A metabolic labeling method detects m6A transcriptome-wide at single base resolution

Abstract

Transcriptome-wide mapping of N6-methyladenosine (m6A) at base resolution remains an issue, impeding our understanding of m6A roles at the nucleotide level. Here, we report a metabolic labeling method to detect mRNA m6A transcriptome-wide at base resolution, called ‘m6A-label-seq’. Human and mouse cells could be fed with a methionine analog, Se-allyl-l-selenohomocysteine, which substitutes the methyl group on the enzyme cofactor SAM with the allyl. Cellular RNAs could therefore be metabolically modified with N6-allyladenosine (a6A) at supposed m6A-generating adenosine sites. We pinpointed the mRNA a6A locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. We identified a few thousand mRNA m6A sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of m6A-label-seq with available m6A sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered m6A sites and holds promise to locate nuclear nascent RNA m6A modifications.

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Fig. 1: The rationale of m6A-label-seq, a metabolic labeling method to pinpoint RNA m6A at base resolution.
Fig. 2: Establishment of an in vitro assay for identifying a6A in RNAs.
Fig. 3: Cellular labeling of mRNAs with a6A to substitute m6A under natural metabolism.
Fig. 4: High-throughput m6A-label-seq results for human HeLa and HEK293T and mouse H2.35 cell mRNAs.
Fig. 5: Comparison of m6A sites identified by m6A-label-seq and other high-throughput sequencing methods in HEK293T cells and validation of selected m6A-label-seq sites by an independent orthogonal method.

Data availability

The high-throughput sequencing data reported in this paper have been deposited in the Gene Expression Omnibus database at https://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE131316). All data supporting the findings are available in the paper and Supplementary Information files.

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Acknowledgements

We acknowledge support from the National Key Research and Development Program of China (2017YFA0506800), the National Natural Science Foundation of China (91853110, 21977087), the Fundamental Research Funds for the Central Universities, and Dabeinong Funds for Discipline Development and Talent Training in Zhejiang University. We thank B. Zhao at Life Sciences Institute of Zhejiang University for offering the H2.35 cell line. We thank L. Zhang at Shanghai Jiaotong University for providing FTO protein. We thank S.F. Reichard for editing the manuscript. C.H. is an investigator at the Howard Hughes Medical Institute.

Author information

Affiliations

Authors

Contributions

J.L. conceived the project. X.S. and J.C. designed and performed most experiments. J.C. worked on the bioinformatics analysis and Z.L., L.M. and X.C. offered guidance. M.C. and S.X. worked on the synthesis of Se-allyl-l-selenohomocysteine. Q.D., T.L. and X.Y. provided help with building in vitro biochemical assays. M.G. helped with subcloning. F.W. and Y.W. performed mass spectrometry measurements. J.L. supervised the project. J.L., X.S. and J.C. wrote the manuscript. Y.Y., C.H. and X.F. contributed to the manuscript. All authors commented on the manuscript.

Corresponding author

Correspondence to Jianzhao Liu.

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Competing interests

C.H. is a scientific founder of Accent Therapeutics and a member of its scientific advisory board. All other authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–22 and Tables 1–4

Reporting Summary

Supplementary Data Set 1:

The detailed information of a6A peaks from the a6A-RIP sequencing in HeLa cells.

Supplementary Data Set 2:

The detailed information of a6A peaks from the a6A-RIP sequencing in HEK293T cells.

Supplementary Data Set 3:

The detailed information of a6A peaks from the a6A-RIP sequencing in H2.35 cells.

Supplementary Data Set 4:

The detailed information of m6A peaks from the m6A-RIP sequencing in HEK293T cells

Supplementary Data Set 5:

The detailed information of m6A peaks from the m6A-RIP sequencing in H2.35 cells

Supplementary Data Set 6:

The detailed information of mutation sites called from m6A-label-seq in HeLa cells.

Supplementary Data Set 7:

The detailed information of mutation sites called from m6A-label-seq in HEK293T cells.

Supplementary Data Set 8

The detailed information of mutation sites called from m6A-label-seq in H2.35 cells.

Supplementary Data Set 9

Differential gene expression results in HeLa cells under methionine versus Se-derived analog.

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Shu, X., Cao, J., Cheng, M. et al. A metabolic labeling method detects m6A transcriptome-wide at single base resolution. Nat Chem Biol 16, 887–895 (2020). https://doi.org/10.1038/s41589-020-0526-9

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