PONE-D-23-00650The RNA-binding KH-domain in the unique transcription factor of the malaria parasite is responsible for its transcriptional regulatory activityPLOS ONE

Dear Dr. Komaki-Yasuda,

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.

You'll see that referee #1 only raised minor issues, whereas referee #2 raised three points regarding Figures 1 & 3 and clarification of a section of the Methods. None of the issues raised seem too difficult to rectify, and to give you the chance to improve Figures 1 & 3 I have marked your submission down for major revision.

Please submit your revised manuscript by Mar 30 2023 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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PLOS ONE

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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: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: No

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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: No

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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

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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: The authors present a well done work on the identification of functional markers of a transcription factor that they had previously described (PREBP). This protein has 4 KH domains known to bind to either RNA or single stranded DNA. By working on plasmids expressing a luciferase activity regulated by PREBP, the authors show that the presence of these domains promotes the efficiency of expression and that they are therefore important in the regulation of transcription. Based on these results, they searched in the annotation of the genome of three Apicomplexa parasites for the existence of genes coding for proteins carrying KH motif. They identified about twenty of them which they classified into three groups according to their homologies, group 1 seeming the most promising in terms of transcription regulation potential. To reinforce this theoretical information, they did express the three proteins newly identified in the genome of P. falciparum, which they coupled to GFP in order to know their location in the infected red blood cell. One of these proteins co-localizes with the nucleus throughout the erythrocyte cycle. The authors conclude that it could be a new transcription factor.

The work presented by the authors is of good quality, well described and provides substantial data. The material and method part is well documented and allows a good understanding of how the data was obtained. The results are clearly presented and the discussion interesting even if the chapter lines 560-581 could be shortened.

I have two minor remarks

The first concerns the use of qPCR to study the association between PREBP and the promoter already identified. It would have been more appropriate to do RNAseq on the CHIPs. In addition to confirming the PREBP target, it could have revealed other targets on other parts of the genome and made it possible to identify a gene transcription cascade regulated by PREBP.

The second concerns the bioinformatics part of the manuscript. For example when we test on Plasmodb the "KH_1" search for P. falciparum (isolate 3D7), we find 5 genes as authors did but not exactely the same : the PF3d7_0302800 gene does not come out (because the denomination in interpro domain is "K Homology domain, type 1" and not "KH_1" as for the others) on the other hand we find the Pf3D7_1469300 gene which the authors do not talk about… is there an explanation for this?

Reviewer #2: Komaki-Yasuda and Kano profile Prx regulatory element-binding protein (PREBP) in P. falciparum blood stage parasites using various tools and approaches including parasite transgenic lines, confocal microscopy, and luciferase-based activity assays. The authors also perform a bioinformatic search for KH-domain proteins across various Apicomplexan parasites to identify new putative transcription factors. They then infer the roles of these domain-containing proteins as putative transcriptional regulators in Plasmodium spp. and beyond. They then use confocal imaging to profile one of these factors (PF3D7_0302800), showing its localization the nucleus P. falciparum blood stage parasites.

The study is related to- and builds on the authors’ previous work [17,18]. The methods chosen to profile PREBP are well thought out, however for PF3D7_0302800, a more thorough analysis could have been done. The researchers acknowledge that follow-up studies for this factor are necessary. The study has the potential to serve as a value resource to the malaria researcher community, but in the manuscript’s current format, the authors fall short of disseminating the findings in a manner that meet PLOS ONE's criteria for publication.

My major queries and concerns include the following:

Using confocal imaging, the authors follow the cellular localization of PREBP throughout the parasite's IDC. The results here support their previous findings showing the differential localization of PREBP using a complementary technique ([18] Figure 5). In Figure 1, the authors present representative images of the PREBP-GFP localization at different stages of the parasite's IDC. The current format of the data does not satisfy criterion #3 of PLOS ONE's publication requirements. To meet the replication standards, the authors should provide information as to how many parasites were assessed at each stage to allow them arrive at their conclusions. On Lines 270-278 the authors use terms such "throughout the cytoplasm" and "a particular strong region of green fluorescence...which coincided with the location of nuclear localization". These descriptions are rather subjective.The authors should perform co-localization analysis (for instance, using co-localization coefficients such as Pearson's correlation or Mander's split coefficients) to provide readers with quantitative support for your claims. Reporting both the number of parasites and the correlation assessment will add rigor to this analysis. In terms of controls, the authors generate a PfPREBP peptide antibody in [18] and use this antibody for ChIP analysis in the current study. I am curious as to why they do not use this antibody as a control in their confocal imaging analysis? This would provide visual support that addition of the GFP-tag to PREBP does not alter localization patterns. Presumably the co-localization coefficients would be the same when comparing the endogenous PREBP stained with anti-PREBP and a fluorescent secondary antibody to PREBP-GFP.

The authors use ChIP to assess the recruitment of PREBP on the cis-enhancer of the pf1-cys-prx gene. While this approach is powerful in assessing protein-DNA interactions, optimization is critical. The methods described pertinent to this analysis are not clear, making it very difficult for researchers to reproduce the protocol. For example, the authors report volumes in their methods section; however, these values are largely meaningless to the research community. It would be more informative to know what the quantity of material used for each of the steps in the workflow (i.e. ug, ng, mg, grams)? The authors should provide kit product numbers when applicable (i.e What is the Product number for the "The CHiP Assay Kit" (Thermo Fisher))? The authors fail to report any quality control steps for the assay. What were the positive and negative antibody controls used? A western blot showing that PREBP has been immunoprecipitated, along with the appropriate controls should be provided in the manuscript. Were positive and negative control loci used in RT-PCR? If so, the authors should provide the primer sequences. In Figure 2, the results related to the enrichment of target DNA using primers A and E are misleading. Presenting the data with error bars representing the standard deviation of 2 biological replicates is insufficient.The authors should overlay the raw data points. Likewise, the authors should perform a statistical test to compare relative enrichment of target DNA to primer set C. These modifications will satisfy #3 of PLOS ONE's publication criterion.

For the luciferase assay, the data represented in Figure 3 show: (1) the deletion of all 4 KH-domains negatively impacts pf1-cys-prx expression when the protein is truncated from the N-terminus. When all 4 KH-domains are deleted from a C-terminus, a decrease in pf1-cys-prx is not observed—unless the -cis enhancer in pf1-cys-prx is deleted. On lines 348-351, the authors indicated that "As a result, PREBP activity was not significantly reduced until it lacked 3 of 4 KH domains. Then, the activity was significantly decreased when it lacked all the putative KH domains (p < 0.01) (Fig 3C, right panel)"; however, this is not consistent with Figure 3. In the right panel they show that the activity is not significantly decreased unless the pf1-cys-prx -cis enhancer is deleted (i.e "I"). Please clarify these findings. Additionally, the authors should plot the individual data points on the graph (not just the bar plot +/- SD).

In the bioinformatic search for KH-domain containing proteins that may encode for transcription factors, the authors indicate that they removed several proteins that are orthologs of well-known proteins (Lines 401-402). Both PF3D7_0302800 (RNA-binding protein, putative) and PF3D7_1415300(RNA-binding protein Nova-1 putative) are annotated as RNA-binding proteins in PlasmoDB (release 61). The authors should clarify why these genes were not filtered out and are included in the search list. Additionally, the authors should provide the supplementary files with the genes that were included and excluded in the bioinformatic analysis. These lists will allow the research community to reproduce the workflow should it be required to re-run their search pipeline.

For the confocal microscopy assay assessing the localization of PF3D7_0302800, the authors should include the number of parasites assessed, and perform a co-localization analysis. Unlike PREBP [18], there is no supporting evidence for protein localization other than the confocal imaging data presented in the study. Since the PF3D7_0302800 is GFP-tagged, a western blotting of cytoplasmic and nuclear fractions of the parasite lysate would serve as complimentary method to support the authors' claim that the protein is localized to the nucleus over the course of its IDC.

Minor comments and questions:

Please adhere to PLOS' minimum data set definition for all analyses performed in this study. Along these lines, please indicate whether the plasmids generated in the study will be available, and if so, which repository the research community can find them. Same goes for the PREBP antibody and -GFP parasite lines, are these available to the research community? If so, how can they be obtained?

The authors indicate that PREBP can bind RNA (Line 19). When performing the ChIP analysis was an RNase used to digest RNA? Is it possible that the transcription factor could be binding nascent RNA or pre-mRNA in the nucleus? A bioanalyzer electropherogram for both DNA and for RNA would provide support for your ChIP approach.

Line 57: Please add the gene number (i.e PF3D7_XXXXXXX) when first mentioning pf1-cys-prx. Searching for pf1-cys-prx on PlasmoDB brings up four different genes (https://plasmodb.org/plasmo/app/search?q=pf1-cys-prx%20&documentType=gene&organisms=Plasmodium%20falciparum%203D7). Which one is correct?

Line 88: "thorough" should be "throughout".

Line 93: Instead of "some Apicomplexan parasites" it is better to state: "various Apicomplexan parasites".

Lines 104-171: The methods section describing the plasmid construction can be reduced significantly. Simply generate a table for the primers used and, in the text, direct the reader to this supplementary table in the event they would like to find these sequences.

Lines 383-385: The authors indicate that the left panel shows activity of the PREBP deletion series from the N terminus and the right panel shows that of the deletion series from the C terminus. However, "E:p1-10R-PREBP deltaC3" is a C-terminus deletion and it is being represented in the left panel instead of the right panel.

Please correct Figure Legend 3 to be consistent with what is described on Lines 383-385.

In the methods describing the bioinformatic analysis, please provide the database release # and date for each organism assessed. For example, Line 248 should read (PlasmoDB, Release #, day month year).

Lines 248-249: Were all Plasmodium spp. Toxoplasma spp. and Cryptosporidium spp. used in the screen? If not, please add strain information for those that were used

Line 258: which strains of the parasite species were used?

Line 390: I recommend using the word "putative" instead of "hypothetical" when describing the potential role of KH-domain containing proteins as transcription factors.

Line 439: What do the authors mean by "almost the same". It would be better to indicate the % overlap to provide the readers with a better indication of the similarity/dissimilarity between the approaches. A Venn Diagram would be useful here.

Line 451: Please provide clarification as to why ortholog group 1 was prioritized.

Lines 458 and 463: Please modify. As per their minimum data set definition, PLOS does not permit references to “data not shown.” PF3D7_1415300-GFP confocal data should be included as a supplementary file.

Line 508: "melts" is not the correct word here.

Lines 517: Please change pfGNC5 to italicized P. falciparum (Pf) GNC5; then, on lines 521 and 529 use PfGNC5.

Lines 532-525: Is this data generated in the current study? If so, please provide the alignment data and scores in a supplementary file.

Line 535-536: The statement here is incorrect. In PlasmoDB, PF3D7_0302800 is annotated as "RNA-binding protein, putative".

Line 538 should read "in the parasite nucleus throughout the ring, trophozoite, and schizont stages."

Line 562: State "were reported previously [40]" instead of "were reported [40] in 2015".

Line 576: What is a "typical" transcription factor?

Line 582-583: Please rephrase. There is extensive evidence showing AP2s as transcriptional regulators in Plasmodium species.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-23-00650R1The RNA-binding KH-domain in the unique transcription factor of the malaria parasite is responsible for its transcriptional regulatory activityPLOS ONE

Dear Dr. Komaki-Yasuda,

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. I have marked you revised manuscript down for minor revision to allow you to correct the small number of grammatical errors, as they is no proof reading stage with PONE. These are listed below:

N 16: “The PREBP had been shown” should be “PREBP has been shown..”

LN 18: “The PREBP…” should be “PREBP…”

LN 27-28: “Bioinformatic analysis suggested…” should be “Bioinformatic analysis revealed…”

LN 75: “…PREBP was expected…” should be “…PREBP was predicted from…”

LN 84: “The finding of PREBP suggested…” should be “ These findings suggest…”

LN 95: “unknown” should be “putative”

LN 371: “(see Fig 4A)” should be “(Fig 4A)”

LN 409-410: “…organisms, unknown KH-domain containing proteins might act as unknown transcription factors” should be “…organisms, KH-domain containing proteins might act as transcription factors.”

LN 611-612: “…in the Apicomplexan…” should be “…in Apicomplexan…”Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

For Lab, Study and Registered Report Protocols: These article types are not expected to include results but may include pilot data. 

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Please submit your revised manuscript by Jan 20 2024 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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• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Gordon Langsley

Academic Editor

PLOS ONE

Journal Requirements:

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. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. 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.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. 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 #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

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4. 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 #2: Yes

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5. 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 #2: Yes

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6. 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 #2: The resubmission of the manuscript entitled “The RNA-binding KH-domain in the unique transcription factor of the malaria parasite is responsible for its transcriptional regulatory activity" was submitted by Komaki-Yasuda and Kano in response to two reviewer comments and recommendations. This manuscript is focused on understanding the role of KH-domains in transcriptional regulation of Plasmodium falciparum and potentially other Apicomplexans.

The amended manuscript has many strengths. The authors both re-analyze, and generate additional data to better support their claims. The resubmission is highly responsive to the reviewer comments. Based upon the evaluation of scientific and technical merit, this manuscript is acceptable for publication in its current format.

In terms of the general readability and accessibility of the manuscript: prior to publication, I highly recommend that the authors (perhaps with the guidance of editorial team at PLOS one) modify the title of their manuscript to make it more fitting to the body of work presented. For example, the authors argue that the KH-domain is important for gene regulation at the DNA level (i.e. as a transcription factor). The usage of “RNA-binding” in the title is somewhat confusing and takes away from the authors’ argument. Likewise, the use of “the unique transcription factor” in the title is very vague. Consider using the specific name of the transcription factor(s) that you are referring (i.e. PREBP etc). A more specific title will facilitates the retrieval of the paper from bibliographic databases.

Some grammatical errors that should be fixed prior to publication:

LN 16: “The PREBP had been shown” should be “PREBP has been shown..”

LN 18: “The PREBP…” should be “PREBP…”

LN 27-28: “Bioinformatic analysis suggested…” should be “Bioinformatic analysis revealed…”

LN 75: “…PREBP was expected…” should be “…PREBP was predicted from…”

LN 84: “The finding of PREBP suggested…” should be “ These findings suggest…”

LN 95: “unknown” should be “putative”

LN 371: “(see Fig 4A)” should be “(Fig 4A)”

LN 409-410: “…organisms, unknown KH-domain containing proteins might act as unknown transcription factors” should be “…organisms, KH-domain containing proteins might act as transcription factors.”

LN 611-612: “…in the Apicomplexan…” should be “…in Apicomplexan…”

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7. 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 #2: No

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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.

The RNA-binding KH-domain in the unique transcription factor of the malaria parasite is responsible for its transcriptional regulatory activity

PONE-D-23-00650R2

Dear Dr. Kanako Komaki-Yasuda,

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.

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Kind regards,

Gordon Langsley

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: