The landscape of m1A modification and its posttranscriptional regulatory functions in primary neurons

  1. Chi Zhang
  2. Xianfu Yi
  3. Mengfan Hou
  4. Qingyang Li
  5. Xueying Li
  6. Lu Lu
  7. Enlin Qi
  8. Mingxin Wu
  9. Lin Qi
  10. Huan Jian
  11. Zhangyang Qi
  12. Yigang Lv
  13. Xiaohong Kong
  14. Mingjun Bi
  15. Shiqing Feng
  16. Hengxing Zhou

  • Curated by eLife

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    eLife assessment

    This study presents a valuable finding on the critical features and patterns of m1A modification and in neurons and OGD/R-treated neurons. Moreover, the authors identified m1A modifications on different RNAs and explored the possible effects of m1A modification on the functions of different RNAs via an integrated approach of omics and bioinformatics. The evidence supporting the claims of the authors is solid, although inclusion of more in-depth studies to analyze the transcription factors for the upstream regulation would have strengthened the study. The work will be of interest to neurobiologist and scientists in the field of RNAs.

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Abstract

Cerebral ischaemia‒reperfusion injury (IRI), during which neurons undergo oxygen-glucose deprivation/reoxygenation (OGD/R), is a notable pathological process in many neurological diseases. N1-methyladenosine (m 1 A) is an RNA modification that can affect gene expression and RNA stability. The m 1 A landscape and potential functions of m 1 A modification in neurons remain poorly understood. We explored RNA (mRNA, lncRNA, and circRNA) m 1 A modification in normal and OGD/R-treated mouse neurons and the effect of m 1 A on diverse RNAs. We investigated the m 1 A landscape in primary neurons, identified m 1 A-modified RNAs, and found that OGD/R increased the number of m 1 A RNAs. m 1 A modification might also affect the regulatory mechanisms of noncoding RNAs, e.g., lncRNA–RNA binding proteins (RBPs) interactions and circRNA translation. We showed that m 1 A modification mediates the circRNA/lncRNA‒miRNA–mRNA competing endogenous RNA (ceRNA) mechanism and that 3' untranslated region (3’UTR) modification of mRNAs can hinder miRNA–mRNA binding. Three modification patterns were identified, and genes with different patterns had intrinsic mechanisms with potential m 1 A-regulatory specificity. Systematic analysis of the m 1 A landscape in normal and OGD/R neurons lays a critical foundation for understanding RNA modification and provides new perspectives and a theoretical basis for treating and developing drugs for OGD/R pathology-related diseases.

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  1. Version published to 10.7554/elife.85324 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    N1-methyladenosine (m1A) is a rather intriguing RNA modification that can affect gene expression and RNA stability etc. The manuscript presented the exploration of RNAs m1A modification in normal and OGD/R-treated neurons and the effects of m1A on diverse RNAs. The authors showed that m1 modification can mediate circRNA/LncRNA-miRNA-mRNA mechanism and 3'UTR methylation of mRNAs can disturb miRNA-mRNA binding.

    The manuscript provides evidence for the following,

    1. The OGD/R can have impacts on various functions of m1A mRNAs and neuron fates.
    1. The m1A methylation of mRNA 3'UTRs disturbs the miRNA-mRNA binding.
    1. The authors identified three possible patterns of m1A modification regulation in neurons.

    The main merit of the manuscript is that the authors identified some critical features and patterns of m1A modification and in neurons and OGD/R-treated neurons. Moreover, the authors identified m1A modifications on different RNAs and explored the possible effects of m1A modification on the functions of different RNAs and the overall posttranscriptional regulation mechanism via an integrated approach of omics and bioinformatics. The major weakness of the manuscript is that technique details for many results are missing. Moreover, language inconsistences can be found throughout the manuscript. My general feeling about the manuscript is that some conclusions are rather superficial and therefore require validation and discussion.

    We appreciate your endorsement and constructive opinion concerning our work. Our study provides a comprehensive exploration of the characteristics of m1A modifications in neurons. According to your suggestions, we have specified the technique details in the revised manuscript have included our perspectives on some of the conclusions in the Discussion section. In addition, we have made changes to language inconsistences throughout the manuscript. We hope that the revisions made are acceptable and meet your requirements.

    Reviewer #2 (Public Review):

    In this manuscript, investigators explore the m1A modification, an important post-transcriptional regulatory mechanism, in primary normal neuron and OGD/R treated neuron. As far as I know, the regulatory m1A modification remains poorly characterized in neuron. This is an interesting topic in the context of epitranscriptomics. This paper not only provided us with a landscape of m1A modifications in neuron, but also explored the impact of m1A modifications on the biological functions of different RNA (mRNA, lncRNA, circRNA). In addition, the argument that m1A modification affects miRNA binding to other RNAs is of interest to reader, and the authors have performed a dual luciferase validation here to add feasibility to this conclusion.

    Thank you for your careful review of our study, and thank you for your appreciation on our work. The aim of this work was to explore the characteristics of m1A modification in neurons. We believe that incorporating your advice into the revised manuscript has enhanced the quality of our article.

    Reviewer #3 (Public Review):

    Overall, this is an interesting and well performed study that described a comprehensive landscape of m1A modification in primary neuron and investigated the role of m1A in the circRNA/lncRNA‒miRNA-mRNA regulatory network following OGD/R. The focus on the two different complex regulatory networks for differential expression and differential methylation is important and it will be a valuable resource for the research community that focuses on epitranscriptomics and central nerve system diseases. Collectively, the authors present an exciting piece of work that certainly adds to the literature regarding epitranscriptomic features in neuron. While interesting results obtained and the paper is nicely written, I have the following suggestions for minor revisions to improve the paper.

    We are grateful for your many positive comments and recognition of the potential of our work. Due to your suggestion, we found some shortcomings in our current manuscript. These suggestions were introduced and added value to our article. Our future research will continue to explore some conclusions obtained from this work. And we will continue to contribute our research outcomes in this field. Thank you again for your excellent suggestions!

    1. The authors have explored the role of m1A modification in neuron, but it would have been helpful if the authors described the significance of these findings in depth in some sections (Figure 5 and Figure 6) to enhance the value of the article.

    Thank you for your insightful suggestion. We agree to the comment that the significance of these findings should be described in detail. As such, we have added corresponding content to the Results (line 407-424) and Discussion (line 532-550) sections respectively.

    1. The authors should describe in detail the current research state of m1A modification and the significance of this study to the field of epitranscriptomics in the introduction and Discussion section.

    Thank you for your insightful suggestion. There is relatively little knowledge in the m1A modification area. It is really important to summarize the existing knowledge and research progress in a comprehensive and detailed manner. We conducted a comprehensive latest literature search and added corresponding content to the Introduction (line 78-83) and Discussion section (line 505-511, line 532-562) as you suggested.

  3. eLife

    eLife assessment

    This study presents a valuable finding on the critical features and patterns of m1A modification and in neurons and OGD/R-treated neurons. Moreover, the authors identified m1A modifications on different RNAs and explored the possible effects of m1A modification on the functions of different RNAs via an integrated approach of omics and bioinformatics. The evidence supporting the claims of the authors is solid, although inclusion of more in-depth studies to analyze the transcription factors for the upstream regulation would have strengthened the study. The work will be of interest to neurobiologist and scientists in the field of RNAs.

  4. eLife

    Reviewer #1 (Public Review):

    N1-methyladenosine (m1A) is a rather intriguing RNA modification that can affect gene expression and RNA stability etc. The manuscript presented the exploration of RNAs m1A modification in normal and OGD/R-treated neurons and the effects of m1A on diverse RNAs. The authors showed that m1 modification can mediate circRNA/LncRNA-miRNA-mRNA mechanism and 3'UTR methylation of mRNAs can disturb miRNA-mRNA binding.

    The manuscript provides evidence for the following,
    1. The OGD/R can have impacts on various functions of m1A mRNAs and neuron fates.
    2. The m1A methylation of mRNA 3'UTRs disturbs the miRNA-mRNA binding.
    3. The authors identified three possible patterns of m1A modification regulation in neurons.

    The main merit of the manuscript is that the authors identified some critical features and patterns of m1A modification and in neurons and OGD/R-treated neurons. Moreover, the authors identified m1A modifications on different RNAs and explored the possible effects of m1A modification on the functions of different RNAs and the overall posttranscriptional regulation mechanism via an integrated approach of omics and bioinformatics. The major weakness of the manuscript is that technique details for many results are missing. Moreover, language inconsistences can be found throughout the manuscript. My general feeling about the manuscript is that some conclusions are rather superficial and therefore require validation and discussion.

  5. eLife

    Reviewer #2 (Public Review):

    In this manuscript, investigators explore the m1A modification, an important post-transcriptional regulatory mechanism, in primary normal neuron and OGD/R treated neuron. As far as I know, the regulatory m1A modification remains poorly characterized in neuron. This is an interesting topic in the context of epitranscriptomics. This paper not only provided us with a landscape of m1A modifications in neuron, but also explored the impact of m1A modifications on the biological functions of different RNA (mRNA, lncRNA, circRNA). In addition, the argument that m1A modification affects miRNA binding to other RNAs is of interest to reader, and the authors have performed a dual luciferase validation here to add feasibility to this conclusion.

  6. eLife

    Reviewer #3 (Public Review):

    Overall, this is an interesting and well performed study that described a comprehensive landscape of m1A modification in primary neuron and investigated the role of m1A in the circRNA/lncRNA‒miRNA-mRNA regulatory network following OGD/R. The focus on the two different complex regulatory networks for differential expression and differential methylation is important and it will be a valuable resource for the research community that focuses on epitranscriptomics and central nerve system diseases. Collectively, the authors present an exciting piece of work that certainly adds to the literature regarding epitranscriptomic features in neuron. While interesting results obtained and the paper is nicely written, I have the following suggestions for minor revisions to improve the paper.

    1. The authors have explored the role of m1A modification in neuron, but it would have been helpful if the authors described the significance of these findings in depth in some sections (Figure 5 and Figure 6) to enhance the value of the article.
    2. The authors should describe in detail the current research state of m1A modification and the significance of this study to the field of epitranscriptomics in the introduction and discussion section.

  7. Version published to 10.1101/2023.01.25.525483v1 on bioRxiv