Abstract
N6-methyladenosine (m6A), the most abundant internal messenger RNA modification in higher eukaryotes, serves myriad roles in regulating cellular processes. Functional dissection of m6A is, however, hampered in part by the lack of high-resolution and quantitative detection methods. Here we present evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology that detects and quantifies m6A by global adenosine deamination. With eTAM-seq, we analyze the transcriptome-wide distribution of m6A in HeLa and mouse embryonic stem cells. The enzymatic deamination route employed by eTAM-seq preserves RNA integrity, facilitating m6A detection from limited input samples. In addition to transcriptome-wide m6A profiling, we demonstrate site-specific, deep-sequencing-free m6A quantification with as few as ten cells, an input demand orders of magnitude lower than existing quantitative profiling methods. We envision that eTAM-seq will enable researchers to not only survey the m6A landscape at unprecedented resolution, but also detect m6A at user-specified loci with a simple workflow.
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Data availability
All eTAM-seq data have been deposited to the National Center for Biotechnology Information’s GEO and can be accessed through accession no. GSE201064.
Code availability
Codes for processing eTAM-seq data are available in the following GitHub repository (https://github.com/shunliubio/eTAM-seq_workflow).
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Acknowledgements
The authors thank K. M. Watters for scientific editing of the manuscript. We thank L. Yang and Y. Xiao for maintaining and characterizing Mettl3 cKO mESCs. We thank P. Faber of the University of Chicago Genomics Facility for sequencing support. Funding: C.H. is supported by the National Institutes of Health (NIH; grant no. RM1 HG008935) and is a Howard Hughes Medical Institute Investigator. M.C. is supported by the NIH (grant nos. R01 GM126553 and R01 HG011883), the National Science Foundation (grant no. NSF 2016307), the Sloan Research Fellowship Program and the Chan Zuckerberg Initiative. W.T. is supported by the Searle Scholars Program, a pilot award under grant no. RM1 HG008935.
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Contributions
Y.L.X. and W.T. conceived the project and designed the experiments. Y.L.X. screened deaminases, purified and characterized TadA8.20, and carried out site-specific m6A quantification experiments. S.L. performed all bioinformatic analyses. R.G. designed the workflow for eTAM-seq, optimized the protocol for generating IVT RNA and prepared the libraries. Y.W. assisted with enzyme purification and characterization as well as site-specific m6A quantification. Y.W. analyzed site-specific m6A quantification results. M.C. supervised bioinformatic analyses. C.H. and W.T. supervised the study. Y.L.X., C.H., M.C. and W.T. wrote the manuscript with input from all authors.
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Patent application no. 63/417,245 has been filed for eTAM-seq by the University of Chicago. C.H. is a scientific founder and a scientific advisory board member of Accent Therapeutics, Inc. and Aferna Bio, Inc. The remaining authors declare no competing interests.
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Nature Biotechnology thanks Rui Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Supplementary Notes 1–9, Tables 1–10 and Figs. 1–24.
Supplementary Table 1
TadA8.20-enabled A-to-I conversion in different sequence contexts as reported by nonmethylated RNA probes.
Supplementary Table 2
The sequencing and processing statistics of eTAM-seq libraries.
Supplementary Data 1
Unprocessed western blot for Supplementary Fig. 20a.
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Xiao, YL., Liu, S., Ge, R. et al. Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination. Nat Biotechnol 41, 993–1003 (2023). https://doi.org/10.1038/s41587-022-01587-6
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DOI: https://doi.org/10.1038/s41587-022-01587-6
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