An independently tunable dual control system for RNAi complementation in Trypanosoma brucei

Raveen Armstrong; Matt J. Romprey; Henry M. Raughley; Stephanie B. Delzell; Matthew P. Frost; James Chambers; Grace G. Garman; David Anaguano; Michele M. Klingbeil

doi:10.1371/journal.pone.0321334

Peer Review History

Original SubmissionMarch 5, 2025
5 Mar 2025 Author Response https://doi.org/10.1371/journal.pone.0321334.r001
25 Mar 2025 Decision Letter - Vyacheslav Yurchenko, Editor PONE-D-25-11956An independently tunable dual control system for RNAi complementation in Trypanosoma bruceiPLOS ONE Dear Dr. Klingbeil, 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. Please submit your revised manuscript by May 09 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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This work was financially supported by Bridge Funding from the University of Massachusetts College of Natural Sciences to M.M.K., a UMass Amherst Graduate School Pre-Dissertation Research Grant awarded to RA and the Donald P. Reed Legacy Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing financial interest.” We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: The manuscript on an independently tunable dual control system for RNAi complementation in Trypanosoma brucei was reviewed by 2 independent referees and they both were very positive about this work. Please add a few final touches (as requested by one of the reviewers) and it'll get accepted without re-review. [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? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This is a very nice contribution from two different perspectives - it establishes a dual inducible and tunable system for functional studies of Trypanosoma brucei genes, a major upgrade of a tool box that is in place for a almost 25 years and thanks to which this parasitic protist was lifted among top model organisms. So this reviewer simply applauds this and hopes mane labs will start using it. Next, the authors used the newly developed system for smart functional dissection of POLIB, an unusual mitochondrial polymerase that has most likely specialized functions due to its association with the complex kinetoplast DNA network. The insight they present is novel, interesting and provide further solid grounds for acceptance of this study. Finally, they introduce Kinetometric - a new program to exactly evaluate the size of the kinetoplast in T. brucei, certainly another valuable addition to the field. I am missing at least some newer references to kinetoplast studies; the authors cite old papers which is fine, but a fresh look into the literature (f.e. Michieletto NAR 2025 and below) would refresh the intro and/or discussion (f.e. when listing the kinetoplast associated proteins (around line 530 in the discussion), the authors ignore a paper by Pyrih J. et al. (Cell Reports), which identified a range of such proteins, and or the contribution by Cadena et al Curr. Biol. 2024 that associate first protein with the nabelschnur. Minor issues: 56 - unicellular protist does not sound good, as by most definitions protist = unicellular 63 and 74 - I suggest the authors replace "novel trypanosome briology" (used twice), as the combination of words is somewhat awkward. 102 - a reference seems to be missing here 496 and 497 - hours are missing 564 - Sunter and colleagues would be correct 587 - Bruhn 2010 - kind of forgotten here Reviewer #2: One of the established approaches for studying the structure-function analysis of proteins in Trypanosoma brucei is RNAi complementation in the single inducer tetracycline-On system. However, it is well known in the field that this single inducer system has not been successful for studies of several proteins. Here the authors present the establishment of an alternative vanillic acid-tetracycline dual inducible system for procyclic form T. brucei. In addition, to track the earliest reduction in kinetoplast DNA (kDNA) size, they developed a semiautomated 3D image analysis tool to measure kDNA volume which will be helpful to study kDNA replication defects. Overall, this is an excellent manuscript and the dual inducer system is a very valuable addition to the available toolkit for T. brucei and will also allow temporal regulation to modulate the timing of gene expression. ****** 6. PLOS authors have the option to publish the peer review history of their article (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: Julius Lukes Reviewer #2: 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". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0321334.r002
Revision 1
2 Apr 2025 Author Response Vyacheslav Yurchenko, Ph.D. Rebuttal responses for PONE-D-25-11956 April 2, 2025 Dear Dr. Yurchenko, Thank you for your letter and the referee's comments on our manuscript. We were happy to learn that both reviewers were very positive about our work, indicated that we report interesting and novel findings that will be of value to the wider community. We thank the reviewer's for their time and overall supportive comments. We also greatly appreciate the suggestions of Reviewer 1 to provide better references and rewording in some areas of the manuscript. We believe we have addressed all of their concerns in a satisfactory way as outlined below. Our responses are highlighted in blue and modifications to the manuscript are highlighted with yellow with appropriate line references for the revised manuscript. Responses to Academic Editor 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Thank you for pointing out our manuscript did not adhere to PLOS ONE's style requirements. We have addressed the following: 1. font size for headings and subheading has been corrected; 2. each paragraph is now indented; 3. titles for headings and subheadings have been corrected to sentence case; and 4. in text referencing to figure and supplemental files has been corrected. All changes have been addressed throughout the manuscript. 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Thank you for pointing this out to us. We have removed all text related to support from the Acknowledgments section and provided our amended funding statement to be submitted on our behalf in the cover letter; new Acknowledgments lines 655-660. 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. In your cover letter, please note whether your blot/gel image data are in Supporting Information. We have provided all original blot/gel data as a supplemental pdf file (S1_raw_images). We mention this in our cover letter. 4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. We have carefully reviewed all references from our original submission and those that we have added for the revised version. None of the references have been retracted. Responses to Reviewer # 1: (reviewers comments are italicized) 1. I am missing at least some newer references to kinetoplast studies; the authors cite old papers which is fine, but a fresh look into the literature (f.e. Michieletto NAR 2025 and below) would refresh the intro and/or discussion (f.e. when listing the kinetoplast associated proteins (around line 530 in the discussion), the authors ignore a paper by Pyrih J. et al. (Cell Reports), which identified a range of such proteins, and or the contribution by Cadena et al Curr. Biol. 2024 that associate first protein with the nabelschnur. 1a. We have added the Michieletto NAR 2025 reference to line 79 when referring to the overall structure of the kDNA. 1b.We have added the following text to highlight the unusual nabelschnur structure and 2 essential proteins for kDNA segregation (line 90-94) with the appropriate references; Another distinctive feature is the final physical connection between daughter kDNA networks at the later stages of segregation called the nabelschnur (umbilical cord). This filamentous bridge contains maxicircles threads (Gluenz et al Mol Cell Biol 2011) with at least two essential proteins for kDNA segregation, a leucine aminopeptidase (LAP1) and NAB70 (Pena-Diaz et al PLos Path 2017; Cadena et al Curr Biol 2024). 1c. We have added two references (Billington et al Nature Microbio 2023; Pyrih et al Cell Reports 2023) to line 532 when referring to localization of proteins to multiple subcompartments around the kDNA network. 2. 56 - unicellular protist does not sound good, as by most definitions protist = unicellular We agree and have removed the redundant word “unicellular”; line 57. 3. 63 and 74 - I suggest the authors replace "novel trypanosome briology" (used twice), as the combination of words is somewhat awkward. We agree and replaced novel trypanosome with “eukaryotic”; line 63. Additionally, we replaced novel trypanosome biology with “divergent trypanosome features”; line 75. 4. 102 - a reference seems to be missing here Yes, we inadvertently omitted the following reference (Delzell et al Biochemistry 2022); line 107. 5. 496 and 497 - hours are missing We have not made any changes here because we are referring to the total number of doublings and not the doubling time; lines 499 and 500. 6. 564 - Sunter and colleagues would be correct. Thank you for suggesting this fix. However, when we carefully read through the Sunter MBP 2016 paper, pJ1173 was generated in this paper in which Jack Sunter was the sole author. Therefore, we will keep the original language of “original approach reported by Sunter”; line 569. 7. 587 - Bruhn 2010 - kind of forgotten here Again thank you for catching this incorrectly formatted reference. This has now been updated appropriately; line 592. Responses to Reviewer #2: Thank you for your very positive comments! Again thank for your time and very positive comments on our manuscript for publication in PLoS One. Attachments Attachment Submitted filename: Response to ReviewersPLoSOne2025.docx https://doi.org/10.1371/journal.pone.0321334.r003
4 Apr 2025 Decision Letter - Vyacheslav Yurchenko, Editor An independently tunable dual control system for RNAi complementation in Trypanosoma brucei PONE-D-25-11956R1 Dear Dr. Klingbeil, 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. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. 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, Vyacheslav Yurchenko, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): In my opinion, this is a very interesting and important work Reviewers' comments: https://doi.org/10.1371/journal.pone.0321334.r004

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