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Temporal resolution of NAIL-MS of tRNA, rRNA and Poly-A RNA is overcome by actinomycin D
Round 1
Manuscript submitted on 06 Dec 2022![](https://www.rsc-cdn.org/pubs-core/2022.0.153/content/NewImages/icon-letter.png)
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12-Jan-2023
Dear Professor Kaiser,
dear Stefanie:
Manuscript ID: CB-ART-12-2022-000243
TITLE: Temporal limitation of NAIL-MS of tRNA, rRNA and Poly-A RNA is overcome by actinomycin D
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Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg
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Reviewer 1
Hagelskamp and co-workers studied the temporal limitations of a mass spectrometry-based method to used metabolic labeling to profile RNA modifications (NAIL-MS). The method is performed by treating cells with isotopically labeled metabolites to monitor the timing of RNA synthesis and decoration with chemical modifications (ref 17). In their prior report, they found RNA species in which the core structure (ribose and bases) were not isotopically labeled while the methyl groups added were labeled. They termed these species hybrids and assumed they were RNA transcripts synthesized prior to media exchange with the isotopically labeled compounds and the maturation process completed after media exchange to introduce labels for the methyl groups added during maturation. They applied the compound actinomycin D to suppress transcription to monitor the fate of these hybrid species for support or refute of their hypothesis regarding the hybrid species. This paper appears to be an addendum to their prior work, and as such appears to be written that way in hast. The studies and conclusions are good, and the work should be published, but the document needs a significant number of details added, clarifications, and general proof reading to be a stand-alone document.
The data are not publicly available. Proteomics data based on RSC’s author guideline should be deposited at a minimum.
On page 2 second paragraph a description of the MS method is outlined to describe the signals and which RNA type they are assigned. They describe how the +3 signal from (D3)-L-methionine differentiates methyl group marks before and after media exchange; however, in the next sentence there is no description of what was added to give the +8/+10 signal differences for pyrimidine and purine RNA nucleotides, respectively, synthesized before or after media exchange. Readers without knowledge of this field need to read ref 17 in detail to understand this.
The term transcription rate and modification rate are confusing especially with the unit of percentage. In chemistry, rate is used to describe change over time with time as a unit. Modification rate is defined in the caption to Fig. S1 to be “modification abundance of new RNAs compared to steady stat modification abundance.” How were they compared? I did not find the definition for transcription rate.
In Fig 3D, what are the green stars?
In Fig 1B, C, and D, why are the y-axes broken? There are no data above the breaks.
In Fig 1, why were the tRNA and rRNA analyzed at 6 h while the mRNA were analyzed at 2, 4, and 8 h?
On page 2, third paragraph the % hybrid in mRNA at 2, 4, and 8 h were 8, 35, and 98%, but in the next sentence it is stated that 78% post-methylation in poly-A RNA after 8 h was observed. Where did the 78% come from? Moreover, in the next sentence the authors describe this as high as a result of the short half-life of mRNA. For readers unfamiliar with RNA half-lives in HEK293 cells, adding the values for clarification would be beneficial. There are other instances where these values could be added.
Examples of missing references. Page 2 third paragraph the sentence regarding proven co-transcriptionl and nuclear methylation of rRNA. Who proved this? On page 3 paragraph two there is description of the addition of m6A and m7G and the unknown stage of addition during maturation. Who’s work are you referring?
Typos
The final sentence of the first paragraph on page 2 needs rewritten. Many other little typos throughout that can be fixed during proofreading.
Reviewer 2
The manuscript by Hagelskamp et al. describes a new set of experiments using actinomycin D to suppress transcription for isotope labelling of RNAs. The authors indicate that this addition of actinomycin D may be an important control for future biological experiments.
I have a number of queries for the authors to address:
1) Overall, I believe the manuscript is very difficult to follow (this comes up in many of my other points). It took quite a while for me to understand what the authors were suggested as the original hypothesis and the new hypothesis. I am still not sure I understand what the authors mean by ‘post methylated’. For this point, I believe the authors need to rewrite the introduction and include a new schematic in the introduction.
2) In the introduction the authors talk about their development of ‘NAIL-MS’, but do not refer to any of the other substantive literature on adding labelled nucleosides, SAM, etc for labelling experiments of nucleic acids.
3) It is not clear what labelled species are being added at any time. At one point it is mentioned that deuterated methionine is added. But in Fig 1A it also looks like labelled nucleosides and nucleobases are added. Please give a description of this in the results section.
4) On page 2 “Thus, 13% abundance of hybrid species might not be connected to late maturation and postmethylation as previously hypothesized. Our doubts are further supported by the abundance of 8%, 35% and 93% hybrid m6A in Poly-A RNA after, 2h, 4 h and 8 h respectively.” It is not obvious where any of this data or numbers are coming from. Please cite and explicitly refer.
5) On page 2 “Thus, it is unlikely to find 78% post-methylation in Poly-A RNA after 8 hours, especially given the on average short halflife of mRNA.” If this is from Fig 1E, then I don’t see where 78% is from?
6) In the ‘AcmD does not impact abundance of RNA writers’ section (page 5), two contradictory points are made:
1st: “We are confident that the loss of hybrid modifications in rRNA and Poly- A RNA are not connected to a sudden loss of RNA writers under AcmD,”
2st: “Overall, the inhibition of mRNA transcription through AcmD leads to a general, significant decrease in protein abundance. Many RNA writers are found in the average area of reduced abundance.”
The authors only say three were not significantly changed, are these the only ones that can affect the methylation they are studying?
7) On page 6 the authors state “Interestingly, AcmD is a substantial stress to the cells which might result in an adaptation of the translation apparatus towards higher efficiency”. Could this mean that the effect the authors see if stress related and not transcriptionally related? Could a different stress-inducing non-transcriptional repressed have the same effect?
Minor:
snoRNAs in introduction is not given a full abbreviation
Half life of the different RNAs is mentioned but no details are given. Please give literature numbers to compare each species
Fig 1 B-E, why 6 hour time point used for B-D, but no 6 hour time point for E?
Fig S4, I cannot see the morphology in the pictures given, please use higher quality images
On page 3 “The abundance of hybrid m6A in Poly-A RNA is roughly twice as high as the remaining transcription rate at the 8 h time point (Fig. 3C).” twice compared to what?
On page 6 “As a side note, the Poly-A RNA studied here is mainly composed of mature mRNA used for active transcription.” Do the authors mean translation?
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Dear Claudia,
thank you for the positive assessment of our work. Both reviewers have made excellent suggestions which we have incorporated throughout the manuscript. The manuscript has greatly improved and for better readability we have rephrased various sections of the manuscript. We believe it is now better accessible to the general audience.
We look forward to the results of the 2nd evaluation.
Best
Stefanie
This text has been copied from the Microsoft Word response to reviewers and does not include any figures, images or special characters:
We thank the reviewers for their expert opinion and on point statements which have improved our work. A detailed response, indicated by *, is found below.
Referee: 1
Comments to the Author
Hagelskamp and co-workers studied the temporal limitations of a mass spectrometry-based method to used metabolic labeling to profile RNA modifications (NAIL-MS). The method is performed by treating cells with isotopically labeled metabolites to monitor the timing of RNA synthesis and decoration with chemical modifications (ref 17). In their prior report, they found RNA species in which the core structure (ribose and bases) were not isotopically labeled while the methyl groups added were labeled. They termed these species hybrids and assumed they were RNA transcripts synthesized prior to media exchange with the isotopically labeled compounds and the maturation process completed after media exchange to introduce labels for the methyl groups added during maturation. They applied the compound actinomycin D to suppress transcription to monitor the fate of these hybrid species for support or refute of their hypothesis regarding the hybrid species. This paper appears to be an addendum to their prior work, and as such appears to be written that way in hast. The studies and conclusions are good, and the work should be published, but the document needs a significant number of details added, clarifications, and general proof reading to be a stand-alone document.
* We thank the reviewer for the positive assessment of the manuscript and the critical but yet correct statements made. We have followed all suggestions as indicated below.
The data are not publicly available. Proteomics data based on RSC’s author guideline should be deposited at a minimum.
* We apologize for not uploading the data earlier. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD039549. It is accessible for reviewers using the username: reviewer_pxd039549@ebi.ac.uk and password: FokrTX6K until it becomes publicly available after publication. A statement has been placed in the manuscript.
[1] Perez-Riverol Y, Bai J, Bandla C, Hewapathirana S, García-Seisdedos D, Kamatchinathan S, Kundu D, Prakash A, Frericks-Zipper A, Eisenacher M, Walzer M, Wang S, Brazma A, Vizcaíno JA (2022). The PRIDE database resources in 2022: A Hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res 50(D1):D543-D552 (PubMed ID: 34723319).
On page 2 second paragraph a description of the MS method is outlined to describe the signals and which RNA type they are assigned. They describe how the +3 signal from (D3)-L-methionine differentiates methyl group marks before and after media exchange; however, in the next sentence there is no description of what was added to give the +8/+10 signal differences for pyrimidine and purine RNA nucleotides, respectively, synthesized before or after media exchange. Readers without knowledge of this field need to read ref 17 in detail to understand this.
* We apologize for being unclear. We have added a detailed description in the introduction and re-structures the results section as mentioned by the reviewer.
The term transcription rate and modification rate are confusing especially with the unit of percentage. In chemistry, rate is used to describe change over time with time as a unit. Modification rate is defined in the caption to Fig. S1 to be “modification abundance of new RNAs compared to steady stat modification abundance.” How were they compared? I did not find the definition for transcription rate.
* We agree with the reviewer and thank him/her for pointing out our inappropriate phrase. After thorough consideration, we have come to the conclusion that the “transcription rate” is in fact the “new transcript ratio”. We have changed all figures, figure legends and text accordingly. We have further discussed the term “modification rate” which is actually better reflected by the term “modification status*” with * indicating: absolute modification abundance in new transcripts compared to the steady state modification abundance. A detailed equation was added to the manuscript. We thank the reviewer for this statement which has substantially improved the manuscript.
In Fig 3D, what are the green stars?
* The green stars indicate modification. A statement has been made in the figure legend.
In Fig 1B, C, and D, why are the y-axes broken? There are no data above the breaks.
* The axes were broken to highlight the low abundance of hybrid species in the long-lived rRNAs and tRNA compared to the mRNA. We agree with the reviewer that the break is not necessary. It has been removed.
In Fig 1, why were the tRNA and rRNA analyzed at 6 h while the mRNA were analyzed at 2, 4, and 8 h?
* Due to the shorter half-life of Poly-A RNA (on average 9 hours), we chose narrow time points for Poly-A RNA and a time point in between for the long lived tRNA and rRNA (6 hours). For Poly-A RNA the 6 hour time-point was omitted due to the already expensive experiment design (NAIL + 2x Poly-A enrichment + Ribo-depletion ~ 500 € per time point per replicate) as we belief there is no additional gain in information from this time point.
On page 2, third paragraph the % hybrid in mRNA at 2, 4, and 8 h were 8, 35, and 98%, but in the next sentence it is stated that 78% post-methylation in poly-A RNA after 8 h was observed. Where did the 78% come from? Moreover, in the next sentence the authors describe this as high as a result of the short half-life of mRNA. For readers unfamiliar with RNA half-lives in HEK293 cells, adding the values for clarification would be beneficial. There are other instances where these values could be added.
* We thank the reviewer for catching this writing error which has previously escaped our attention. The typo has been corrected and we have added the half-lives of Poly-A RNA, tRNA and rRNA in the text.
Examples of missing references. Page 2 third paragraph the sentence regarding proven co-transcriptionl and nuclear methylation of rRNA. Who proved this? On page 3 paragraph two there is description of the addition of m6A and m7G and the unknown stage of addition during maturation. Who’s work are you referring?
* We thank the reviewer for pointing out the missing references. They were added.
Typos
The final sentence of the first paragraph on page 2 needs rewritten. Many other little typos throughout that can be fixed during proofreading.
* We thank the reviewer for catching this typo. We have thoroughly read the manuscript and all typos have been removed.
Referee: 2
Comments to the Author
The manuscript by Hagelskamp et al. describes a new set of experiments using actinomycin D to suppress transcription for isotope labelling of RNAs. The authors indicate that this addition of actinomycin D may be an important control for future biological experiments.
I have a number of queries for the authors to address:
1) Overall, I believe the manuscript is very difficult to follow (this comes up in many of my other points). It took quite a while for me to understand what the authors were suggested as the original hypothesis and the new hypothesis. I am still not sure I understand what the authors mean by ‘post methylated’. For this point, I believe the authors need to rewrite the introduction and include a new schematic in the introduction.
* We thank the reviewer for pointing out the difficulties in reading our manuscript. We have rewritten several aspects of it which has improved the manuscript.
2) In the introduction the authors talk about their development of ‘NAIL-MS’, but do not refer to any of the other substantive literature on adding labelled nucleosides, SAM, etc for labelling experiments of nucleic acids.
* We apologize and have added appropriate citations.
3) It is not clear what labelled species are being added at any time. At one point it is mentioned that deuterated methionine is added. But in Fig 1A it also looks like labelled nucleosides and nucleobases are added. Please give a description of this in the results section.
* Thank you for pointing out this gap. It has been fixed.
4) On page 2 “Thus, 13% abundance of hybrid species might not be connected to late maturation and postmethylation as previously hypothesized. Our doubts are further supported by the abundance of 8%, 35% and 93% hybrid m6A in Poly-A RNA after, 2h, 4 h and 8 h respectively.” It is not obvious where any of this data or numbers are coming from. Please cite and explicitly refer.
* The manuscript has been rewritten and the figures and graphs are now cited more explicitly.
5) On page 2 “Thus, it is unlikely to find 78% post-methylation in Poly-A RNA after 8 hours, especially given the on average short halflife of mRNA.” If this is from Fig 1E, then I don’t see where 78% is from?
*We apologize. This was a typo in the manuscript, that escaped our attention at the stage of submission.
6) In the ‘AcmD does not impact abundance of RNA writers’ section (page 5), two contradictory points are made:
1st: “ We are confident that the loss of hybrid modifications in rRNA and Poly- A RNA are not connected to a sudden loss of RNA writers under AcmD,”
2st: “Overall, the inhibition of mRNA transcription through AcmD leads to a general, significant decrease in protein abundance. Many RNA writers are found in the average area of reduced abundance.”
The authors only say three were not significantly changed, are these the only ones that can affect the methylation they are studying?
* Thank you for pointing out this contradiction caused by poor writing and explanation. The 1st statement was referring to the absolute abundance of RNA writers and global protein abundances after 8 hours of AcmD which is indeed unchanged. This makes sense as most proteins are stable within the 8 hour time frame of the experiment. The 2nd statement only refers to the cells reaction to the AcmD treatment and the remaining translation of potentially important proteins measured by a pulse chase SILAC experiment. In contrast to statement one it does not regard absolute protein abundance but only abundance of new proteins made in the SILAC context. Due to the lack of mRNAs in AcmD-treated cells, the global abundance of newly synthesized proteins drops. Yet, some proteins, such as NSun2, drop less strongly compared to others. We have revised and rewritten the manuscript accordingly.
7) On page 6 the authors state “Interestingly, AcmD is a substantial stress to the cells which might result in an adaptation of the translation apparatus towards higher efficiency”. Could this mean that the effect the authors see if stress related and not transcriptionally related? Could a different stress-inducing non-transcriptional repressed have the same effect?
* We thank the reviewer for this intriguing questions. Our data on AcmD indicates that although AcmD is a tool to enhance the temporal resolution of NAIL-MS experiments, it is also a stressor. In a next step, we will now study how stressors (oxidative, methylating, drugs…) change the hybrid species abundance and modification status of new transcripts in absence and presence of transcription. In this next study, we will learn, which epitranscriptome changes are transcription-related and which are truly adaptive.
Minor:
snoRNAs in introduction is not given a full abbreviation
* The abbreviation has been added.
Half life of the different RNAs is mentioned but no details are given. Please give literature numbers to compare each species
* The half-lives and citations are now given in the text.
Fig 1 B-E, why 6 hour time point used for B-D, but no 6 hour time point for E?
* Due to the shorter half-life of Poly-A RNA (on average 9 hours), we chose narrow time points for Poly-A RNA and a time point in between for the long lived tRNA and rRNA (6 hours). For Poly-A RNA the 6 hour time-point was omitted due to the already expensive experiment design (NAIL + 2x Poly-A enrichment + Ribo-depletion ~ 500 € per time point per replicate) as we belief there is no additional gain in information from this time point.
Fig S4, I cannot see the morphology in the pictures given, please use higher quality images
* We apologize, but we have no other pictures available. The purpose of these pictures is to show, that high concentrations of AcmD lead to cell death and thus lower density in the flask. We have re-phrased accordingly.
On page 3 “The abundance of hybrid m6A in Poly-A RNA is roughly twice as high as the remaining transcription rate at the 8 h time point (Fig. 3C).” twice compared to what?
* This very unclear section has been completely revised. It now reads: “While most hybrid species vanish upon blocking transcription, it is noteworthy that 4.5% hybrid species formation remains during AcmD treatment. For comparison, the remaining transcription under AcmD treatment is ~ 2% and thus hybrid species abundance is roughly double the amount of remaining transcription.”
On page 6 “As a side note, the Poly-A RNA studied here is mainly composed of mature mRNA used for active transcription.” Do the authors mean translation?
* Yes, embarassingly, we did. Thank you for pointing out this mistake.
Round 2
Revised manuscript submitted on 03 Feb 2023![](https://www.rsc-cdn.org/pubs-core/2022.0.153/content/NewImages/icon-letter.png)
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15-Feb-2023
Dear Professor Kaiser,
Dear Stefanie:
Manuscript ID: CB-ART-12-2022-000243.R1
TITLE: Temporal resolution of NAIL-MS of tRNA, rRNA and Poly-A RNA is overcome by actinomycin D
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Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg
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Reviewer 1
This is now ready for publication.
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