Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms

Giulia Venturi; Federico Zacchini; Cinzia Lucia Vaccari; Davide Trerè; Lorenzo Montanaro

doi:10.1371/journal.pone.0261476

Peer Review History

Original SubmissionSeptember 21, 2021
25 Oct 2021 Decision Letter - Baisong Lu, Editor PONE-D-21-30501Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoformsPLOS ONE Dear Dr. Montanaro, 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. In addition to carefully addressing the concerns and comments raised by the reviewers, please take care of the following issues: 1) Line 104, please specify A260 for RNA quantification.2) Please consider adding a diagram in Fig.1 (possibly as Fig.1B and put current Fig.1B as 1C) to illustrate the mechanism of the method, including the labeling of the 5.8S rRNA (three forms) and the 5S rRNA by respective labeled primers in reverse transcription, the separation of the cDNA, the detection and the quantification.3) Please check sentence 241 for readability.4)Fig4B, line 254, please check whether the ratio decreases or rises. Please submit your revised manuscript by Dec 09 2021 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. 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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: Yes 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, Venturi and colleagues describe a useful primer extension technique based on fluorescently labeled primers, with reaction products analyzed by the capillary gel electrophoresis. This method does not require radioactively labeled probes, conventional denaturing polyacrylamide gels, and autoradiography. The described experimental protocol is applicable for the analysis of alternatively processed 5.8S forms and also provides a starting point for a similar type of analysis of other rRNAs. However, from what I see, there is one potentially significant methodological issue concerning the interpretation of key results, and a missing link with the previous literature directly relevant to the RNAs in question. These issues need to be corrected. There are also some statements regarding the processing pathway that are not fully accurate if one considers recent developments in the literature. Major issues. 1. Lines 49-51: "Two isoforms of 5.8S rRNA have been described: a short form (5.8S short) and a longer one with 5 extra nucleotides at the 5’ of the sequence (5.8S long) (2, 3)". The authors cite 2010-2011 studies here. I wonder if the authors are familiar with a more recent study, which indicated the presence of 3 isoforms of 5.8S in both mouse and human cells, with two forms seemingly dependent on exonuclease activity (Wang et al., RNA 2015, 21(7):1240-8. doi: 10.1261/rna.051169.115). From looking at the data in Fig 1, it appears the authors are also seeing three isoforms: the electropherogram shows nicely an additional peak, at the expected position for where the 5' end of the 5.8S-cropped is supposed to be, as described by Wang et al (2015). This should be addressed in the discussion of the data, and the Abstract modified as well. 2. Reverse transcriptases are known to efficiently append nontemplated nucleotides to the 3' end of RNA/cDNA duplexes, typically adding a single residue when working on a 5' uncapped RNA template, which is the case here (Biotechniques 2001, 30(3):574-80, 582; Scientific Reports 2017, 7:41769; J Biol Chem. 2019, 294(48):18220–18231). The protocol the authors used does not appear to include blunting of the ends of the RT reaction products. This will affect both the size of the RT products detected in the capillary electrophoresis setting (Fig 1), and the heterogeneity of any detected 5' ends in the sequencing analysis (Fig 2). Because this directly bears on the interpretation of the key results, the authors need, at the very least, discuss this caveat and re-evaluate their data with regard to the precise 5' end identification of different isoforms, taking into account this RT activity. Minor issues. 1. The principal protocol (line 119 and the following) would be more valuable to other researchers if some additional details were provided to facilitate its application in other labs. In addition to the range of RNA concentrations tested in this study, please indicate the RNA amount you consider optimal for the technique. What kind of tubes did you use to minimize evaporation during incubation of these small volumes? Please indicate the source of the fluorescently labeled primers and the type of purification (if any was used) after the synthesis of these oligos. 2. 48-49: "Ribosome biogenesis has been widely studied in yeast and only a few studies have been conducted on human cell lines." While it is true that this pathway used to be more intensely studied in yeast, there has been steady progress in human cells in recent years, certainly not 'only a few' studies. Consider, for example, the recent work on the human small subunit assembly, no less detailed than any work done in yeast (Sameer Singh et al, Science (2021). DOI:10.1126/science.abj5338). 3. Lines 51-55 "the cleavage at the internal transcribed spacer 1 (ITS1) operated by RNAse MRP", "the long isoform derives from direct ITS1 cleavage by a still uncharacterized enzyme (8)". The model of ITS1 cleavages to which the authors are referring is debatable, and as the recent literature shows, is very likely incorrect, please see Li et al., Int. J. Mol. Sci. 2021, 22(13), 6690; https://doi.org/10.3390/ijms22136690. Nick Watkins' group also showed that MRP deletion had no appreciable effects on ITS1 cleavage in human cells (Ref 7). 5. Refs 13 and 14 are identical. Reviewer #2: In the manuscript, Venturi and colleagues developed a method based on primer extension and fragment analysis for the peak in the electropherogram to evaluate 5.8S rRNA isoforms. The authors optimized the method by adjusting the RNA and primer amount and confirmed the 5.8rRNA isoforms by reverse transcription and 5’RACE. The results showed that the newly established method was able to analyze the abundance of two isoforms of 5.8S rRNA in samples with small amount or low quality rapidly and safely. It can be a very useful 5.8S rRNA isoform analysis method for basic research and clinical diagnostic applications. The authors provided a clear description for the methodology. However, the manuscript can be improved with some small changes. Minor concerns 1. In the manuscript, the RNA samples were from cell lines or XpressRef Universal Total RNA. It will be great if the author provide normal patient frozen tissue data to confirm that the method can be used in clinical application. 2. The authors mention that common technique such as Northern Blot requires high quantities of good quality RNA. The authors are recommended to provide the range of RNA amount used in traditional method to show that the new method is more sensitive. 3. Where is Box1 in the Introduction part? I can only find it from the reference paper. 4. In Materials and Methods, line98, the concentration of L-Glutamine and antibiotics should use specified (e.g. 2mM L-Gln) rather than stating 1%. 5. In Materials and Methods, line108, “1X106 cells” 6 should be superscript. 6. A and B labels were missing from Figure1. 7. In Fig 1. legend, it is unnecessary to repeat the details which are already described in method part. 8. There is no statistical analysis in the Materials and Methods. It will be better to do a statistical analysis for figure 4A and B. ****** 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: No 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. https://doi.org/10.1371/journal.pone.0261476.r001
Revision 1
23 Nov 2021 Author Response Academic Editor’s comments: 1) Line 104, please specify A260 for RNA quantification. we added to the text absorbance 260 nm 2) Please consider adding a diagram in Fig.1 (possibly as Fig.1B and put current Fig.1B as 1C) to illustrate the mechanism of the method, including the labeling of the 5.8S rRNA (three forms) and the 5S rRNA by respective labeled primers in reverse transcription, the separation of the cDNA, the detection and the quantification. thank you for your suggestion. we preferred to implement Fig.1A with additional details illustrating the mechanism of the method. Fig1B shows a typical electropherogram resulting from its application. 3) Please check sentence 241 for readability. we changed the sentence: We observed that in two independent experiments 5.8S long/short value is similar in each sample, and that the average ratio value of the ratio results is stable when RNA amount decreases. 4)Fig4B, line 254, please check whether the ratio decreases or rises. we changed the approach to calculate the amount of the different 5.8S rRNA isoforms, figures and text has been changed accordingly. Reviewer #1 major 1. Lines 49-51: "Two isoforms of 5.8S rRNA have been described: a short form (5.8S short) and a longer one with 5 extra nucleotides at the 5’ of the sequence (5.8S long) (2, 3)". The authors cite 2010-2011 studies here. I wonder if the authors are familiar with a more recent study, which indicated the presence of 3 isoforms of 5.8S in both mouse and human cells, with two forms seemingly dependent on exonuclease activity (Wang et al., RNA 2015, 21(7):1240-8. doi: 10.1261/rna.051169.115). From looking at the data in Fig 1, it appears the authors are also seeing three isoforms: the electropherogram shows nicely an additional peak, at the expected position for where the 5' end of the 5.8S-cropped is supposed to be, as described by Wang et al (2015). This should be addressed in the discussion of the data, and the Abstract modified as well. Thank you for the comment. We amended the text, the figure and the bibliography mentioning the additional cropped isoform and included its evaluation in our study. 2. Reverse transcriptases are known to efficiently append nontemplated nucleotides to the 3' end of RNA/cDNA duplexes, typically adding a single residue when working on a 5' uncapped RNA template, which is the case here (Biotechniques 2001, 30(3):574-80, 582; Scientific Reports 2017, 7:41769; J Biol Chem. 2019, 294(48):18220–18231). The protocol the authors used does not appear to include blunting of the ends of the RT reaction products. This will affect both the size of the RT products detected in the capillary electrophoresis setting (Fig 1), and the heterogeneity of any detected 5' ends in the sequencing analysis (Fig 2). Because this directly bears on the interpretation of the key results, the authors need, at the very least, discuss this caveat and re-evaluate their data with regard to the precise 5' end identification of different isoforms, taking into account this RT activity. The issue regarding RT-mediated extra nucleotide addition was considered in the discussion (lines 302-308). Minor 1. The principal protocol (line 119 and the following) would be more valuable to other researchers if some additional details were provided to facilitate its application in other labs. In addition to the range of RNA concentrations tested in this study, please indicate the RNA amount you consider optimal for the technique. What kind of tubes did you use to minimize evaporation during incubation of these small volumes? Please indicate the source of the fluorescently labeled primers and the type of purification (if any was used) after the synthesis of these oligos. we added these details in the protocol. The optimal RNA amount is 250 ng. tubes multiply-Pro cup 0.2ml PP sarstedt. The source of fluorescently labeled primers is IDT and they have been purified by HPLC. 2. 48-49: "Ribosome biogenesis has been widely studied in yeast and only a few studies have been conducted on human cell lines." While it is true that this pathway used to be more intensely studied in yeast, there has been steady progress in human cells in recent years, certainly not 'only a few' studies. Consider, for example, the recent work on the human small subunit assembly, no less detailed than any work done in yeast (Sameer Singh et al, Science (2021). DOI:10.1126/science.abj5338). thank you for the suggestion, we changed the sentence accordingly. 3. Lines 51-55 "the cleavage at the internal transcribed spacer 1 (ITS1) operated by RNAse MRP", "the long isoform derives from direct ITS1 cleavage by a still uncharacterized enzyme (8)". The model of ITS1 cleavages to which the authors are referring is debatable, and as the recent literature shows, is very likely incorrect, please see Li et al., Int. J. Mol. Sci. 2021, 22(13), 6690; https://doi.org/10.3390/ijms22136690. Nick Watkins' group also showed that MRP deletion had no appreciable effects on ITS1 cleavage in human cells (Ref 7). We thank the reviewer, we changed the text in the introduction and in the box1 to show how this specific issue is currently under debate. 4. Refs 13 and 14 are identical. We corrected the error. Minor concerns 1. In the manuscript, the RNA samples were from cell lines or XpressRef Universal Total RNA. It will be great if the author provide normal patient frozen tissue data to confirm that the method can be used in clinical application. We agree with the reviewer on the importance of the issue. We considered that using a limited number of pathological samples could not provide a definitive confirmation of the possibility to use the method on clinical samples. To test the method in controlled conditions we preferred to perform the experiment reported in Figure 4C. 2. The authors mention that common technique such as Northern Blot requires high quantities of good quality RNA. The authors are recommended to provide the range of RNA amount used in traditional method to show that the new method is more sensitive. We reported RNA concentration typically used in northern blot analysis in the discussion paragraph. 3. Where is Box1 in the Introduction part? I can only find it from the reference paper. The text of box1 is reported in the text file while the image is uploaded as a figure. 4. In Materials and Methods, line98, the concentration of L-Glutamine and antibiotics should use specified (e.g. 2mM L-Gln) rather than stating 1%. We modified the text specifying the exact concentration of L-Glutamine, Penicillin and Streptomycin. 5. In Materials and Methods, line108, “1X106 cells” 6 should be superscript. We corrected the error. 6. A and B labels were missing from Figure1. We corrected the figure. 7. In Fig 1. legend, it is unnecessary to repeat the details which are already described in method part. We modified the figure legend accordingly. 8. There is no statistical analysis in the Materials and Methods. It will be better to do a statistical analysis for figure 4A and B. We added paragraph to the Materials and methods indicating the statistical analysis performed. We included statistical analysis in figure 4A and B. Attachments Attachment Submitted filename: Response to Reviewers - 2021-11-26T005323.482.docx https://doi.org/10.1371/journal.pone.0261476.r002
29 Nov 2021 Decision Letter - Baisong Lu, Editor PONE-D-21-30501R1Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoformsPLOS ONE Dear Dr. Montanaro, 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. ============================== Legend for Fig.1A should define the short names for the three isoforms. Line 28 and 76, ”two 5.8S isoforms” should be “three 5.8s isoforms”. ============================== Please submit your revised manuscript by Jan 13 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, Baisong Lu, Ph.D Academic Editor PLOS ONE Journal Requirements: 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. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: [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. https://doi.org/10.1371/journal.pone.0261476.r003
Revision 2
30 Nov 2021 Author Response Academic Editor’s comments: Legend for Fig.1A should define the short names for the three isoforms. We amended the figure legend Line 28 and 76, ”two 5.8S isoforms” should be “three 5.8s isoforms”. We corrected the errors Attachments Attachment Submitted filename: Response to Reviewers2.docx https://doi.org/10.1371/journal.pone.0261476.r004
3 Dec 2021 Decision Letter - Baisong Lu, Editor Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms PONE-D-21-30501R2 Dear Dr. Montanaro, 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, Baisong Lu, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0261476.r005
Formally Accepted
7 Dec 2021 Acceptance Letter - Baisong Lu, Editor PONE-D-21-30501R2 Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms Dear Dr. Montanaro: 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 Dr. Baisong Lu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0261476.r006

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