The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes

Jessie A. C. Altieri; Klemens J. Hertel

doi:10.1371/journal.pone.0257503

Peer Review History

Original SubmissionSeptember 1, 2021
4 Oct 2021 Decision Letter - Ravindra N Singh, Editor PONE-D-21-28397 The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes PLOS ONE Dear Dr. Hertel, 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. Both reviewers liked your study. However, they had minor concerns that could be addressed without any new experiments. Please be as detailed as possible in your explanations. Please submit your revised manuscript by Nov 18 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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Please upload a new copy of Figure 5 as the detail is not clear. Please follow the link for more information: " ext-link-type="uri" xlink:type="simple">https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/" " ext-link-type="uri" xlink:type="simple">https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/" [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions 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: Yes Reviewer #2: Yes ******** 2. 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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 the authors tested whether metabolic labeling with 4-thiouridine (4sU), a tool commonly used to investigate different aspects of RNA metabolism, creates any biases/negative effect on pre-mRNA splicing. To do so, they performed in vitro experiments with well controlled levels of 4sU incorporation (from 0 to 100%) as well as cell culture experiments with commonly used 4sU concentrations. The presented study is significant, since until now consequences of 4sU labeling on nascent pre-mRNA splicing have not been evaluated. The results of the study indicate that pre-mRNA splicing can be impacted by the incorporation of 4sU into pre-mRNA. The effect was particularly pronounced in in vitro experiments (splicing, transcription, and pre-mRNA stability) at elevated levels of 4sU incorporation. In addition, how much 4sU influenced splicing depended on the strength of the splice sites in question. Overall, the results showed that in case of splicing, the incorporation of 4sU into pre-mRNAs is not without consequences and can affect pre-mRNA splicing. The consequences become detectable at elevated levels of 4sU incorporation and in the context of suboptimal splice sites. Therefore, the study suggests that appropriate controls should be included when metabolic labeling is used. 1. Check for mistakes. For example, (a) in “Materials and Methods” it is stated that in in vitro transcription the relative amount of 4sU was 0%, 2.5%, 15%, 30%, 100%. However, in the Results “15%” sample is absent. (b) in “Material and Methods”, page 6, “HEK293 metabolic labeling” there should be a space between “protocol was followed” and “[6]” (line 134). (c) In “Results”, line 231, a bracket is missing in front of “A” 2. Why write “In vitro splicing was carried out …for 90 min …” if the time points were “1 hour” and “2 hours” and there is no “90 min” time point at all (in “Materials and Methods”). 3. In Figure 1A, please, mark the 3’ and 5’ splice sites in addition to just indicating their strength. 4. Is there any explanation to why the total signal is dramatically weaker at “1 hour” as compared to “t=0” input RNA specifically at 0% and 2.5% labeling (in Figure 1B)? Also, why the pre-mRNA band at t=0 is significantly weaker at 100% 4sU incorporation as compared to 0%, 2.5% and 30%? If different amount of pre-mRNA was used for in vitro splicing, please, specify in “Materials and Methods”. 5. Why “24 h” ZNF711 signal is so weak in the presence of 4sU? 6. In Figure 4B, please, provide quantitation for ADAP2 similarly as you did for DOLPP1 since visually (gel image) 4sU labelling at 24 hr appears to cause a difference in ADAP2 splicing pattern. Maybe even provide quantitation for all five exon skipping events. Reviewer #2: The manuscript “The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes” by Altieri and Hertel describes how the commonly used 4SU modified base has limited effect on splicing in cells at the concentrations employed for most experiments. This result should give other researchers confidence that use of 4SU should not strongly bias their experimental outcomes for constitutive splicing. However, it also is a reminder that the splicing events most responsive to regulation, are also most likely to be sensitive to experimental conditions. The authors also examine in vitro RNA splicing and find some changes in splicing efficiency and stability with very high incorporation of 4SU. Overall, the data are clearly presented, and their conclusions are generally well supported. The authors should address the following minor points to improve the presentation of the studies. 1- For in vitro splicing assays, the methods should explicitly describe the calculation to determine splicing efficiency, which is typically band intensity of the product mRNA divided by the summed band intensity for pre-mRNA, one of the 1st intermediate products, and mRNA- each corrected for background and the number of radiolabeled nucleotides incorporated in each species. 2-Changes in in vitro splicing efficiency can also be due to differential stability of the RNA species. Indeed, the data shown in Fig. 1B indicates that the pre-mRNA is apparently disappearing at higher rate than mRNA formation. The authors also show that high 4SU incorporation stabilizes the pre-mRNA for the AdML transcript. Is it possible that the calculated decrease in splicing with high 4SU may instead reflect increased pre-mRNA stability? Discussion of this potentially confounding issue is needed. 3-What type of variation is seen with degradation assay shown if Figure 2? 4- The nature of experimental replicates for Figures 4 and 5 should be stated. Are they biological replicates of RNA extracted from cells or technical replicates of RT-PCR analysis? The former would better support the conclusions related to 4SU induced splicing changes for the same reason that they are examining sensitive splicing events. 5- Is there a possibility that 4SU induces aberrant splicing? There are some new bands that appear at 24 hr with RSU appearing with DOLP11, TRA2B and DDX5. ****** 6. 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Revision 1
22 Nov 2021 Author Response We thank the reviewers for their thorough and careful reading of the manuscript. We have addressed their comments and concerns to the best of our abilities. We have made the necessary changes to the manuscript per the reviewers’ feedback, and we have addressed the questions raised in our responses. The reviewer’s critiques are listed first and our responses are headed by the word “Response”: Reviewer #1: The presented study is significant, since until now consequences of 4sU labeling on nascent pre-mRNA splicing have not been evaluated. The results of the study indicate that pre-mRNA splicing can be impacted by the incorporation of 4sU into pre-mRNA. The effect was particularly pronounced in in vitro experiments (splicing, transcription, and pre-mRNA stability) at elevated levels of 4sU incorporation. In addition, how much 4sU influenced splicing depended on the strength of the splice sites in question. Overall, the results showed that in case of splicing, the incorporation of 4sU into pre-mRNAs is not without consequences and can affect pre-mRNA splicing. The consequences become detectable at elevated levels of 4sU incorporation and in the context of suboptimal splice sites. Therefore, the study suggests that appropriate controls should be included when metabolic labeling is used. 1. Check for mistakes. For example, (a) in “Materials and Methods” it is stated that in in vitro transcription the relative amount of 4sU was 0%, 2.5%, 15%, 30%, 100%. However, in the Results “15%” sample is absent. Response: We thank the reviewer for his/her positive feedback on our work and their careful reading of our manuscript. We are sorry for the oversight mistake. In the revised manuscript we have fixed this error. (b) in “Material and Methods”, page 6, “HEK293 metabolic labeling” there should be a space between “protocol was followed” and “[6]” (line 134). Response: We are sorry for the oversight mistake. In the revised manuscript we have fixed this error. (c) In “Results”, line 231, a bracket is missing in front of “A” Response: We are sorry for the oversight mistake. In the revised manuscript we have fixed this error. 2. Why write “In vitro splicing was carried out …for 90 min …” if the time points were “1 hour” and “2 hours” and there is no “90 min” time point at all (in “Materials and Methods”). Response: Thank you for pointing out this mistake. In the revised manuscript we have fixed this error. 3. In Figure 1A, please, mark the 3’ and 5’ splice sites in addition to just indicating their strength. Response: The revised Figure 1 now defines the 3’ and 5’ splice sites and their strength. 4. Is there any explanation to why the total signal is dramatically weaker at “1 hour” as compared to “t=0” input RNA specifically at 0% and 2.5% labeling (in Figure 1B)? Also, why the pre-mRNA band at t=0 is significantly weaker at 100% 4sU incorporation as compared to 0%, 2.5% and 30%? If different amount of pre-mRNA was used for in vitro splicing, please, specify in “Materials and Methods”. Response: At optimized in vitro splicing conditions (energy source, energy regeneration source, MgCl2, DTT) T7 polymerase-generated pre-mRNA (only cap analog, no poly A tail) is attacked and degraded by RNases contained within nuclear extract. This happens within the first ~15 minutes of the splicing reaction as spliceosomal components and RNases compete for the pre-mRNA that has been added to the reaction. RNAs that associate with the spliceosome are protected from this degradation. In a typical splicing reaction ~50-75% of the input RNA is degraded quickly. This is a well-documented phenomenon that is often countered by loading different amounts of the splicing reaction onto the gel – with the result that time courses do not show the visual loss of RNA intensity. As splicing efficiencies at various time points are calculated based on the relative representation of pre-mRNA and mRNA, loading only a fraction of a time point will not alter the calculated splicing efficiency. We have opted not to load fractions of time points, but the entire reaction for each time point. As a result, our in vitro splicing gels show differential total counts in each of the time point lanes. In the revised manuscript, we state our loading approach in the ‘Materials and Methods’. 5. Why “24 h” ZNF711 signal is so weak in the presence of 4sU? Response: We thank the reviewer for pointing out this anomaly, which was caused by a loading error. We have re-run the experiments and now show an updated RT-PCR image. The corresponding quantitation was updated accordingly. 6. In Figure 4B, please, provide quantitation for ADAP2 similarly as you did for DOLPP1 since visually (gel image) 4sU labelling at 24 hr appears to cause a difference in ADAP2 splicing pattern. Maybe even provide quantitation for all five exon skipping events. Response: As suggested, in the revised manuscript all images showing RT-PCR results are now accompanied with quantitation. Reviewer #2: The manuscript “The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes” by Altieri and Hertel describes how the commonly used 4SU modified base has limited effect on splicing in cells at the concentrations employed for most experiments. This result should give other researchers confidence that use of 4SU should not strongly bias their experimental outcomes for constitutive splicing. However, it also is a reminder that the splicing events most responsive to regulation, are also most likely to be sensitive to experimental conditions. The authors also examine in vitro RNA splicing and find some changes in splicing efficiency and stability with very high incorporation of 4SU. Overall, the data are clearly presented, and their conclusions are generally well supported. The authors should address the following minor points to improve the presentation of the studies. 1- For in vitro splicing assays, the methods should explicitly describe the calculation to determine splicing efficiency, which is typically band intensity of the product mRNA divided by the summed band intensity for pre-mRNA, one of the 1st intermediate products, and mRNA- each corrected for background and the number of radiolabeled nucleotides incorporated in each species. Response: As suggested, the revised manuscript now contains a description how the efficiency of splicing was calculated. This information is included in the ‘Materials Methods’ section. 2-Changes in in vitro splicing efficiency can also be due to differential stability of the RNA species. Indeed, the data shown in Fig. 1B indicates that the pre-mRNA is apparently disappearing at higher rate than mRNA formation. The authors also show that high 4SU incorporation stabilizes the pre-mRNA for the AdML transcript. Is it possible that the calculated decrease in splicing with high 4SU may instead reflect increased pre-mRNA stability? Discussion of this potentially confounding issue is needed. Response: The reviewer pointed out a plausible alternative explanation for the changes in splicing efficiency, namely that differential degradation rates of the pre-mRNA might influence calculated splicing efficiencies. To understand the influence of degradation differences on splicing differences it is best to evaluate the profiles of our �-globin substrate. It shows the most striking splicing differences when comparing fully and not modified RNA substrates (Fig. 1B,C). However, as stated in the manuscript, we did not detect statistically significant differences in the stability of the 0% and 100% 4sU modified pre-mRNAs. Thus, for the �-globin substrate the difference in splicing is not influenced by stability differences – it is all splicing. A similar analysis of the compounding splicing/degradation factors for the AdML substrate shows minimal, but not statistically significant splice differences (Fig. 1D), but stability differences (Fig. 2). Thus, it is possible that the trend of reduced splicing efficiency observed for the AdML constructs (although not statistically significant) could be a consequence of stability differences. So, for AdML, these potentially confounding effects cannot be uncoupled, as hinted to by the reviewer. In the revised manuscript now discuss the potential of these confounding factors. 3-What type of variation is seen with degradation assay shown if Figure 2? Response: We apologize for not showing the variation of the data shown in Figure 2. This has been corrected and the revised figure now shows that variation with error bars. We thank the reviewer for pointing out this oversight. 4- The nature of experimental replicates for Figures 4 and 5 should be stated. Are they biological replicates of RNA extracted from cells or technical replicates of RT-PCR analysis? The former would better support the conclusions related to 4SU induced splicing changes for the same reason that they are examining sensitive splicing events. Response: All replicates are biological replicates, and the revised manuscript now explicitly states this. 5- Is there a possibility that 4SU induces aberrant splicing? There are some new bands that appear at 24 hr with 4SU appearing with DOLP11, TRA2B and DDX5. Response: Yes, it is possible that 4sU induces aberrant splicing. The reviewer is correct in pointing out that additional RT-PCR bands appear in the 24hrs time point for the genes listed. Without sequencing the PCR product, it is difficult to assign identities to these bands, meaning assigning whether the bands represent aberrant splicing. In the revised manuscript we now point out the additional bands as possible, but unconfirmed evidence of aberrant splicing. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0257503.r002
24 Nov 2021 Decision Letter - Ravindra N Singh, Editor The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes PONE-D-21-28397R1 Dear Dr. Hertel, 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, Ravindra N Singh, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0257503.r003
Formally Accepted
3 Dec 2021 Acceptance Letter - Ravindra N Singh, Editor PONE-D-21-28397R1 The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes Dear Dr. Hertel: 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. Ravindra N Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0257503.r004

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