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

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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

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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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.

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Reviewer #1: Yes: Sebastian Baumgarten

Reviewer #2: No

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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.

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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: