Modification of Adenosine196 by Mettl3 Methyltransferase in the 5’-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation
, Pavel Melnikov 2, Tatiana Prikazchikova 1, Olga Dontsova 1,3 and Timofei Zatsepin 1,3Round 1
Reviewer 1 Report
The authors describe an interesting new feature for Mettl3 in ribosome biogenesis. Mettl3 methylates 5'ETS at position 196. This methylation may decrease rRNA processing.
Mettl3 has been shown in certain cases to act without its catalytic activity. Though the authors show that m6A196 methylation seems dependent of Mettl3 and Mettl3 binds to 5'ETS, it cannot completely ruled out with the experiments presented that Mettl3 could act on 5'ETS independently of its methyltransferase function.
The authors cite in introduction diseases associated with ribosome biogenesis. Mettl3 is associated with various cancers either as oncogene or tumor suppressor. Downregulation of Mettl3, by increasing rRNA processing, could be associated to cell proliferation. The authors could discuss this point.
Author Response
Dear Reviewer,
We would like to thank you for your careful review of our manuscript. Positive comments include that this work is comprehensive, well-written, and technically convincing with important implications in cancer therapy. Your comments were highly relevant and contributed to improve the quality of this manuscript. The authors and I would like to thank you for the time you spent reviewing this manuscript. Below, you will find answers to your comments. In addition, we have highlighted the changes directly in the manuscript.
Point 1: Mettl3 has been shown in certain cases to act without its catalytic activity. Though the authors show that m6A196 methylation seems dependent of Mettl3 and Mettl3 binds to 5'ETS, it cannot completely ruled out with the experiments presented that Mettl3 could act on 5'ETS independently of its methyltransferase function.
- We agree with this viewpoint. Mettl3 binding and/or adenosine methylation (or both events) can influence on the rate of rRNA processing. However, any of these events results in changes and somehow influence of 47S pre-RNA interactions with unknown proteins.
Point 2: The authors cite in introduction diseases associated with ribosome biogenesis. Mettl3 is associated with various cancers either as oncogene or tumor suppressor. Downregulation of Mettl3, by increasing rRNA processing, could be associated to cell proliferation. The authors could discuss this point.
- Thank you for the suggestion. We add this point in the discussion section.
Reviewer 2 Report
See uploaded file
Comments for author File: 
Comments.pdf
Author Response
Dear Reviewer,
We would like to thank you for your careful review of our manuscript. Positive comments include that this work is comprehensive, well-written, and technically convincing with important implications in cancer therapy. Your comments were highly relevant and contributed to improve the quality of this manuscript. The authors and I would like to thank you for the time you spent reviewing this manuscript. Below, you will find answers to your comments. In addition, we have highlighted the changes directly in the manuscript.
Point 1: The present manuscript describes antibody-based approaches to identify 6-methyladenosine at position 196 of 47S pre-rRNA and identify Mettl3 as the methylating enzyme. Various assays suggest that the methylation affects the rate of 5’ pre-rRNA processing and eventually the amount of mature cytoplasmic rRNA. The experimental data are generally compelling and demonstrates the claimed effect of Mettl3 on (pre-)rRNA. One important aspect, however, is not discussed in the manuscript, namely the extent to which the pre-rRNA population is methylated at A196. Mettl3 only methylates a small fraction of a given mRNA target, but rRNA modifications are normally present at a (close to) stoichiometric ratio. Which of the two scenarios are we closest to? The (pre-)rRNA effects are of less interest if only a small fraction of the pre-rRNA is methylated, and the methylation is then possible just a side effect of Mettl3’s “lax” substrate recognition. Is there anyway to give a quantitative estimate from the data?
- Based on our m6A IP data we can evaluate that around 65% of 47S pre-rRNA is methylated by the estimation the ration of RNA after immunoprecipitation and before in the control sample. Accurate estimation is limited due to variations in enrichment and losses during immunoprecipitation and subsequent procedures. We found that downregulation of Mettl3 resulted in significant drop of methylation in 47S pre-rRNA. Thus, we propose that main portion of 47S pre-rRNA is methylated in normal cells.
Point 2: The issue as a minimum demands a thorough discussion. Several minor points should also be addressed: p. 2, line 67 -69. A lot is known about m6A in prokaryotic rRNA, both at the level of “housekeeping” modifications and as acquired antibiotic resistance.
- We added this point in the Introduction section, p.2 line 68-69.
Point 3: p.3, line 111 and 118: Define content of PBS and DMEM; a lot of modified recipes exist. p. 4 line 157 (and elsewhere): annotation of “degrees Celsius”.
- We introduced vendors of PBS and DMEM and changed the annotation in the manuscript.
Point 4: Figure 1 (and other figures): What do the asterisks indicate? Text to figure 1C: I suppose that the “fold enrichment” compares KD and control cells, but that is not what the text says. Which primers are used for the IgG control pull-down?
-The asterisks indicate the p-value meaning. We added information in the description of figures.
In figure 1C we showed RNA-immunoprecipitation data for HEK293 cell extract using anti-Mettl3 antibodies. This data additionally proves the interaction of Mettl3 methyltransferase with 5’-ETS of 47S pre-rRNA. We added correction that IP was done for HEK293 cell extract.
Sincerely,
Dr. Olga Sergeeva
