PONE-D-22-12422Transcriptional changes in Plasmodium falciparum upon conditional knock down of mitochondrial ribosomal proteins RSM22 and L23PLOS ONE

Dear Dr. Hangjun Ke,

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Reviewers' comments:

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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: No

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: No

Reviewer #2: Yes

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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: In this manuscript, Dass et al. investigate the function of two putative components of the Plasmodium falciparum mitoribosome. Using conditional knock-downs, the authors convincingly show that both RSM23 and L23 are mitochondrially localized and are important fo parasite growth. They then go on to analyze RNA accumulation after 2 days of KD and after 4 and 6 days of KD (the latter only for PfMRPL23 KD). The study is rather descriptive and does not deliver insights into the molecular function of the two putative mitoribosomal proteins.

Major concerns

1. The authors make a number of conclusions based on mitochondrial RNA accumulation. This is problematic since RNA Seq is carried out using polyA-selected RNA. However, it is still an open question in the field, to what extend mitochondrial mRNA and rRNA are polyadenylated and what function polyadenylation has here. This may also be the reason for the failure to detect several known mitochondrial rRNA fragments. Thus, the bias introduced into the RNA seq data of mitochondrial transcripts is potentially huge. This is true if different time points are compared, but is even more relevant if comparisons between different genes are made. All comparisons between the accumulation of rRNA fragments from the small versus the large subunit are difficult. Therefore, it is essential to confirm conclusions on differential expression with orthogonal methods (qRT-PCR; RNA gel blot hybdirization).

2. Depleting mitoribosomes is expected to lead to massive problems with mitochondrial respiration, which in turn affects the cell on all levels. This obviously includes the accumulation of RNAs in all three genetic compartments. Thus, the RNA pattern observed by RNA seq is the secondary product of a number of problems these KD cells face and is not helpful to understand the primary function of the two proteins investigated. There are some interesting differences in RNA accumulation between the two knock-outs, which might be explained by the different severity of the mutants on mitochondrial translation, but in the end, no insights into the translational capacity of the mutants is shown. A targeted ribosome footprinting analysis or polysome analysis would be needed to understand the impact of the proteins on translation.

3. The two proteins investigated are presumed to be mitoribosome components based on sequence comparisons, but this has not been experimentally shown. In this light, the differential effects on the transcriptome might indicate different functions.

4. The idea that PfRSM22 is an assembly factor of the mitoribosome is not substantiated by any data. There is no evidence shown for “mitoribosome synthesis inhibition” (lines 146-148).

Minor concern

The authors do not provide access to the raw data which precludes any analysis on the reviewers’ site. Usually, SRA accessions (or the like) are made available for the reviewing process.

Reviewer #2: The authors present an article on the study of Plasmodium falciparum, of which two nuclear genes coding for mitochondrial proteins have been KD. Their choices fell on the PfRSM22 and PfMRPL23 genes without any reason being known (continuation of their previous studies ?). The authors used a conditional KD system to explore the transcriptomic bulk response of erythrocyte stages at two stages : early (day 2 post KD) and late (More than 6 days post KD) phases. By comparing the differential expression of other proteins, the authors propose a series of new potentially MRP-like proteins.

The manuscript is well written and very clear. The technical approach is convincing and the results are well presented. They attest to the localization of these two proteins in the mitochondria and also show that the KD of these genes leads to the death of the parasites at 8 and 6 days post KD for PfRSM22 and PfMRLP23 respectively.

However, I have two remarks :

The first concerns the methodology adopted in the analysis of the sequencing results. In their materials and methods the authors state that transcriptomic data are analyzed in duplicate using a set of tools including clusterProfiler R Package. The results presented are not exactly comparable with those found using PlasmoDB algorithm from Gene Ontology Enrichment pages using their avaliable data. It would seem relevant to me to discuss the convergences found between these two methodologies and how they could reinforce the robustness of the data presented.

The second remark concerns the relevance of analyzing the transcriptome at the late phase where it seems at least that a majority of the parasites are already dead (figure 1 E) and 1 F)), the data collected are therefore in my opinion difficult if not impossible to analyze and certainly subject to controversy.

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Reviewer #1: No

Reviewer #2: No

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Transcriptional changes in Plasmodium falciparum upon conditional knock down of mitochondrial ribosomal proteins RSM22 and L23

PONE-D-22-12422R1

Dear Dr. Hangjun Ke,,

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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Reviewers' comments:

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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: (No Response)

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Reviewer #2: No

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