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m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome

Nature Biotechnology volume 40, pages 1210–1219 (2022)Cite this article

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An Author Correction to this article was published on 30 November 2022

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Abstract

Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, here, we introduce m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. We mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. We observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.

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Fig. 1: m6A-SAC-seq strategy and development.
Fig. 2: Characteristics of quantitative m6A maps in poly(A)-tailed RNAs from HeLa, HEK293 and HepG2 cells.
Fig. 3: Effects of m6A on the modified RNAs in cell lines.
Fig. 4: m6A dynamics across hematopoietic stem cell differentiation into monocytes.
Fig. 5: m6A modification impacting gene expression during monocytopoiesis.

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Data availability

Data have been deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE162357.

Code availability

For m6A-SAC-seq data processing, the code is available in the following GitHub repositories: https://github.com/shunliubio/m6A-SAC-seq and https://github.com/CTLife/m6A-SAC-seq.

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Acknowledgements

We thank P. Faber of the University of Chicago Genomics Facility for sequencing support and Q. Jin for helping with UHPLC–QQQ–MS/MS. We also thank T. Wu, H.L. Shi and Z.J. Zhang for discussions. L.H. was supported by a Chicago Fellows Program, Chicago Biomedical Consortium (CBC) postdoctoral award and a Leukemia & Lymphoma Society Special Fellow Award. B.T.H. was supported by an NIH fellowship (F32 CA221007). We thank support from National Institutes of Health (NIH) grants RM1 HG008935 (C.H.), R01 GM126553 (M.C.), R01 CA243386 (J.C.), R01 CA214965 (J.C.), R01 CA236399 (J.C.), R01 CA211614 (J.C.) and R01 DK124116 (J.C.), The Simms/Mann Family Foundation (J.C.) and The Margaret Early Medical Research Trust (R.S.). M.C. is supported by a Sloan Foundation Research Fellowship and a Human Cell Atlas Seed Network grant from the Chan Zuckerberg Initiative. C.H. is an investigator of the Howard Hughes Medical Institute. J.C. is a Leukemia & Lymphoma Society (LLS) Scholar.

Author information

Author notes

  1. These authors contributed equally: Lulu Hu, Shun Liu, Yong Peng, Ruiqi Ge, Rui Su.

  2. These authors jointly supervised this work: Chuan He, Jianjun Chen, Mengjie Chen, Lulu Hu.

Authors and Affiliations

  1. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    Lulu Hu, Shun Liu, Yong Peng, Ruiqi Ge, Bryan T. Harada, Qing Dai, Jiangbo Wei, Lisheng Zhang, Ziyang Hao, Liangzhi Luo, Huanyu Wang, Yuru Wang & Chuan He

  2. Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Lulu Hu, Shun Liu, Yong Peng, Ruiqi Ge, Bryan T. Harada, Qing Dai, Jiangbo Wei, Lisheng Zhang, Ziyang Hao, Liangzhi Luo, Huanyu Wang, Yuru Wang & Chuan He

  3. Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA

    Lulu Hu, Shun Liu, Yong Peng, Ruiqi Ge, Bryan T. Harada, Qing Dai, Jiangbo Wei, Lisheng Zhang, Ziyang Hao, Liangzhi Luo, Huanyu Wang, Yuru Wang & Chuan He

  4. Fudan University Institutes of Biomedical Sciences, Shanghai Cancer Center, Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism (Ministry of Science and Technology), Shanghai Medical College of Fudan University, Shanghai, China

    Lulu Hu

  5. Section of Genetic Medicine, Department of Medicine, The University of Chicago, Chicago, IL, USA

    Shun Liu, Yong Peng & Mengjie Chen

  6. Department of Human Genetics, The University of Chicago, Chicago, IL, USA

    Shun Liu, Yong Peng & Mengjie Chen

  7. Department of Systems Biology, Beckman Research Institute of City of Hope, Monrovia, CA, USA

    Rui Su & Jianjun Chen

  8. Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Chamara Senevirathne & Minkui Luo

  9. Program of Pharmacology, Weill Cornell Medical College of Cornell University, New York, NY, USA

    Minkui Luo

  10. City of Hope Comprehensive Cancer Center, City of Hope, Duarte, CA, USA

    Jianjun Chen

  11. Gehr Family Center for Leukemia Research, City of Hope, Duarte, CA, USA

    Jianjun Chen

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Contributions

L.H. and C.H. conceived the study. M.C. supervised the bioinformatic analysis. J.C. supervised the sample preparation for HSPC differentiation into monocytes. L.H. designed the experiments. S.L. and Y.P. performed the bioinformatic analysis. L.H. and R.G. prepared the libraries. R.S. prepared the samples for HSPC differentiation with J.C. C.S. synthesized the allyl-SAM cofactor under the supervision of M.L. B.T.H. edited the manuscript. Q.D. synthesized the RNA probes, a6A and a6m6A standards. J.W. and H.W. helped with RNA sample preparation. L.Z. helped with method design. Z.H. helped with cell culture. L.L. and Y.W. helped with FTO purification. L.H., S.L, Y.P., M.C. and C.H. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Lulu Hu, Mengjie Chen, Jianjun Chen or Chuan He.

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Competing interests

A patent application for m6A-SAC-seq has been filed by the University of Chicago. C.H. is a scientific founder and a scientific advisory board member of Accent Therapeutics, Inc., and Inferna Green, Inc. The remaining authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–14, Table 1 and descriptions of Supplementary Data 1–8.

Reporting Summary

Supplementary Data 1

m6A sites identified in cell lines and HSPC samples.

Supplementary Data 2.

m6A-modified gene transcripts during HSPC differentiation into monocytes.

Supplementary Data 3

Differentially expressed gene transcripts with m6A modifications during HSPC differentiation into monocytes.

Supplementary Data 4

Gene transcripts with significant changes in both m6A stoichiometry and transcript level during HSPC differentiation into monocytes.

Supplementary Data 5

m6A sites from HSPC samples overlapped with ENCODE RBP eCLIP peaks from the K562 cell line.

Supplementary Data 6

Differential AS events identified in HSPC samples during differentiation.

Supplementary Data 7

Differential AS events with significant changes of m6A stoichiometry during HSPC differentiation into monocytes.

Supplementary Data 8

Differential alternative splicing events identified in HSPC METTL3/METTL14 knockdown samples.

Supplementary Data 9

Unprocessed gel for Supplementary Fig. 1a.

Supplementary Data 10

Unprocessed gel for Supplementary Fig. 4a.

Supplementary Data 11

Unprocessed western blot for Supplementary Fig. 14e.

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Hu, L., Liu, S., Peng, Y. et al. m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome. Nat Biotechnol 40, 1210–1219 (2022). https://doi.org/10.1038/s41587-022-01243-z

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