the muscle atrophy in FAM210A knockout mice caused by the defects of fusion, or the degradation of protein, or both?

    Answer: We thank the reviewer for this question. To answer this question, we isolated myofibers from WT and Fam210aMKO mouse at 4-week-old and quantify the myonuclei number. We did not observe a significant reduction of myonuclei number per myofiber in the Fam210aMKO mouse, suggesting that the myoblast fusion into myofibers was not affected in the Fam210aMKO model. (Response Figure 2)

    Response Figure 2. DAPI staining and quantification in the single myofiber isolated from WT and Fam210aMKO mice.

    The number of myonuclei in the WT and Fam210aMKO was not different, suggesting normal fusion of satellite cells in Fam210aMKO mice.

    We also did western blot to check the atrophy related protein expression in WT and Fam210aMKO mouse at different ages. Interestingly, we did not observe a significant induction of these proteins (Atrogin-1, MuRF1) in the Fam210aMKOmuscle. Therefore, we conclude that the muscle atrophy was due to protein translation defects in the Fam210aMKO, independent of myoblast fusion and protein degradation (Figure S4C).

    Are the growth curves of muscle mass growth in EDL and SOL the same in FAM210A knockout mice?

    Answer: We thank the reviewer for the question. In the Myl1Cre mediated Fam210a KO model, Fam210a was deleted in both fast (EDL) and slow (SOL) muscles (see response to Reviewer 3, second point). We think that the “growth curve” of the EDL and SOL muscle should be same (stagnant and even reduced) upon Fam210a KO as the mouse grows from 4-week to 8-week.

    The oxygen consumption and carbon dioxide production are higher in FAM210A knockout mice, suggesting a high metabolism rate. In contrast, the heat production of FAM210A knockout mice is lower, suggesting a low metabolism rate. Any explanation?

    Answer: The VCO2 and VO2 values were normalized to the body weight, and the KO value appeared high because their body weights were much lower at the time of test. While for heat production (unit: Kcal/hr), body weight was not a factor in the calculation. The seemingly contradicting/surprising result that a weak KO mouse could have higher VCO2 and VO2could be recapitulated in other mouse models (for example PMID: 22307625).

    Given the high glucose consumption in FAM210A, why is the clearance rate of blood glucose low?

    Answer: We believe there is a misunderstanding here. A smaller AUC (as seen in the KO) suggest faster blood glucose clearance. The circulating glucose level after fasting is lower in the KO mice, which suggests that the Fam210aMKO mice were consuming more glucose compared to the WT mice. In the GTT test, the Fam210aMKO mice showed a lower AUC after the injection of glucose, implying that the Fam210aMKO mice cleared the injected glucose at a faster rate, probably due to a pseudo-fasting state which would promote the uptake of circulating glucose when available.

    Are there any changes of the abilities for the FAM210A knockout mice in running endurance?

    Answer: Indeed, the Fam210aMKO mice ran less distance, shorter time, and at a lower speed when tested on a treadmill endurance running program (Figure 3)

    In page 5, the last sentence of the 2nd paragraph, the authors concluded "There results suggest that Fam210aMKO induces a metabolic switch to a more oxidative state." It is better to describe it as muscle metabolic since the whole-body metabolism has not been carefully examined.

    Answer: We thank the reviewer for pointing this out, we will change the wording to better reflect the changes observed in the Fam210aMKO mouse regarding the metabolism.

    In Fig. 6, what is the link between increased transcription level of Fgf21 and the elevated level of aberrant acetylation of proteins?

    Answer: We thank the reviewer for this interesting question! However, we did not pursue a direct causal relationship between Fgf21 level and aberrant protein acetylation. In our model, we are proposing that mitochondrial defects in the Fam210aMKO model can trigger the integrated stress response which leads to a higher Fgf21 transcript level in the muscle. This is coinciding with the acetylation increase in the muscle due to the excessive production of acetyl-CoA. A potential relationship between Fgf21 and protein acetylation warrant examination in a future study.

    After careful considerations on the mechanism proposed in the study, we decided to remove qPCR data showing the modest increase of Fgf21 mRNA level. The removal of this data will not change the conclusions we draw nor lessen the significance of the mitochondria transfer experiment.

    Is there any link between the increased acetylation level of rebolsome proteins and the translation defects?

    Answer: Indeed, there are ample studies showing that ribosomal proteins can be acetylated, and that the acetylation of ribosomal proteins can affect the protein synthesis process, for example in PMID: 35604121 and PMID: 37742082. Here in this paper, we showed by ribosome profiling assay that the muscle has defects in the polysome formation (at 4-week and 6-week), when the protein acetylation was significantly increased in the Fam210aMKO mice (Figure 4D-4G).

    How do the abnormal mitochondria lead to increased protein acetylation? And how do these defects further cause translation problem?

    Answer: As elaborated in the discussion, we propose that upon Fam210a KO in mature myofiber, the TCA cycle in the mitochondria was disrupted, blocking utilization of acetyl-CoA and resulting in the accumulation of acetyl-CoA in the muscle. The excess acetyl-CoA lead to increased protein acetylation in the cytosol. We identified that ribosomal proteins are hyperacetylated in the muscle. We also observed that the polysome formation in the muscle was impaired, which exacerbates the translation efficiency.

    Consistently, when we treated C2C12 during in vitro culture with sodium acetate to mimic the increase of acetylation of proteins, we showed that excessive levels of acetyl-CoA can block the differentiation of C2C12 cells (Response Figure 3).

    Response Figure 3. The effect of sodium acetate on the differentiation of C2C12 myoblasts.

    The differentiation of C2C12 myoblasts into myotubes were probed by the protein abundance of Myog and MF20, which showed a decrease in the expression level when sodium acetate was added in increasing amounts.

    The defects in translation will cause general problems besides mitochondria defects. Are there any phenotypes related to the overall translation inhibition observed? If not, why?

    Answer: Just to clarify, our model suggests that mitochondrial defects in the Fam210a KO causes cytosolic translation defects, not the other way around. We showed by SUnSET experiment that the global translation was indeed reduced in the Fam210aMKO muscle at 4-week. We also observed that the p-S6 level which indicates the global protein translation was decreased. It is also true that the global translational arrest can exacerbate the mitochondrial defects and fewer mitochondrial proteins can be synthesized. This feed forward loop can explain the aggravating phenotype in the Fam210aMKO mouse as the mouse gets older.

    Are the abnormal mitochondria, increased protein acetylation, and translation inhibition observed in 2-6 weeks old mice? When were these defects first found? Are they correlated with muscle atrophy?

    Answer: At 2-week-old, the protein synthesis or degradation was not changed between WT and Fam210aMKO mice (Figure S4C). The mitochondria abnormality was first observed at 4 weeks of age, concomitant with the decrease of protein translation (decreased p-S6), polysome formation, and protein hyperacetylation. The acetylation increase was apparent at 6-week together with decreased p-S6 level, polysome assembly and mitochondrial defects. Decreased protein translation has been shown to cause muscle atrophy (PMID: 19046572).

    Reviewer #1 (Significance (Required)):

    This manuscript described many interesting phenotypes of Fam210a knockout mice. However, the links between these phenotypes are obscure. The logic of the manuscript will be greatly improved if the authors could provide explanations to logically link the phenotypes.

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

    Summary: In this manuscript, Chen et al., investigate the functions of FAM210A in skeletal muscle physiology and metabolism. FAM210A is a mitochondria-localized protein in which mutations have been associated with sarcopenia and osteoporosis. Using publicly available gene expression datasets from human skeletal muscle biopsies the authors first demonstrate that the expression of FAM210 is reduced in muscle atrophy-associated diseases and increased in muscle hypertrophy conditions. Based on this, they show that a muscle specific Fam210a deletion leads to muscle atrophy/weakness, systemic metabolic defects, and premature lethality in mouse. Further examination of the knockout myofibers reveals impaired mitochondrial respiration and translation program. Additionally, the authors demonstrate that the flow of TCA cycle is disrupted in the FAM210A-deleted myofibers, which causes abnormal accumulation of acetyl-coA and hyperacetylation of a subset of proteins. The authors claim that Fam210a deletion in skeletal muscle induces the hyper-acetylation of several small ribosomal proteins that leads to ribosomal disassembly and translational deficiency. However, this conclusion is not supported by adequate experimentation and rigorous analysis of ribosomal proteins acetylation and ribosome assembly.

    Major comments:

    -In general, figure legends are lacking information regarding number of biological replicates used and details about statistical analysis. What does three * vs. one * mean in terms of p-value? Exact p-values should be indicated.

    Answer: We thank the reviewer for pointing this out, we have added the information to the revised figure legends.

    -The mechanistic studies linking muscle phenotypes with ribosomal protein hyperacetylation and mRNA translation defects are underdeveloped and not rigorously carried.

    Answer: We agree with the reviewer and have added new data in the revised manuscript to strengthen this link. For example, we have now provided direct evidence on the defective polysome assembly in the Fam210a KO muscles (Figure 4D-4G), which should profoundly impact mRNA translation. In addition, other groups have also shown that ribosomal protein acetylation can impact mRNA translation and polysome formation (PMID: 35604121).

    We also explored the effect of acetylation on differentiation (a process accompanied by extensive protein synthesis) related to our mouse model. We used sodium acetate to elevate acetylation during C2C12 differentiation. We found that increased acetylation indeed impaired the differentiation as can be seen by the reduced expression of MF20 (myosin protein) by WB and IF. The differentiation marker Myogenin was also reduced (Response Figure 3, 4).

    Response Figure 4. Immunofluorescence staining of Myog and MF20 in the differentiated C2C12 myotubes treated with different amounts of sodium acetate.

    The number of MF20 (green) positive myotubes and Myog (red) positive nuclei was significantly reduced in the cells treated with 15mM and 30mM sodium acetate.

    -Fig S1: The validation WB of FAM210A KO is not the most convincing. Why are the FAM210A levels so low in TA compared to other tissues?

    Answer: This is due to the insufficient proteins loaded as it was obvious from the Tubulin marker. We have replaced the WB blot with more convincing blots as requested (Figure S1C).

    -Fig 2G: The authors state "Hematoxylin and eosin (H&E) staining did not reveal any obvious myofiber pathology in the Fam210a KO mice up to 8 weeks". However there seems to be a progressive increase in nuclei up to 8-weeks in the KO. What is the significance of this?

    Answer: Thank you for pointing this out. We have now changed the wording and quantified the myonuclei number per myofiber. The increase of myonuclei in the H&E images is likely due to the smaller myofiber size in the Fam210aMKOmouse compared to the WT (Response Figure 5).

    Response Figure 5. Quantification of the myonuclei number in the H&E images.

    -IP-MS analysis for FAM210A interacting proteins requires validation with IP and reverse IP + WB experiment.

    Answer: We did perform the co-IP with SUCLG2 and FAM210A antibodies to try to confirm the interaction. To be more specific, we transduced C2C12 myoblasts cells with an Fam210a overexpression virus and differentiated the cells for 3 days. The myotubes were used to test the interaction by pulling down Fam210a with a myc antibody (FAM210A has a myc tag) and blot with SUCLG2 antibody. Unfortunately, the results were not promising (Response Figure 6). We reasoned that the interaction might be indirect or too transient to be reliably detected.

    Response Figure 6. co-IP of SUCLG2 and FAM210A.

    • Figure 4A requires quantification of the SDH signals from multiple samples.

    Answer: We thank the reviewer for this suggestion. We have added the quantification of the staining (Figure S5B).

    • Figure 6F: To clearly demonstrate an increase in protein acetylation in the FAM210 MKO, the authors must provide quantification data generated with more then N=1. Please add the molecular weights markings on the side of the blots.

    Answer: We thank the reviewer for this suggestion, we have provided the quantification of the Acetylated-lysine blots, and added the molecular weight markers (Figure 6F, Figure S6C).

    • Figure 6H and S5: The mitochondria transfer experiment appears to be quite efficient compared to previously published studies. It would be important to control that the signal observed in the recipient cells is not due to the leakage of the MitoTracker dye from the donor mitochondria.

    Answer: This is an interesting point though MitoTracker dye is not supposed to leak as it covalently binds to mitochondrial proteins. Even though the dye may leak to mark the endogenous mitochondrial, it does not affect our goal to demonstrate that transfer of Fam210aMKO mitochondria into healthy cells can induce protein hyperacetylation. Additional evidence argues against the leakiness of Mitotracker dye to subsequently mark other mitochondria in the recipient cells: 1) mtDNA and MitoTracker signal both increase linearly with the increasing amounts of mitochondria transferred (Figure S7A); 2) We have now also included confocal images to show the presence of both MitoTracker labeled and non-labeled mitochondria in the recipient cells. We reason that if MitoTracker leaks within a cell then it would have labeled all mitochondrial in that cell (Figure 7C).

    • Figure 6J: The increase in Fgf21 is modest. Although the difference is statistically significant, is it biologically important?

    Answer: We thank the reviewer for this question; indeed, the increase is modest. We think the reason of the modest increase compared to the drastic increase seen in vivo was because when we transplanted the WT and Fam210aMKOmitochondria to the recipient cell, the original mitochondria in the recipient were not depleted, which could explain the milder effect. However, we were able to show that the recipient cells readily increase the acetylation of proteins after receiving the Fam210aMKO mitochondria, recapitulating the phenotype we saw in the Fam210aMKO muscle.

    After careful considerations on the mechanism proposed in the study, we decided to remove qPCR data showing the modest increase of Fgf21 mRNA level. The removal of this data will not change the conclusions we draw nor lessen the significance of the mitochondria transfer experiment.

    • Figure 6C: How significant is the difference in acetylation of RPL30 in WT vs. KO. RPS13 was not found in the WT MS? Was this normalized to Input?

    Answer: the MS was performed with same loading. The mass spectrometry results for protein identification after AcK-IP were from pooled samples from 3 independent replicates (as the KO muscles are very scarce). Therefore, there was not a significance test.

    • Figure 7D: What are the MW of the bands shown on this blot? This experiment is by no means sufficient to demonstrate and confirm that ribosomal proteins are acetylated. An increase in RPL30 and RPS13 acetylation must be directly assessed.

    Answer: We thank the reviewer for suggesting the more direct assays to look at RPL30 and RPS13 acetylation. We have shown that the ribosome fractions were indeed hyperacetylated in the Fam210aMKO mouse compared to the WT control (Figure S6D). We agree that this result cannot lead to the conclusion that the RPL30 and RPS13 are specifically hyperacetylated. Indeed, we have tried to use Acetylated lysine antibody pull down and RPS13/RPL30 blot to show the increase in the acetylated RPS13/RPL30 protein. However, we cannot show a robust increase in the acetylation, potentially due to the low number of acetylation sites on RPS13 and RPL30 protein. We therefore have reworded the conclusion in the revised manuscript to better reflect the results.

    • Fig7E: This experiment is not properly executed and in its current state does not rigorously support that "hyper-acetylation of several small ribosomal proteins leads to ribosomal disassembly". A) UV profiles of the fractionation must be provided to assess the quality of the profile. B) Provide MW markers. Which band is RPL30? The Input and free fraction bands are not at the same size. RPL30 should at least be visible on the 60S and polysomes from the WT. C) These results do not match the acetylation MS data, which seem to show that the increase in acetylation is much greater for RPS13. However, RPS13 presence on polysomes (assuming they are polysomes) is not affected in the KO. D) This type of experiment must be done for three independent biological replicates, blots from single lanes must be quantified and normalized to total signal (from all the lanes) for the same antibody.

    Answer: we appreciate the great advice on improving the experiment. As suggested, we have now added proper experimentation (UV profile, and better WB), with the help of Dr. Kotaro Fujii (included as co-author in the revised manuscript). The following results showed that in the 4-week sample, there was a decrease in the 80S monosome and polysome in the Fam210aMKO mice compared to the WT. The change was more drastic at 6-week (Figure 4D-4G). Similarly, due to the scarce amount of muscle in the KO mice, we need to pool samples from the 6-week-old mice for the experiment, and hope the reviewer can understand the situation. With the clear peaks shown in the UV profile as well as the WB results, we provide more convincing evidence that the polysome assembly was indeed impaired in the Fam210aMKO (Figure 4D-4G).

    • Fig 7F: Global translation rates are assessed by puro incorporation at week 4, a time point when differences in protein acetylation were not observed. This does not support the hypothesis that increased acetylation of ribosomal proteins causes defect in protein translation. (Referencing the authors statement p.7 lines 321-24.).

    Answer: We thank the reviewer for this question. When we quantified the protein acetylation increase in the muscle at 4-weeks, we showed that there was a significant increase. But like the reviewer said, the ribosomal fractions were not significantly acetylated by WB at 4-week. We reasoned that, at early stages (4-weeks), the ISR signaling can lead to the translational arrest, along with the polysome formation defects, leading to the decreased protein translation. These are included in the discussion.

    • Other studies have implicated Fam210A in the regulation of mitochondrial protein synthesis through an interaction with EF-Tu. The authors also identified EF-Tu as an interactor in their LC-MS analysis (FigS4). A role for this interaction accounting for mitochondrial and translation defects seems to be underestimated and unexplored here.

    Answer: We agree with this point and believe the cytoplasmic translation defects are in addition to the mitochondrial translational defects. We have shown that FAM210A KO leads to the decrease of the MTCO1 which is encoded by the mitochondrial genome. Besides, we also showed by mitochondrial proteomics that TUFM was reduced in the KO, which also contributed to translational arrest in the mitochondria (Figure 5J). To answer whether mitochondrial encoded proteins are decreased in upon Fam210a KO, we blotted the protein lysates at different stages with antibodies for a few mitochondrial encoded proteins and showed that they decreased with ages (Response Figure 7).

    Response Figure 7. WB analysis and quantification of mitochondrial encoded proteins in WT and Fam210aMKO muscle at different ages.

    The mitochondrial proteins were indeed decreased in Fam210aMKO starting from 6-weeks of age compared to the WT.

    Minor comments:

    -What is known about FAM210A, other studies assessing its role, and the rational for studying its function should be better introduced.

    Answer: We thank the reviewer for the suggestion to have more information of FAM210A functions/mechanisms in the introduction. We have added more background to the introduction.

    -In the discussion the authors states: "Moreover, when the proportion of ribosomal protein phosphorylation buildup in the Fam210aMKO, the assembly of the translational machinery is impaired therefore further dampen the cellular translation". Do they mean acetylation and not phosphorylation?

    Answer: We are sorry about the typo and have changed it. We thank the reviewer for catching this.

    • Please use the term "mRNA translation" or "protein synthesis" instead of "protein translation" in the text.

    Answer: We thank the reviewer for the suggestion to properly refer to these processes. We have changed the terms in the manuscript.

    -The methods section for RT-qPCR: It should ne M-MLV RT and not M-MLC. If the qPCR data was normalized with 18S, please provide the sequence of the primers in the table. Information on how primer efficiency was tested must be included in the method section.

    Answer: We thank the reviewer for pointing this out. We have changed the texts. We also have provided 18S sequence and provide texts about how primer efficiency was tested.

    Reviewer #2 (Significance (Required)):

    General assessment: Previous genome-wide association studies have found that mutations in FAM210A were associated with sarcopenia and osteoporosis. Because FAM210A is not expressed in the bone and highly expressed in skeletal muscle, it suggests that FAM210A likely plays an important role in muscle, which could also affect bone regulation. The authors here provide further evidence of an important role for FAM210A in diseases affecting muscle function by demonstrating that the expression of FAM210A decreases with age and in patients affected by Pompe disease, Duchenne muscular dystrophy and hereditary recessive myopathy. FAM210A is a mitochondria-localized protein and given the crucial role of mitochondria in supporting muscle metabolism, elucidating the molecular function of FAM210A may provide important insights into diseases biology that could lead to the development of therapeutic approaches. Thus, a significant protein and regulatory pathway are explored in this study that can potentially impact human health. In this manuscript, the authors provide compelling evidence of the importance of Fam210a in muscle homeostasis with their newly generate mouse model. The experiments looking at muscle physiology, function and metabolism are well-executed and for the most part rigorous, which are the strengths of this manuscript. However, the conclusion that Fam210a deletion in skeletal muscle induces the hyper-acetylation of several small ribosomal proteins, which leads to ribosomal disassembly and translational deficiency is not supported by the data presented here. As noted in the comments above, these experiments need major improvement. Additionally, there are other concerns about general scientific rigor and conclusions inconsistent with the data presented as also noted in the comments section.

    Advance: Although a previous study explored the role of FAM210A using a skeletal muscle-specific KO induced at postnatal 28 days under a HSA promoter, the model used by the authors here provide a cleaner approach and more insights into the molecular functions of FAM210A in muscle physiology. The findings that Fam210a MKO disrupts the flow of TCA cycle, which leads to an abnormal accumulation of acetyl-CoA is interesting and provide new conceptual advance on the roles of FAM210A in mitochondria function in muscle. Acetyl-CoA production is an important source of acetyl-group that can be transferred to proteins and regulate gene expression programs. Thus, this is an important finding. However, molecular mechanism by which FAM210A regulates this process through an interaction with SUCLG2 is not provided and the nature this interaction is superficially explored.

    Audience: Findings from this manuscript are likely to interest both basic research and translational/clinical audiences as it explores the physiological and molecular function of a disease-linked protein. The findings are also likely to impact the fields of metabolism, mitochondria function and regulation of gene expression by protein acetylation (if concerns raised regarding these experiments are addressed).

    The fields of expertise of this reviewer are protein and RNA modifications, ribosome biogenesis and mRNA translation.

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

    The authors state that in their manuscript "the role of mitochondria in regulating cytosolic protein translation in skeletal muscle cells (myofibers)" has been explored (Line 19-20). As experimental model, they used mice expressing Cre recombinase under the control of the myosin light chain 1 promoter. The first conclusion was that "FAM210A is positively associated with muscle mass in mice and humans". The authors say that the presented data "reveal a novel crosstalk between the mitochondrion and ribosome mediated by FAM210A".

    I recognize the potential of this work since the role of FAM210a has been more deeply investigated in skeletal muscle. In fact, the study by Tanaka et al, 2018 presented only a preliminary characterization of the role of FAM210a in muscle. However, I think that this work is not complete and each aspect that has been investigated is not well connected with each other. In particular, it is not clear whether the disrupted ribosomal assembly by hyperacetylation causes muscle atrophy or it is altered under catabolic states during atrophy (primary cause or consequence of?).

    Answer: We thank the reviewer for recognizing the importance of the study that characterizes the effect of FAM210A in muscle mass maintenance. In this study, we have shown that polysome formation was impaired at 4-week and therefore the translational efficiency was reduced in the muscle. This translational decrease coincides with the acetylation increase. Moreover, we showed by mitochondrial transfer experiment that the mitochondria from the Fam210aMKO mice can carry the phenotype and lead to acetylation increase in the recipient cells. Since muscle protein synthesis defects have been known to lead to muscle dystrophy, and we have shown that in the Fam210aMKO model, protein synthesis was indeed defective while there was not an induction of atrophy. Therefore, we conclude that the in the KO model, the protein synthesis defects lead to muscle atrophy.

    The other major point is represented by the fact that the Myl1-CRE expressing model provides selectivity in fast muscle fibers (see for example Barton PJR, Harris AJ, Buckingham M. Myosin light chain gene expression in developing and denervated fetal muscle in the mouse. Development. 1989;107: 819-824). Then the authors knocked out FAM210a only in fast fibers and they never take in consideration this key point! This is crucial since fast and slow muscles have different content of mitochondria with different size, shape, and metabolism! The muscle fibers can be classified based on the mitochondrial metabolism (see for example Chemello et al., 2019; PMID: 30917329).

    Regarding this point, they simply wrote at Line 75-76 "using a skeletal muscle specific Myl1 (myosin, light polypeptide 1) driven Cre recombinase specifically expressed in post-differentiation myocytes and multinucleated myofibers,...". It would be more correct to write multinucleated type 2 myofibers showing the reduction of FAM210a in different fiber types.

    I think that the authors must solve these aspect and then organize the findings accordingly. The data are in general interesting for broad type of audience.

    Answer:

    We appreciate the reviewer’s comment on the Myl1 knock-in Cre (Myl1Cre) model, which prompted us to more explicitly clarify some of the confusions around this model. We fully respect the validity of the 1989 study by Dr. Buckingham and other studies showing fast muscle specific expression of Myl1. However, we and others have shown that Myl1 not only mark the fast but also the slow myofibers (elaborated below). The discrepancy can be explained by the fact that using the Myl1Cre as a lineage marker is different from directly examining Myl1 expression at static timepoints by in situ hybridization (ISH). This is because Cre recombinase can accumulate and diffuse to all the myonuclei in a multinucleated myofiber, subsequently leading to deletion of LoxP-flanked DNA in all nuclei. Also, in the Cre/LoxP system, only a small amount of Cre recombinase is needed to induce the recombination of the target loxP sites and lead to gene KO. Another example of the discrepancy between the static mRNA pattern and the dynamic gene expression during development is the Hox gene expression. When the corresponding author (SK) of this manuscript was trained with Dr. Joshua R Sanes, he developed 3 Cre lines driven by three different Hox genes– that have been shown by ISH to be expressed in a specific rostral to caudal domain in the spinal cord during development. However, each of these Cre model ended up marking all the spinal cord without any domain specificity. In the case of Myl1Cre mouse model, we have previously published a paper on the lineage-tracing results using the Myl1Cre and showed that Myl1Cre marked all fast AND slow myofibers in mice (Wang et al, 2015, PMID: 25794679). In another lineage tracing study using nuclear GFP reporter, we report that Myl1Cre marks 96% nuclei in myofibers regardless of fiber types (Bi et al., 2016, PMID: 27644105), the remainder 4% non-marked nuclei potentially represent satellite cells. Other groups have also used the Myl1Cre model to induce KO in both fast and slow muscles (Pereira et al, 2020, PMID: 31916679). Therefore, we believe that the Myl1Cre mouse model allows us to efficiently knockout the Fam210a gene in both slow and fast muscle.

    To directly confirm that Fam210a was efficiently knocked out in both slow and fast muscles using the Myl1Cre mouse model, we isolated different muscle groups (Soleus and diaphragm that contains a large fraction of slow myofibers, TA and EDL that contain predominantly fast myofibers) and checked the expression level and the KO efficiency of Fam210a by WB. We have shown that even in slow muscles like diaphragm and SOL, the KO was very efficient, as there were no visible FAM210A bands in the WB (Figure S1C).

    In more detail:

    The data must be analyzed and discussed based on the fact that FAM210a has been deleted specifically in fast fibers. First the authors must show the protein levels of FAM210a in both fast, slow and mixed fast-slow muscles. Then for example in Figure S1C EDL, GAS and SOL muscles must be included.

    Answer: This is related to the misunderstanding of the Myl1Cre model. We understand the reviewer’s concern and therefore isolated proteins from different muscles in WT and Fam210aMKO mice at 4-weeks and checked the expression level of FAM210A. We have shown that regardless of fast or slow muscles, FAM210A was deleted.

    The blot in general must be repeated since it has poor quality (continuum of FAM210a band in the samples).

    Answer: We thank the reviewer for this suggestion and increase the data quality. We have changed the original blot with the following blots showing that FAM210A was not deleted in other non-muscle tissues (Figure S1C).

    Please provide staining of TA, GAS and SOL muscles to show how Myl1CRE-directed deletion of FAM210a affect the different myofibers.

    Answer: This point is also related to assumption that Myl1Cre only induce deletion in fast myofibers. We have done staining in both EDL and SOL muscle to show the relative changes in myofiber compositions. We found that the myofibers in EDL and SOL muscle have shifted to a more oxidative type upon Fam210a KO (Figure S3).

    In Figure 2F where decreased TA muscle weight was showed in the Fam210aMKO mice, the authors must include also the other muscles (EDL, GAS and SOL).

    Answer: We thank the reviewer for helping us be more rigorous on the phenotype examination. We understand that the reviewer initially raised this question because of the concern on Myl1Cre model. Now that we have shown the MylCremarks both the fast and slow muscles, we believe this question is no longer a concern. Besides, to indirectly answer the question, we would like for the reviewer to appreciate the size difference of the EDL as well as the SOL muscle in Figure S3 in the manuscript. As can be seen from the images, the size of the SOL muscles in the KO was significantly reduced compared to the WT, speaking in favor of the KO effect on slow muscles.

    In general, since the HSA-CRE model is generally used for gene manipulation in skeletal muscles the authors must characterize their model considering that the myosin light chain 1 promoter Myl1-Cre is mainly active in postmitotic type II myofibers. The last model can also give advantage for mosaic gene manipulation in muscles with mixed fiber types.

    Answer: We thank the reviewer for bringing this point up. We hope by the multiple lines of evidence that we provided in the previous questions, we can convince the reviewer that the KO model using the Myl1Cre does not lead to a mosaic gene manipulation in the muscle. On the contrary, the KO model is a homogeneous KO in both fast and slow muscles.

    Line 118-119 Fam210a level is positively corelated with muscle mass, as it is reduced in muscle atrophy conditions and increased in muscle hypertrophy conditions. Fig 1: I don't like since there are many different models in which the muscle mass reduction is associated with different mechanisms. Then independently of mechanisms associated with changes in muscle mass Fam210a is always linked to? Which common mechanism can explain this?

    Answer: We understand that the reviewer would like to pursue a conserved mechanism governing muscle mass maintenance, however, we by no means wanted to make a direct causal relationship between FAM210A level and different muscle disease/atrophy conditions. Indeed, the atrophic conditions presented have different mechanisms leading to muscle mass reduction, yet we wanted to present the possible connection that Fam210a level and muscle mass are co-regulated, and we later confirmed by KO mouse model that FAM210A KO indeed reduces muscle mass.

    Line 144-146 Hematoxylin and eosin (H&E) staining did not reveal any obvious myofiber pathology in the Fam210aMKO mice up to 8 weeks (Figure 2G). I totally disagree! It seems that there is more inflammation upon deletion of Fam210aMKO. Please check it.

    Answer: We thank the reviewer for pointing this out to help us more rigorously describe our results. We have changed the wording to better reflect the changes observed with H&E images.

    Fig3E-L there is a huge difference between EDL and SOL. The authors can't avoid to discuss their data considering the real expression of CRE upon Myl promoter: specific deletion in fast fibers. I think that the data in FIGS3 are very important and must be linked to data in Fig3. Organize in a different way all the presented data to really describe what is happening upon deletion of Fam210a.

    Again, the authors MUST organize better their data in the manuscript: to each paragraph must correspond data in the main figures. For example: at Line 189 Fam210aMKO mice exhibit systemic metabolic defects and at Line 208 Fam210aMKO increases oxidative myofibers and decreases glycolytic myofibers. These two paragraphs discuss data showed only in supplementary figures.

    Answer: We thank the reviewer for this suggestion. As shown in the previous responses, the Myl1Cre indeed induce efficient deletion of Fam210a in slow muscles. Therefore, we did not consider this to be a myofiber-specific deletion model. We consider these two results as the effect of a mitochondrial protein (FAM210A) on the myofiber metabolism (independent of myofiber type specific deletion), and that the deletion of Fam210a results in mitochondrial stress, which can lead to myofiber switch (Figure S3).

    Physical activity mast be monitored. Show respiratory exchange ratio (RER = VCO2/VO2) and discuss the results.

    Answer: We thank the reviewer for this suggestion. By these results, we would like to demonstrate that muscle homeostasis is important for the systemic metabolism, disruption of muscle mass maintenance in the Fam210aMKO mice leads to defects in the whole-body metabolism. We have now included the RER results (Figure S2F, S2G). The results show that the Fam210aMKO mice had significantly lower RER (VCO2/VO2) value at daytime, indicating that the mice rely more on utilizing fat as the fuel source. This is consistent with the proteomics results (Figure 5K) that the Fam210aMKOmice have increased FAO pathway. Unfortunately, our metabolic chamber does not have the capacity to monitor activity. We instead include data on heat production (Figure S2E).

    "Fam210aMKO increases oxidative myofibers and decreases glycolytic myofibers". The data mast be associated with the evaluation of the expression levels of FAM210 in different fiber type to really understand what is happening upon FAM210a loss.

    Answer: We understand the reviewer’s concern on the different expression level of Fam210a as well as the KO efficiency using the *Myl1Cre *model. We have shown that Fam210a is knocked out in fast and slow muscles, therefore, we did not consider the effects on fast and slow myofibers separately.

    As SDH activity in type 1 fibers is higher than type 2 the and since the authors are using a model in which Fam210a is deleted only in type 2 fiber they should understand what is happening: fiber 1

    Answer: We agree with the reviewer that the SDH activity is different in different myofibers. We have shown by western blot that FAM210A was similarly KO in both fast and slow muscles. When we performed fiber type staining in EDL and SOL muscle, we saw that there was a shift towards the slower myofiber types both in the EDL and SOL muscle, due to mitochondrial defects.

    Associate a cox assay with the sdh assay

    Answer: We thank the reviewer for this suggestion. We have shown by SDH staining as well as seahorse experiments using isolated mitochondria that the complex II activity was impaired in the muscle. We understand the reviewer would like to see a COX assay to show the defects of the mitochondrial function. Though we were not able to perform the COX assay, we showed from other aspects that the mitochondrial function was impaired by running WB of the mitochondrial encoded proteins (ATP6, MTCO2, mtCYB) and showed these proteins were decreased with ages. Along with the morphological changes of the mitochondria shown by electron microscope (Figure 5 and Figure S5), we conclude that these changes must have impacted mitochondrial function.

    Figure 4b blot tubulin and FAM210a look strange. Look especially at first and second and fourth form the left side.

    Answer: We are sorry about the mistake in the images, we have changed the Tubulin blot in the Oxphos blots.

    Figure 4B OXPHOS protein levels look similar between wt and KO. Include the quantification with the significance (min 3-5 mice per genotype).

    Answer: we have quantified the change between WT and KO on different proteins (Reponse Figure 8).

    Response Figure 8. Quantification of the OxPHOS proteins in WT and Fam210aMKO muscle at different ages.

    Quantification of the blots showed that indeed the mitochondrial proteins were decreased in the Fam210aMKO. The change of mitochondrial encoded protein MTCO1 was earlier detected in the Fam210aMKO.

    Provide TEM analysis for SOL muscle. I would understand whether mitochondria are differently affected in fast and slow muscles.

    Answer: We understand the reviewer was originally concerned about the KO efficiency of Fam210a in fast and slow muscles, based on the assumption about the MylCre model. We have shown that the FAM210A protein was similarly depleted in both fast and slow muscles by western blot. In this case, we would speculate that the mitochondrial change in fast and slow muscles would be similar because the mitochondrial changes were due to the inherent defects in the mitochondria.

    In all experiments must be clear which muscle type or types was/were used:

    Line 268: "isolated from WT and Fam210aMKO muscles at 6 weeks of age".

    Line 587 "Muscle lysate acetyl-CoA contents"

    For Seahorse Mitochondrial Respiration Analysis at Line 599 "isolated mitochondria from muscle"

    For TCA cycle metabolomics at Line 615 "muscle tissue was weighed and homogenized"

    For SCS activity assay at Line 632 "mitochondria from muscles were isolated"

    For LC-MS/MS at Line 647 "Mitochondria were purified from skeletal muscles and subjected to proteomics analysis".

    For Ribosome isolation at Line 676 "Skeletal muscle from mice"

    For Polysome profiling experiment at Line 696 "muscle tissues from mice were dissected"

    It is important to know which muscles were used since confounding effects of the specific deletion of FAM210a in type 2 fibers must be identified and discussed.

    Answer: We thank the reviewer for considering the different muscle groups in our mouse model. For experiments requiring a large amount of muscle tissue, such as ribosome isolation, mitochondrial isolation and polysome profiling, we used all the muscles from the mouse. For WB experiments, we used the TA muscle. We have included this information in the method section in the manuscript. Since we have shown that FAM210A was similarly depleted in different muscles (see previous responses), we think it is justified to pool muscles from the same mouse.

    Line 296-297 The authors wrote "Consistently, the mRNA levels of Atf4, Fgf21 and the associated transcripts were highly induced in the Fam210aMKO 296 both in the 4-week and 6-week-old muscle samples". Is Fgf21 responsible for the reduction of body weight? (see for example PMID: 28552492, PMID: 28607005 and PMID: 33944779). Measure the circulating Fgf21 protein in Ko and wt mice.

    Answer: We thank the reviewer for this great suggestion. Indeed, Fgf21 can potentially lead to body weight reduction, and this can explain the smaller body weight in our mouse model as well. However, we are more concerned about the muscle changes in our mouse model, therefore we did not further validate the changes of Fgf21 in the circulation.

    After careful considerations on the mechanism proposed in the study, we decided to remove qPCR data showing the modest increase of Fgf21 mRNA level. The removal of this data will not change the conclusions we draw nor lessen the significance of the mitochondria transfer experiment.

    Moreover the authors must check Opa1 total protein level and also the ratio between long and short isoforms. Is Fam210a interacting with Opa1?

    Answer: We thank the reviewer for this interesting question. Another publication from our lab has shown that Fam210a can modulate the cleavage of OPA1 in brown adipose tissue and influence the cold-induced thermogenesis (PMID: 37816711). Indeed, OPA1 deletion in muscle can lead to muscle atrophy and postnatal death at about day 10 (PMID: 28552492) through the induction of UPR (ISR) and the induction of Fgf21. We did not check the interaction between FAM210A and OPA1 in the muscle context, and FAM210A was not found to be interacting with OPA1 in brown adipose tissue (PMID: 37816711). However, the focus of this study was the acetylation change and the FAM210A effect on muscle mass maintenance. Therefore, we did not pursue the OPA1 related mechanism in skeletal muscle.

    The final part of the paper is really interesting but need to be discussed knowing exactly the used experimental model. Then check in which fiber types FAM210a is loss.

    Answer: We thank the reviewer for the stringency on the model used. Indeed, the mitochondria can be different from different muscle groups. However, since the muscle isolated from WT and KO mice was properly controlled and therefore can balance the effects of different mitochondria. We have consistently observed the increased acetylation when mutant mitochondria were transferred.

    Regarding the mitochondrial transplantation I'm surprise to see that it happens in the direction of unhealthy mitochondria to healthy cells. Are you able to rescue the phenotype of Fam210a KO cells with healthy mitochondria?

    Answer: We thank the reviewer for bringing this interesting yet important question up! Our mitochondrial transfer results support a “gain-of-function” model where excessive Acetyl CoA produced by the Fam210a-KO mitochondrial induces hyperacetylation. Regarding the question to transfer healthy mitochondria to rescue the KO cells, we reason that even when we transfer the healthy mitochondria to the KO cells, the healthy mitochondria will not stop the mutant mitochondria from making excessive amounts of acetyl-CoA and thus protein acetylation. A clean transfer would require depletion of the mitochondria in the KO cells and concomitant restoring FAM210A level in the KO cells (as the lack of Fam210a gene in the KO cells will eventually convert the transferred mitochondrial into mutants with the normal turnover of FAM210A). This is technically highly challenging and nearly impossible to do. We hope that the reviewer can understand the difficulties.

    Reviewer #3 (Significance (Required)):

    In conclusion, the strength of the presented paper is the novelty: the authors explored the role of FAM210a in skeletal muscle. However, the major limitation is represented by the fact that they did not show in which fiber types Fam210a is knocked out. In fact, the used CRE recombinase expressing model is well-known to be specific for type 2 fibers. Then since mitochondria and metabolism are central in this manuscript and they are different in the fast and slow fiber types, the authors must dissect in details this point.

    Moreover, there are many data but they are not linked each other and discussed properly. The paper must be completely re-organized.

    This manuscript can be interesting for a broad type of audience.

    I'm an expert on mitochondria, metabolism and skeletal muscle.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors state that in their manuscript "the role of mitochondria in regulating cytosolic protein translation in skeletal muscle cells (myofibers)" has been explored (Line 19-20). As experimental model, they used mice expressing Cre recombinase under the control of the myosin light chain 1 promoter. The first conclusion was that "FAM210A is positively associated with muscle mass in mice and humans". The authors say that the presented data "reveal a novel crosstalk between the mitochondrion and ribosome mediated by FAM210A".

    I recognize the potential of this work since the role of FAM210a has been more deeply investigated in skeletal muscle. In fact, the study by Tanaka et al, 2018 presented only a preliminary characterization of the role of FAM210a in muscle. However, I think that this work is not complete and each aspect that has been investigated is not well connected with each other. In particular, it is not clear whether the disrupted ribosomal assembly by hyperacetylation causes muscle atrophy or it is altered under catabolic states during atrophy (primary cause or consequence of?).

    The other major point is represented by the fact that the Myl1-CRE expressing model provides selectivity in fast muscle fibers (see for example Barton PJR, Harris AJ, Buckingham M. Myosin light chain gene expression in developing and denervated fetal muscle in the mouse. Development. 1989;107: 819-824). Then the authors knocked out FAM210a only in fast fibers and they never take in consideration this key point! This is crucial since fast and slow muscles have different content of mitochondria with different size, shape, and metabolism! The muscle fibers can be classified based on the mitochondrial metabolism (see for example Chemello et al., 2019; PMID: 30917329).

    Regarding this point, they simply wrote at Line 75-76 "using a skeletal muscle specific Myl1 (myosin, light polypeptide 1) driven Cre recombinase specifically expressed in post-differentiation myocytes and multinucleated myofibers,...". It would be more correct to write multinucleated type 2 myofibers showing the reduction of FAM210a in different fiber types.

    I think that the authors must solve these aspect and then organize the findings accordingly. The data are in general interesting for broad type of audience.

    In more detail:

    The data must be analyzed and discussed based on the fact that FAM210a has been deleted specifically in fast fibers. First the authors must show the protein levels of FAM210a in both fast, slow and mixed fast-slow muscles. Then for example in Figure S1C EDL, GAS and SOL muscles must be included. The blot in general must be repeated since it has poor quality (continuum of FAM210a band in the samples). Please provide staining of TA, GAS and SOL muscles to show how Myl1CRE-directed deletion of FAM210a affect the different myofibers. In Figure 2F where decreased TA muscle weight was showed in the Fam210aMKO mice, the authors must include also the other muscles (EDL, GAS and SOL). In general, since the HSA-CRE model is generally used for gene manipulation in skeletal muscles the authors must characterize their model considering that the myosin light chain 1 promoter Myl1-Cre is mainly active in postmitotic type II myofibers. The last model can also give advantage for mosaic gene manipulation in muscles with mixed fiber types. Line 118-119 Fam210a level is positively corelated with muscle mass, as it is reduced in muscle atrophy conditions and increased in muscle hypertrophy conditions. Fig 1: I don't like since there are many different models in which the muscle mass reduction is associated with different mechanisms. Then independently of mechanisms associated with changes in muscle mass Fam210a is always linked to? Which common mechanism can explain this? Line 144-146 Hematoxylin and eosin (H&E) staining did not reveal any obvious myofiber pathology in the Fam210aMKO mice up to 8 weeks (Figure 2G). I totally disagree! It seems that there is more inflammation upon deletion of Fam210aMKO. Please check it. Fig3E-L there is a huge difference between EDL and SOL. The authors can't avoid to discuss their data considering the real expression of CRE upon Myl promoter: specific deletion in fast fibers. I think that the data in FIGS3 are very important and must be linked to data in Fig3. Organize in a different way all the presented data to really describe what is happening upon deletion of Fam210a. Again, the authors MUST organize better their data in the manuscript: to each paragraph must correspond data in the main figures. For example: at Line 189 Fam210aMKO mice exhibit systemic metabolic defects and at Line 208 Fam210aMKO increases oxidative myofibers and decreases glycolytic myofibers. These two paragraphs discuss data showed only in supplementary figures. Physical activity mast be monitored. Show respiratory exchange ratio (RER = VCO2/VO2) and discuss the results. "Fam210aMKO increases oxidative myofibers and decreases glycolytic myofibers". The data mast be associated with the evaluation of the expression levels of FAM210 in different fiber type to really understand what is happening upon FAM210a loss. As SDH activity in type 1 fibers is higher than type 2 the and since the authors are using a model in which Fam210a is deleted only in type 2 fiber they should understand what is happening: fiber 1 Associate a cox assay with the sdh assay Figure 4b blot tubulin and FAM210a look strange. Look especially at first and second and fourth form the left side. Figure 4B OXPHOS protein levels look similar between wt and KO. Include the quantification with the significance (min 3-5 mice per genotype). Provide TEM analysis for SOL muscle. I would understand whether mitochondria are differently affected in fast and slow muscles. In all experiments must be clear which muscle type or types was/were used: Line 268: "isolated from WT and Fam210aMKO muscles at 6 weeks of age". Line 587 "Muscle lysate acetyl-CoA contents" For Seahorse Mitochondrial Respiration Analysis at Line 599 "isolated mitochondria from muscle" For TCA cycle metabolomics at Line 615 "muscle tissue was weighed and homogenized" For SCS activity assay at Line 632 "mitochondria from muscles were isolated" For LC-MS/MS at Line 647 "Mitochondria were purified from skeletal muscles and subjected to proteomics analysis". For Ribosome isolation at Line 676 "Skeletal muscle from mice" For Polysome profiling experiment at Line 696 "muscle tissues from mice were dissected" It is important to know which muscles were used since confounding effects of the specific deletion of FAM210a in type 2 fibers must be identified and discussed. Line 296-297 The authors wrote "Consistently, the mRNA levels of Atf4, Fgf21 and the associated transcripts were highly induced in the Fam210aMKO 296 both in the 4-week and 6-week-old muscle samples". Is Fgf21 responsible for the reduction of body weight? (see for example PMID: 28552492, PMID: 28607005 and PMID: 33944779). Measure the circulating Fgf21 protein in Ko and wt mice. Moreover the authors must check Opa1 total protein level and also the ratio between long and short isoforms. Is Fam210a interacting with Opa1? The final part of the paper is really interesting but need to be discussed knowing exactly the used experimental model. Then check in which fiber types FAM210a is loss. Regarding the mitochondrial transplantation I'm surprise to see that it happens in the direction of unhealthy mitochondria to healthy cells. Are you able to rescue the phenotype of Fam210a KO cells with healthy mitochondria?

    Significance

    In conclusion, the strength of the presented paper is the novelty: the authors explored the role of FAM210a in skeletal muscle. However, the major limitation is represented by the fact that they did not show in which fiber types Fam210a is knocked out. In fact, the used CRE recombinase expressing model is well-known to be specific for type 2 fibers. Then since mitochondria and metabolism are central in this manuscript and they are different in the fast and slow fiber types, the authors must dissect in details this point. Moreover, there are many data but they are not linked each other and discussed properly.The paper must be completely re-organized.

    This manuscript can be interesting for a broad type of audience.

    I'm an expert on mitochondria, metabolism and skeletal muscle.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: In this manuscript, Chen et al., investigate the functions of FAM210A in skeletal muscle physiology and metabolism. FAM210A is a mitochondria-localized protein in which mutations have been associated with sarcopenia and osteoporosis. Using publicly available gene expression datasets from human skeletal muscle biopsies the authors first demonstrate that the expression of FAM210 is reduced in muscle atrophy-associated diseases and increased in muscle hypertrophy conditions. Based on this, they show that a muscle specific Fam210a deletion leads to muscle atrophy/weakness, systemic metabolic defects, and premature lethality in mouse. Further examination of the knockout myofibers reveals impaired mitochondrial respiration and translation program. Additionally, the authors demonstrate that the flow of TCA cycle is disrupted in the FAM210A-deleted myofibers, which causes abnormal accumulation of acetyl-coA and hyperacetylation of a subset of proteins. The authors claim that Fam210a deletion in skeletal muscle induces the hyper-acetylation of several small ribosomal proteins that leads to ribosomal disassembly and translational deficiency. However, this conclusion is not supported by adequate experimentation and rigorous analysis of ribosomal proteins acetylation and ribosome assembly.

    Major comments:

    • In general, figure legends are lacking information regarding number of biological replicates used and details about statistical analysis. What does three * vs. one * mean in terms of p-value? Exact p-values should be indicated.
    • The mechanistic studies linking muscle phenotypes with ribosomal protein hyperacetylation and mRNA translation defects are underdeveloped and not rigorously carried.
    • Fig S1: The validation WB of FAM210A KO is not the most convincing. Why are the FAM210A levels so low in TA compared to other tissues?
    • Fig 2G: The authors state "Hematoxylin and eosin (H&E) staining did not reveal any obvious myofiber pathology in the Fam210a KO mice up to 8 weeks". However there seems to be a progressive increase in nuclei up to 8-weeks in the KO. What is the significance of this?
    • IP-MS analysis for FAM210A interacting proteins requires validation with IP and reverse IP + WB experiment.
    • Figure 4A requires quantification of the SDH signals from multiple samples.
    • Figure 6F: To clearly demonstrate an increase in protein acetylation in the FAM210 MKO, the authors must provide quantification data generated with more then N=1. Please add the molecular weights markings on the side of the blots.
    • Figure 6H and S5: The mitochondria transfer experiment appears to be quite efficient compared to previously published studies. It would be important to control that the signal observed in the recipient cells is not due to the leakage of the MitoTracker dye from the donor mitochondria.
    • Figure 6J: The increase in Fgf21 is modest. Although the difference is statistically significant, is it biologically important?
    • Figure 6C: How significant is the difference in acetylation of RPL30 in WT vs. KO. RPS13 was not found in the WT MS? Was this normalized to Input?
    • Figure 7D: What are the MW of the bands shown on this blot? This experiment is by no means sufficient to demonstrate and confirm that ribosomal proteins are acetylated. An increase in RPL30 and RPS13 acetylation must be directly assessed.
    • Fig7E: This experiment is not properly executed and in its current state does not rigorously support that "hyper-acetylation of several small ribosomal proteins leads to ribosomal disassembly". A) UV profiles of the fractionation must be provided to assess the quality of the profile. B) Provide MW markers. Which band is RPL30? The Input and free fraction bands are not at the same size. RPL30 should at least be visible on the 60S and polysomes from the WT. C) These results do not match the acetylation MS data, which seem to show that the increase in acetylation is much greater for RPS13. However, RPS13 presence on polysomes (assuming they are polysomes) is not affected in the KO. D) This type of experiment must be done for three independent biological replicates, blots from single lanes must be quantified and normalized to total signal (from all the lanes) for the same antibody.
    • Fig 7F: Global translation rates are assessed by puro incorporation at week 4, a time point when differences in protein acetylation were not observed. This does not support the hypothesis that increased acetylation of ribosomal proteins causes defect in protein translation. (Referencing the authors statement p.7 lines 321-24.).
    • Other studies have implicated Fam210A in the regulation of mitochondrial protein synthesis through an interaction with EF-Tu. The authors also identified EF-Tu as an interactor in their LC-MS analysis (FigS4). A role for this interaction accounting for mitochondrial and translation defects seems to be underestimated and unexplored here.

    Minor comments:

    • What is known about FAM210A, other studies assessing its role, and the rational for studying its function should be better introduced.
    • In the discussion the authors states: "Moreover, when the proportion of ribosomal protein phosphorylation buildup in the Fam210aMKO, the assembly of the translational machinery is impaired therefore further dampen the cellular translation". Do they mean acetylation and not phosphorylation?
    • Please use the term "mRNA translation" or "protein synthesis" instead of "protein translation" in the text.
    • The methods section for RT-qPCR: It should ne M-MLV RT and not M-MLC. If the qPCR data was normalized with 18S, please provide the sequence of the primers in the table. Information on how primer efficiency was tested must be included in the method section.

    Significance

    General assessment: Previous genome-wide association studies have found that mutations in FAM210A were associated with sarcopenia and osteoporosis. Because FAM210A is not expressed in the bone and highly expressed in skeletal muscle, it suggests that FAM210A likely plays an important role in muscle, which could also affect bone regulation. The authors here provide further evidence of an important role for FAM210A in diseases affecting muscle function by demonstrating that the expression of FAM210A decreases with age and in patients affected by Pompe disease, Duchenne muscular dystrophy and hereditary recessive myopathy. FAM210A is a mitochondria-localized protein and given the crucial role of mitochondria in supporting muscle metabolism, elucidating the molecular function of FAM210A may provide important insights into diseases biology that could lead to the development of therapeutic approaches. Thus, a significant protein and regulatory pathway are explored in this study that can potentially impact human health. In this manuscript, the authors provide compelling evidence of the importance of Fam210a in muscle homeostasis with their newly generate mouse model. The experiments looking at muscle physiology, function and metabolism are well-executed and for the most part rigorous, which are the strengths of this manuscript. However, the conclusion that Fam210a deletion in skeletal muscle induces the hyper-acetylation of several small ribosomal proteins, which leads to ribosomal disassembly and translational deficiency is not supported by the data presented here. As noted in the comments above, these experiments need major improvement. Additionally, there are other concerns about general scientific rigor and conclusions inconsistent with the data presented as also noted in the comments section.

    Advance: Although a previous study explored the role of FAM210A using a skeletal muscle-specific KO induced at postnatal 28 days under a HSA promoter, the model used by the authors here provide a cleaner approach and more insights into the molecular functions of FAM210A in muscle physiology. The findings that Fam210a MKO disrupts the flow of TCA cycle, which leads to an abnormal accumulation of acetyl-CoA is interesting and provide new conceptual advance on the roles of FAM210A in mitochondria function in muscle. Acetyl-CoA production is an important source of acetyl-group that can be transferred to proteins and regulate gene expression programs. Thus, this is an important finding. However, molecular mechanism by which FAM210A regulates this process through an interaction with SUCLG2 is not provided and the nature this interaction is superficially explored.

    Audience: Findings from this manuscript are likely to interest both basic research and translational/clinical audiences as it explores the physiological and molecular function of a disease-linked protein. The findings are also likely to impact the fields of metabolism, mitochondria function and regulation of gene expression by protein acetylation (if concerns raised regarding these experiments are addressed). The fields of expertise of this reviewer are protein and RNA modifications, ribosome biogenesis and mRNA translation.

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    Referee #1

    Evidence, reproducibility and clarity

    In the manuscript entitled "FAM210A mediates an inter-organelle crosstalk essential for protein synthesis and muscle growth in mouse", Chen et al, found that knocking out of FAM210A specifically in muscle using Myl Cre resulted in abnormal mitochondria, hyperacetylation of cytosolic proteins, and translation defects. The manuscript uncovered the new functions of FAM210A in regulating metabolism and translation. I have the following the concerns about the manuscript.

    Comments

    1. One of the major phenotypes of FAM210A is the decrease of muscle mass after 6 weeks after birth. Is this phenotype caused by the accumulation of progressive loss of muscle mass from birth? Are the body weight and muscle mass reduced in FAM210A knocking out new born mice? Is the muscle mass growth curve the same in FAM210A and WT mice from birth to 6 weeks after birth? These results will reveal more mechanism of FAM210A mediated muscle mass control.
    2. Does the muscle mass continue to decrease after 8 weeks?
    3. FAM210A knockout mice displayed high lethal rate. Is there any potential mechanism for the high lethality?
    4. In Figure 2, the muscle mass decreased significantly, while the fat mass only decreased slightly. In FAM210A knockout mice. However, the ratio of the lean mass and fat mass to body mass did not change in FAM210A knockout mice compared to WT mice. How do the authors reconcile this?
    5. Are there changes of the number of nuclei per myotube? Is the muscle atrophy in FAM210A knockout mice caused by the defects of fusion, or the degradation of protein, or both?
    6. Are the growth curves of muscle mass growth in EDL and SOL the same n FAM210A knockout mice?
    7. The oxygen consumption and carbon dioxide production are higher in FAM210A knockout mice, suggesting a high metabolism rate. In contrast, the heat production of FAM210A knockout mice is lower, suggesting a low metabolism rate. Any explanation?
    8. Given the high glucose consumption in FAM210A, why is the clearance rate of blood glucose low?
    9. Are there any changes of the abilities for the FAM210A knockout mice in running endurance?
    10. In page 5, the last sentence of the 2nd paragraph, the authors concluded "There results suggest that Fam210aMKO induces a metabolic switch to a more oxidative state." It is better to describe it as muscle metabolic since the whole body metabolism has not been carefully examined.
    11. In Fig. 6, what is the link between increased transcription level of Fgf21 and the elevated level of aberrant acetylation of proteins?
    12. Is there any link between the increased acetylation level of rebolsome proteins and the translation defects?
    13. How do the abnormal mitochondria lead to increased protein acetylation? And how do these defects further cause translation problem?
    14. The defects in translation will cause general problems besides mitochondria defects. Are there any phenotypes related to the overall translation inhibition observed? If not, why?
    15. Are the abnormal mitochondria, increased protein acetylation, and translation inhibition observed in 2-6 weeks old mice? When were these defects first found? Are they correlated with muscle atrophy?

    Significance

    This manuscript described many interesting phenotypes of Fam210a knockout mice. However, the links between these phenotypes are obscure. The logic of the manuscript will be greatly improved if the authors could provide explanations to logically link the phenotypes.

  5. Version published to 10.1101/2023.08.03.551853v2 on bioRxiv
  6. Version published to 10.1101/2023.08.03.551853v1 on bioRxiv