Peer Review History

Original SubmissionMarch 14, 2022
Decision Letter - Friedrich Frischknecht, Editor

PONE-D-22-07594The in vivo RNA structurome of the malaria parasite Plasmodium falciparum, a protozoan with an A/T-rich transcriptomePLOS ONE

Dear Dr. Merrick,

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 Jun 09 2022 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.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
  • 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 you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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,

Friedrich Frischknecht

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

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 

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. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Thank you for stating the following in the Funding Section of your manuscript: 

"UK Medical Research Council (grants MR/K000535/1 and MR/L008823/1) to CJM, Royal Society Kan Tong Po fellowship, Shenzhen Basic Research Project (JCYJ20180507181642811), Research Grants Council of the Hong Kong SAR, China Projects CityU 11101519, CityU 11100218, N_CityU110/17, CityU 21302317, Croucher Foundation Project No. 9509003, 9500030, State Key Laboratory of Marine Pollution Director Discretionary Fund, City University of Hong Kong projects 6000711, 7005503, 9680261, 9667222 to CKK. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Funding 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. Currently, your Funding Statement reads as follows: 

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. 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. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

  

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

6. 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.

Additional Editor Comments:

Dear Catherine,

thanks for sending your nice paper to Plos One. It has now been seen by two experts in the field, who, as you will see, liked the work but have some suggestions to improve the paper. I would encourage you to revise the manuscript according to their comments.

Sincerely

Freddy

[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: No

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: In this manuscript Dumetz et al. probe RNA secondary structures during the asexual replication of Plasmodium falciparum within human red blood cells. They use the well-established method Strucutre-seq that uses the chemical NAI to probe unpaired nucleotides and subsequent RT fall-off to interrogate RNA secondary structures on a transcriptome wide scale. Comparisons of their in vivo derived structurs to known, well characterized structures (e.g. tRNA) show the robustness of their method and I particularly like the ‘divergence’ score to categorize different RNA classes. Finally, the authors compare their RNA structure to translational efficiencies and RNA decay rates and find that some structures within RNA transcripts can influence those. Overall, the study is well designed, the methodology is clear, their findings well-presented and the study will overall lay the foundation to study dynamics of the RNA structurome across the P. falciparum life-cycle in the future.

Please find my comments to specific points below:

line 344ff: The structure-seq relies on RT fall-off. This also happens very frequently at sites of RNA modifications, especially in hypermodified molecules such as tRNA. For the tRNA where the authors do not find a ‘canonical’ structure, the RT fall-off because of modifications might make the probing of the actual structure impossible. This information should be added to the Discussion.

What about different structures in different regions of mRNA transcripts, i.e. 5’UTR-CDS-3’UTR. UTRs in P. falciparum are substantially more A/U rich than the CDS. Please provide a paragraph/table/figure showing where different structures are located and if there is a potential bias.

line 361: The authors do mention the previously published P. falciparum structuromes. How similar are the structures especially of mRNAs identified in the different studies?

line 325: It is not fully clear if all presented structures are derived from the NAI samples. If so, why did the authors chose to present only those in the main text although all samples where sequenced and present in the supplement? How different are the structures of the NAI and DMS samples?

line 502: Different transcript regions have substantially different functions within a mRNA molecule. 5’UTRs are essential for translation and can even initiate protein synthesis cap-idenpendent with secondary structures, i.e. IRES. On the otherhand 3’UTR are crucial for RNA decay. So how does the comparison with translational efficiencies and mRNA decay change when focusing only on structures on certain regions, i.e. 5’UTR and translation and 3’UTR and decay?

Are there certain conserved structures in particular regions present in multiple different transcripts?

Please upload all raw and processed sequencing data to the SRA and GEO databases.

Minor

Please ensure to accurately state “A/U” when talking about the transcriptome (e.g. in the title)

Reviewer #2: Merrick and co-workers used a combination of chemical modification and RNA seq to inform on the secondary structure of mRNAs of the malaria-causing parasite Plasmodium falciparum. The authors were able to provide data on more than half of the parasite’s transcriptome and compare a AT-rich to a AT-balanced genome. The current study complements and expands insights from two recent studies on the same subject.

The presented data appear solid and the conclusions are scientifically sound. But some minor changes (for example Fig. 3) would help the reader to better access the significance of this work. Below are some specific minor comments, which should help to further strengthen this manuscript.

Line 214: Technically the parasite does not live “entirely” inside a host cell, many stages are extracellularly. Please revise.

Sentence starting in line 250 may be clearer if stating that nucleotides were unpaired before labelling reaction.

The labels of Suppl. Fig. 3 are very small.

For Fig. 2C, would it be possible to have the experimentally-determined structures superimposed on the “canonical” folding? This would strengthen the point. While Fig. 2C and Suppl. Fig. 4 clearly show the strength of the approach in detecting the expected canonical structures of some tRNAs, showing some of the non-canonical structures in the main figure would be appreciated.

Revise formatting of the reference in line 358.

End of the sentence in line 403: . instead of ,

Fig. 3, please provide a scale for the different shades of red/fold enrichment. Which P-value cut-off was used to identify significantly enriched GO terms? Also, the figure would be easier to understand, if panels A, B, and C had labels indicating the divergence tiers. It is unclear how the numbers of enriched GO terms in the text correspond to the number of GO terms shown in the figure, please explain.

The Fig. 4A would benefit from a legend describing what the green/red shading and the boxes indicate. Please also explain here what the dot/bracket sequences mean.

Is the term “significant” (line 435) used to indicate statistical significance? If so, please provide information on the tests. If not, please rephrase.

Fig. 5 and 6 caption: It is not the “life cycle” but the development inside erythrocytes.

While the other two studies investigating the structure of P. falciparum mRNAs are mentioned, this manuscript would benefit from an extended discussion of the results from the other studies.

********** 

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: Sebastian Baumgarten

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.

Revision 1

Dear Prof Frischknecht

Thank you for the opportunity to revise this manuscript. Below is a list of revisions (red text) in response to the reviewers’ comments: we hope that the paper will now prove acceptable for publication.

Sincerely,

Catherine Merrick

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

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

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

All checked

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

This has been added in the online submission system

3. Thank you for stating the following in the Funding Section of your manuscript:

"UK Medical Research Council (grants MR/K000535/1 and MR/L008823/1) to CJM, Royal Society Kan Tong Po fellowship, Shenzhen Basic Research Project (JCYJ20180507181642811), Research Grants Council of the Hong Kong SAR, China Projects CityU 11101519, CityU 11100218, N_CityU110/17, CityU 21302317, Croucher Foundation Project No. 9509003, 9500030, State Key Laboratory of Marine Pollution Director Discretionary Fund, City University of Hong Kong projects 6000711, 7005503, 9680261, 9667222 to CKK. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Funding 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. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

The cover letter now contains the correct funding statement, and it has been removed from the actual manuscript.

4. 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. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

There are only 2 gels shown in this manuscript: both were already full-length (uncropped at top and bottom) and the one in supp Fig.1 was already full-width; the one in main Fig.1B has now been additionally provided as a full-width image in supp Fig .1. This is also noted in the cover letter.

5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

The ’data not shown’ have been added as a supplementary table (6) in the revised manuscript.

6. 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.

Done – reference formatting checked & one preprint updated to the published version.

Additional Editor Comments:

Dear Catherine,

thanks for sending your nice paper to Plos One. It has now been seen by two experts in the field, who, as you will see, liked the work but have some suggestions to improve the paper. I would encourage you to revise the manuscript according to their comments.

Sincerely

Freddy

Reviewer #1

In this manuscript Dumetz et al. probe RNA secondary structures during the asexual replication of Plasmodium falciparum within human red blood cells. They use the well-established method Strucutre-seq that uses the chemical NAI to probe unpaired nucleotides and subsequent RT fall-off to interrogate RNA secondary structures on a transcriptome wide scale. Comparisons of their in vivo derived structurs to known, well characterized structures (e.g. tRNA) show the robustness of their method and I particularly like the ‘divergence’ score to categorize different RNA classes. Finally, the authors compare their RNA structure to translational efficiencies and RNA decay rates and find that some structures within RNA transcripts can influence those. Overall, the study is well designed, the methodology is clear, their findings well-presented and the study will overall lay the foundation to study dynamics of the RNA structurome across the P. falciparum life-cycle in the future.

Please find my comments to specific points below:

line 344ff: The structure-seq relies on RT fall-off. This also happens very frequently at sites of RNA modifications, especially in hypermodified molecules such as tRNA. For the tRNA where the authors do not find a ‘canonical’ structure, the RT fall-off because of modifications might make the probing of the actual structure impossible. This information should be added to the Discussion.

This valid point has been added (at line ~400)

What about different structures in different regions of mRNA transcripts, i.e. 5’UTR-CDS-3’UTR. UTRs in P. falciparum are substantially more A/U rich than the CDS. Please provide a paragraph/table/figure showing where different structures are located and if there is a potential bias.

This has now been discussed briefly at line ~370: ‘A second limitation of short-read sequencing was that it resulted in very limited coverage of untranslated regions, which in P. falciparum tend to be extremely A/U-biased (often over 90%), and also highly repetitive with many low-complexity regions. This precluded any comprehensive assessment of RNA structure in the UTRs versus CDSs of mRNAs.’

line 361: The authors do mention the previously published P. falciparum structuromes. How similar are the structures especially of mRNAs identified in the different studies?

Unfortunately, the previously published studies do not provide their reactivity data gene-by-gene - e.g. in dot-bracket format, which would facilitate the comparison of their paired/unpaired nucleotides with ours. (This is a young, developing field and there are not yet any community standards for publishing this type of data). Without this, it would be necessary to take their raw sequencing-read data and re-fold the entire transcriptome. Besides this being a very large task, the resultant comparison could anyway be somewhat flawed if they did not use exactly the same algorithms and parameters as us. Systematic comparisons between structuromes determined by different groups via different methods will likely be an important endeavour as this field develops; however, this is beyond the scope of our current paper.

line 325: It is not fully clear if all presented structures are derived from the NAI samples. If so, why did the authors chose to present only those in the main text although all samples where sequenced and present in the supplement? How different are the structures of the NAI and DMS samples?

This is already addressed (now at line 321): ‘Structural information was more comprehensive (and agreement between replicates was also better), for the NAI-probed datasets because all four unpaired nucleotides were detected. Subsequent analysis was therefore focused on the NAI dataset as the primary in vivo structurome’. We initially tried 2 different probing methods (DMS as well as NAI) because it was unclear whether both chemicals would efficiently access the Plasmodium RNA: this proved to be the case, and the information from the NAI datasets was therefore more complete. Because there is no well-validated way (to our knowledge) to blend data from DMS and NAI together, we chose to focus on the NAI datasets alone.

line 502: Different transcript regions have substantially different functions within a mRNA molecule. 5’UTRs are essential for translation and can even initiate protein synthesis cap-idenpendent with secondary structures, i.e. IRES. On the other hand 3’UTR are crucial for RNA decay. So how does the comparison with translational efficiencies and mRNA decay change when focusing only on structures on certain regions, i.e. 5’UTR and translation and 3’UTR and decay?

As per the point above: our coverage of UTRs, via basic Illumina sequencing, was too patchy to be confident in comparing this with CDSs. In future, some recently published improvements in RNAseq methodologies specifically to preserve A/U-rich Plasmodium sequences (e.g. ‘DAFT-seq’, Chappell et al 2020), could perhaps be used to ameliorate this problem.

Are there certain conserved structures in particular regions present in multiple different transcripts?

We have struggled with this question, which is rather general: i.e. how should we meaningfully define ‘particular regions’ of multiple transcripts? The simplest way to define regions is 5’UTR/CDS/3’UTR – but as noted above, our coverage of UTRs is much lower than of CDSs, precluding a meaningful analysis. We then considered seeking common RNA structures in the codes for common protein domains, but there are many hundreds of protein domains, versus only 4 broad categories of RNA structure – hairpin, stem, bulge and multi-stem-loop, so we did not find this to be meaningful either.

Please upload all raw and processed sequencing data to the SRA and GEO databases.

This is done, apologies for its omission from this manuscript (in a previous submission, it was provided in the metadata rather than the manuscript itself, but it has now been added to Materials & Methods after the section on sequencing). ‘Raw data (FASTQ) from sequencing for each sample is available from the European Nucleotide Archive (ENA) under accession PRJEB44384’.

Please ensure to accurately state “A/U” when talking about the transcriptome (e.g. in the title)

This has been checked/corrected throughout

Reviewer #2

Merrick and co-workers used a combination of chemical modification and RNA seq to inform on the secondary structure of mRNAs of the malaria-causing parasite Plasmodium falciparum. The authors were able to provide data on more than half of the parasite’s transcriptome and compare a AT-rich to a AT-balanced genome. The current study complements and expands insights from two recent studies on the same subject.

The presented data appear solid and the conclusions are scientifically sound. But some minor changes (for example Fig. 3) would help the reader to better access the significance of this work. Below are some specific minor comments, which should help to further strengthen this manuscript.

Line 214: Technically the parasite does not live “entirely” inside a host cell, many stages are extracellularly. Please revise.

This has been corrected

Sentence starting in line 250 may be clearer if stating that nucleotides were unpaired before labelling reaction.

This has been corrected

The labels of Suppl. Fig. 3 are very small.

This has been corrected

For Fig. 2C, would it be possible to have the experimentally-determined structures superimposed on the “canonical” folding? This would strengthen the point. While Fig. 2C and Suppl. Fig. 4 clearly show the strength of the approach in detecting the expected canonical structures of some tRNAs, showing some of the non-canonical structures in the main figure would be appreciated.

In figure 2C, it is the experimentally-determined structures that are shown. In the original figure, we showed only experimentally-determined structures that were canonical (non-canonical versions were in the supp figure). As requested, we have now shown a non-canonical example as well, for direct side-by-side comparison within the main figure.

Revise formatting of the reference in line 358.

All references are checked/updated in the revision.

End of the sentence in line 403: . instead of ,

This has been corrected.

Fig. 3, please provide a scale for the different shades of red/fold enrichment. Which P-value cut-off was used to identify significantly enriched GO terms? Also, the figure would be easier to understand, if panels A, B, and C had labels indicating the divergence tiers. It is unclear how the numbers of enriched GO terms in the text correspond to the number of GO terms shown in the figure, please explain.

We appreciate the reviewer’s pointing out that Figure 3 (a representation of GO terms analysis) was not fully quantitative. We have therefore re-done the analysis, changing the representation to a scatter-plot format, which still represents the relatedness between similar GO terms, but is also quantitative both in terms of enrichment (see rainbow scale) and number of GO terms (see size of circles). We have also labelled the 3 plots with Tiers 1-3.

The Fig. 4A would benefit from a legend describing what the green/red shading and the boxes indicate. Please also explain here what the dot/bracket sequences mean.

This information is all in the legend, but we appreciate the reviewer’s pointing out that the figure is complex to look at without visual legend as well. A visual legend has been added to a revised figure 4.

Is the term “significant” (line 435) used to indicate statistical significance? If so, please provide information on the tests. If not, please rephrase.

This has been corrected to ‘major’

Fig. 5 and 6 caption: It is not the “life cycle” but the development inside erythrocytes.

This has been corrected

While the other two studies investigating the structure of P. falciparum mRNAs are mentioned, this manuscript would benefit from an extended discussion of the results from the other studies.

We do already have within our discussion several paragraphs of comparative insight concerning the two other existing studies:

a) lines 585-594 comparing the overall coverage of our structurome vz that of Qi et al.,

b) lines 636-647 comparing our findings about RNA structure vz translational-efficiency,

c) lines 685-690 concerning the effect of RNA binding proteins, as suggested by Qi et al.

It is difficult to provide much more insightful discussion, particularly of Alvarez et al., since these authors used an entirely different methodology.

Attachments
Attachment
Submitted filename: PlosOne rebuttal.docx
Decision Letter - Friedrich Frischknecht, Editor

The in vivo RNA structurome of the malaria parasite Plasmodium falciparum, a protozoan with an A/U-rich transcriptome

PONE-D-22-07594R1

Dear Dr. Merrick,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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,

Friedrich Frischknecht

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

thanks for providing a constructive revision, in my view the paper can now be accepted. I wonder only if in the table 2 all numbers could be limited to two digits after the . ?

Reviewers' comments:

Formally Accepted
Acceptance Letter - Friedrich Frischknecht, Editor

PONE-D-22-07594R1

The in vivo RNA structurome of the malaria parasite Plasmodium falciparum, a protozoan with an A/U-rich transcriptome 

Dear Dr. Merrick:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Dr. Friedrich Frischknecht

Academic Editor

PLOS ONE

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .