Reduced levels of mitochondrial ribosomal protein MRPL54 does not alter Apc related adenoma formation

Claudia N. Spaan; Eileen Daniels; Wouter L. Smit; Ruben J. de Boer; Joana Silva; Jacqueline L. M. Vermeulen; S. Meisner; Vanesa Muncan; Riekelt H. Houtkooper; Jarom Heijmans

doi:10.1371/journal.pone.0344161

Peer Review History

Original SubmissionMarch 31, 2025
7 May 2025 Decision Letter - Irina Lebedeva, Editor Dear Dr. Heijmans, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. The manuscript set to investigate the plausible role of the mitochondrial ribosomal protein MRPL54 in colorectal tumorigenesis. Using micro-arrays of intestinal organoids with multiple oncogenic mutations, the authors identified upregulation of MRPL54 and other MRPs proteins and used previously established (by their group) Mrpl54 heterozygous mouse model of intestinal carcinoma (Apc-mutant). No detectable effect of MRPL54 heterozygosity was observed compared to the parental mouse model of intestinal carcinoma. The study is based solely on in vivo data and lacks mechanical insight. A few experiments need to be performed to make the story complete. Reduced MRPL54 protein expression or function in the model has to be confirmed. A role of MPRL54 protein in tumorigenesis had been established using limited datasets (microarray and gross adenoma counts). No analysis of patient-derived CRC data for MRPL54 expression or prognostic relevance had been accomplished. A few in vitro functional assays in CRC cell lines (e.g., siRNA knockdown, metabolic flux analysis, ROS measurement) would help to confirm the rationale for the study. Furthermore, functional redundancy among multiple MRPs is not addressed. This is particularly important for metabolically flexible colorectal cancers that use both glycolysis and OXPHOS as energy sources. Minor comments: The manuscript lacks clarity in both writing and data interpretation. For example, the abstract does not clearly reflect the study rationale. Discussion needs to be edited. 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Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Additional Editor Comments (if provided): [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** Reviewer #1: Apart from few gramatical errors, the manuscript is scientifically well written. The methods section and results section is well detailed appropriate for the conducted study. The first paragraph of discussion reads more like results section and need to be edited. Reviewer #2: The authors investigate the potential role of the mitochondrial ribosomal protein MRPL54 in colorectal tumorigenesis. Citing increased MRPL54 expression in colorectal cancer and the known function of mitochondrial ribosomal proteins (MRPs) in metabolic regulation, they hypothesize that reduced expression of Mrpl54 (via heterozygosity) would suppress adenoma formation in a genetically engineered mouse model with Apc mutations. However, they report that Mrpl54 heterozygosity has no detectable effect on adenoma formation or progression, and thus conclude that MRPL54 is not essential for Apc-driven colorectal tumor development. Major Comments 1. Lack of Mechanistic Depth and Novel Insight While MRPL54 has been identified in transcriptomic datasets as upregulated in various cancers, the manuscript does not offer new mechanistic insight into its role or lack thereof in colorectal cancer. The negative finding (no phenotype in heterozygous mice) is not inherently unpublishable, but it must be contextualized with mechanistic rigor, which is absent here. 2. Insufficient Justification of Experimental Strategy The central hypothesis—that MRPL54 is essential for adenoma formation is not adequately supported or tested: Heterozygosity may not be sufficient to significantly impair mitochondrial translation. There is no confirmation of reduced MRPL54 protein expression or function in the model. Functional redundancy among MRPs (of which there are ~82) is not addressed. 3. Overly Simplistic Assumption Regarding Metabolism and Tumorigenesis The authors propose that reducing a single MRP should alter tumorigenesis, but colorectal cancers are metabolically flexible, using both glycolysis and oxidative phosphorylation (OXPHOS). It is therefore not surprising that reducing one MRP does not disrupt tumor initiation. This limitation weakens the conceptual novelty of the work. 4. Incomplete Exploration of MRPL54’s Role The study relies heavily on: A single in vivo model (Apc-mutant mice) Limited datasets (microarray and gross adenoma counts) No in vitro functional assays (e.g., siRNA knockdown, metabolic flux analysis, ROS measurement) No analysis of patient-derived CRC data for MRPL54 expression or prognostic relevance. As such, the manuscript does not sufficiently explore whether MRPL54 has any functional role in tumor metabolism, survival, or progression. 5. Presentation Issues The manuscript lacks clarity in both writing and data interpretation. Several sections are grammatically inconsistent or incomplete. To strengthen future studies, the authors could: Confirm MRPL54 knockdown at the protein and functional level in their model. Perform functional assays in CRC cell lines (e.g., siRNA/CRISPR knockdown with proliferation, OXPHOS, and ROS readouts). Analyze clinical datasets (e.g., TCGA) to correlate MRPL54 expression with patient survival and tumor stage. Explore compensatory responses by other MRPs or mitochondrial stress response pathways. Investigate metabolic consequences of MRPL54 loss using Seahorse or mass spectrometry–based metabolomics. Reviewer #3: This study investigated whether reduced levels of mitochondrial ribosomal protein MRPL54 affect the tumorigenic characteristics of colorectal cancer in mice. MRPL54 was chosen as a target protein because its expression levels correlate with the progression and proliferation stages of this type of cancer, as shown by micro-array mRNA expression results. The authors aimed to test whether reducing MRPL54 levels would inhibit adenoma formation due to its importance in the biogenesis of OXPHOS proteins. Results from the study reveal that deletion of MRPL54 does not alter intestinal adenoma formation in mice. Both WT and KO animals had similar body weight and characteristics at 6 months of age, before euthanasia. Similar numbers of adenomas were recorded in both groups of animals. Overall, this is a concise and clear study. The conclusions are well supported by the results, and data presentations are clear. However, several typos throughout the manuscript need to be corrected before publication. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: Yes: Silpa Gampala Reviewer #2: No Reviewer #3: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". 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Revision 1
7 Jan 2026 Author Response 1. The study is based solely on in vivo data and lacks mechanical insight. A few experiments need to be performed to make the story complete. Reduced MRPL54 protein expression or function in the model has to be confirmed. This research continued the work of Reid and Daniels, using the Mrpl54 bodywide heterozygous mouse model described in Reid et al, 2023. In that study they showed 50% reduction of Mrpl54 gene expression in multiple organs in MRPL54 heterozygous (genotyped) mice. The observation that pups homozygous for the Mrpl54 mutation did not survive as reflected by the Mendelian ratio, is in agreement with the dysfunctional Mrpl54 allele. Tissue of heterozygous and wildtype Mrpl54 mice was used to perform western blots to measure protein expression. Unfortunately, despite using multiple antibodies, these were technically unsuccessful because of nonspecific binding. Our co-authors also tried mass spectrometry, but were not able to obtain a high enough signal. In our manuscript, we included explanation of these results and clarified the missing data of protein expression. 2. A role of MPRL54 protein in tumorigenesis had been established using limited datasets (microarray and gross adenoma counts). No analysis of patient-derived CRC data for MRPL54 expression or prognostic relevance had been accomplished. We agree with the reviewer’s suggestion to investigate multiple publicly available human colorectal cancer (CRC) datasets. We have analyzed these datasets for Mrpl54 expression and found no correlation within the human CRC data. Additionally, we observed no association between MRPL54 and colorectal cancer survival in the Human Protein Atlas. These differences between the organoid and human datasets can be attributed to the inherent complexity of the models used. However, we believe these findings are consistent with the results from our murine experiment. This point has been addressed in our discussion. Due to limitations in word count and figures we chose not to add results of CRC dataset analysis to the result section, but this could be added if the reviewer deems this preferrable. 3. A few in vitro functional assays in CRC cell lines (e.g., siRNA knockdown, metabolic flux analysis, ROS measurement) would help to confirm the rationale for the study. We have added a supplementary figure (2), including experiments with two different human colorectal cancer cell lines transfected with highly specific small interfering RNA targeting MRPL54 as well as a control. In these cells, we demonstrate that although MRPL54 RNA expression is reduced, this does not impact the expression of stem cell markers nor affect metabolic assays as glucose consumption and lactate production. 4. Furthermore, functional redundancy among multiple MRPs is not addressed. This is particularly important for metabolically flexible colorectal cancers that use both glycolysis and OXPHOS as energy sources. We agree with the reviewer and have now addressed this in our discussion. 5. Minor comments: The manuscript lacks clarity in both writing and data interpretation. For example, the abstract does not clearly reflect the study rationale. Discussion needs to be edited. To address this comment we have made textual changes and we have asked colleagues that are native English speakers to give comments on our writing. Textual changes have been made throughout the manuscript. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at We have addressed these style requirements. 2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. We have addressed this. Reviewer 1 1. The first paragraph of discussion reads more like results section and need to be edited. We have revised the discussion and incorporated feedback provided by the editor and reviewers. We feel the revised version of the manuscript has a more comprehensive discussion part compared to the original manuscript. Reviewer 2 1. Lack of Mechanistic Depth and Novel Insight. While MRPL54 has been identified in transcriptomic datasets as upregulated in various cancers, the manuscript does not offer new mechanistic insight into its role or lack thereof in colorectal cancer. The negative finding (no phenotype in heterozygous mice) is not inherently unpublishable, but it must be contextualized with mechanistic rigor, which is absent here. We have expanded our discussion and include finding on Mrpl54 expression in human datasets, as well as new experiments involving a more profound knockdown of Mrpl54 in colorectal cancer cells. We have addressed the fenomenon of redundancy in the discussion. 2. Insufficient Justification of Experimental Strategy The central hypothesis—that MRPL54 is essential for adenoma formation is not adequately supported or tested: Heterozygosity may not be sufficient to significantly impair mitochondrial translation. There is no confirmation of reduced MRPL54 protein expression or function in the model. Functional redundancy among MRPs (of which there are ~82) is not addressed. We agree with the reviewers comments, which have also been addressed by the editor, and we have include redundancy in our revised manuscript. As previousily mentioned, we have added experiments with siRNA’s in cell lines to achieve a more robust knockdown have a more profound knockdown. Furthermore, we emphasize in the manuscript previous experiments with the same Mrpl54 mouse model (Reid et al.), its findings and earlier efforts to confirm decreased MRPL54 protein expression. 3. Overly Simplistic Assumption Regarding Metabolism and Tumorigenesis The authors propose that reducing a single MRP should alter tumorigenesis, but colorectal cancers are metabolically flexible, using both glycolysis and oxidative phosphorylation (OXPHOS). It is therefore not surprising that reducing one MRP does not disrupt tumor initiation. This limitation weakens the conceptual novelty of the work. 4. Incomplete Exploration of MRPL54’s Role The study relies heavily on: A single in vivo model (Apc-mutant mice) Limited datasets (microarray and gross adenoma counts) No in vitro functional assays (e.g., siRNA knockdown, metabolic flux analysis, ROS measurement) No analysis of patient-derived CRC data for MRPL54 expression or prognostic relevance. As such, the manuscript does not sufficiently explore whether MRPL54 has any functional role in tumor metabolism, survival, or progression. We have addressed these comments in our manuscript and have written a more detailed explanation of our revisions in the previous comments of the editor and reviewers. 5. Presentation Issues The manuscript lacks clarity in both writing and data interpretation. Several sections are grammatically inconsistent or incomplete. To address this comment we have made textual changes and we have asked colleagues that are native English speakers to give comments on our writing. Textual changes have been made throughout the manuscript. Reviewer 3 1. Overall, this is a concise and clear study. The conclusions are well supported by the results, and data presentations are clear. However, several typos throughout the manuscript need to be corrected before publication. We have adressed this issue, and textual changes have been made throughout the manuscript. Attachments Attachment Submitted filename: respons to reviewers letter.docx https://doi.org/10.1371/journal.pone.0344161.r002
18 Feb 2026 Decision Letter - Irina Lebedeva, Editor Reduced levels of mitochondrial ribosomal protein MRPL54 does not alter Apc related adenoma formation PONE-D-25-10633R1 Dear Dr. Heijmans, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Irina V. Lebedeva, Ph.D. Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** Reviewer #1: The authors have adequately addressed the reviewer comments, and I recommend acceptance of the revised manuscript. Reviewer #2: all queries answered, authors have corrections. Abstract seems reduced but reads all right. Figures can be refined for the publication. Reviewer #3: (No Response) ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: Yes: Silpa Gampala Reviewer #2: Yes: Narendra Sankpal Reviewer #3: No ******** https://doi.org/10.1371/journal.pone.0344161.r003
Formally Accepted
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