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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All eTAM-seq data have been deposited to the National Center for Biotechnology Information’s GEO and can be accessed through accession no. GSE201064.
Codes for processing eTAM-seq data are available in the following GitHub repository (https://github.com/shunliubio/eTAM-seq_workflow).
Frye, M., Jaffrey, S. R., Pan, T., Rechavi, G. & Suzuki, T. RNA modifications: what have we learned and where are we headed? Nat. Rev. Genet. 17, 365–372 (2016).
Peer, E., Rechavi, G. & Dominissini, D. Epitranscriptomics: regulation of mRNA metabolism through modifications. Curr. Opin. Chem. Biol. 41, 93–98 (2017).
Nachtergaele, S. & He, C. Chemical modifications in the life of an mRNA transcript. Annu. Rev. Genet. 52, 349–372 (2018).
Jiang, X. et al. The role of m6A modification in the biological functions and diseases. Signal Transduct. Target. Ther. 6, 74 (2021).
He, P. C. & He, C. m6A RNA methylation: from mechanisms to therapeutic potential. EMBO J. 40, e105977 (2021).
Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).
Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646 (2012).
Linder, B. et al. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods 12, 767–772 (2015).
Garcia-Campos, M. A. et al. Deciphering the ‘m6A code’ via antibody-independent quantitative profiling. Cell 178, 731–747 (2019).
Zhang, Z. et al. Single-base mapping of m6A by an antibody-independent method. Sci. Adv. 5, eaax0250 (2019).
Wang, Y., Xiao, Y., Dong, S., Yu, Q. & Jia, G. Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine. Nat. Chem. Biol. 16, 896–903 (2020).
Shu, X. et al. A metabolic labeling method detects m6A transcriptome-wide at single base resolution. Nat. Chem. Biol. 16, 887–895 (2020).
Meyer, K. D. DART-seq: an antibody-free method for global m6A detection. Nat. Methods 16, 1275–1280 (2019).
Hu, L. et al. m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome. Nat. Biotechnol. 40, 1210–1219 (2022).
Liu, N. et al. Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA. RNA 19, 1848–1856 (2013).
Xiao, Y. et al. An elongation- and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N6-methyladenosine modification. Angew. Chem. Int. Ed. 57, 15995–16000 (2018).
Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl Acad. Sci. USA 89, 1827–1831 (1992).
Walkley, C. R. & Li, J. B. Rewriting the transcriptome: adenosine-to-inosine RNA editing by ADARs. Genome Biol. 18, 205 (2017).
Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat. Biotechnol. 38, 892–900 (2020).
Grunewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).
Kint, S., De Spiegelaere, W., De Kesel, J., Vandekerckhove, L. & Van Criekinge, W. Evaluation of bisulfite kits for DNA methylation profiling in terms of DNA fragmentation and DNA recovery using digital PCR. PLoS ONE 13, e0199091 (2018).
Benjamini, Y. & Speed, T. P. Summarizing and correcting the GC content bias in high-throughput sequencing. Nucleic Acids Res. 40, e72 (2012).
Liu, J. et al. A METTL3–METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 10, 93–95 (2014).
Hussain, S., Aleksic, J., Blanco, S., Dietmann, S. & Frye, M. Characterizing 5-methylcytosine in the mammalian epitranscriptome. Genome Biol. 14, 215 (2013).
Zhang, Z. et al. Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library. Nat. Methods 18, 1213–1222 (2021).
Piekna-Przybylska, D., Decatur, W. A. & Fournier, M. J. The 3D rRNA modification maps database: with interactive tools for ribosome analysis. Nucleic Acids Res. 36, D178–D183 (2008).
Herbert, Z. T. et al. Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction. BMC Genomics 19, 199 (2018).
Maden, B. E. Identification of the locations of the methyl groups in 18S ribosomal RNA from Xenopus laevis and man. J. Mol. Biol. 189, 681–699 (1986).
van Tran, N. et al. The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112. Nucleic Acids Res. 47, 7719–7733 (2019).
Maden, B. E. Locations of methyl groups in 28S rRNA of Xenopus laevis and man. Clustering in the conserved core of molecule. J. Mol. Biol. 201, 289–314 (1988).
Ma, H. et al. N6-Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation. Nat. Chem. Biol. 15, 88–94 (2019).
Poldermans, B., Roza, L. & Van Knippenberg, P.H. Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3′ end of 16S ribosomal RNA of Escherichia coli. III. Purification and properties of the methylating enzyme and methylase-30 S interactions. J. Biol. Chem. 254, 9094–9100 (1979).
Lafontaine, D., Vandenhaute, J. & Tollervey, D. The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast. Genes Dev. 9, 2470–2481 (1995).
Zorbas, C. et al. The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis. Mol. Biol. Cell 26, 2080–2095 (2015).
Wei, C., Gershowitz, A. & Moss, B. N6, O2′-dimethyladenosine a novel methylated ribonucleoside next to the 5′ terminal of animal cell and virus mRNAs. Nature 257, 251–253 (1975).
Wei, J. et al. Differential m6A, m6Am, and m1A demethylation mediated by FTO in the cell nucleus and cytoplasm. Mol. Cell 71, 973–985 (2018).
Wang, X. et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).
McIntyre, A. B. R. et al. Limits in the detection of m6A changes using MeRIP/m6A-seq. Sci. Rep. 10, 6590 (2020).
Ge, R. et al. m6A-SAC-seq for quantitative whole transcriptome m6A profiling. Nat. Protoc. in press (2022).
Geula, S. et al. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science 347, 1002–1006 (2015).
Wang, Y. et al. N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat. Cell Biol. 16, 191–198 (2014).
Batista, P. J. et al. m6A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15, 707–719 (2014).
Takahashi, K. & Yamanaka, S. A decade of transcription factor-mediated reprogramming to pluripotency. Nat. Rev. Mol. Cell Biol. 17, 183–193 (2016).
Lee, Y., Choe, J., Park, O. H. & Kim, Y. K. Molecular mechanisms driving mRNA degradation by m6A modification. Trends Genet. 36, 177–188 (2020).
Wang, X. et al. N6-methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).
Shi, H. et al. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA. Cell Res. 27, 315–328 (2017).
Shi, H., Wei, J. & He, C. Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers. Mol. Cell 74, 640–650 (2019).
Kluesner, M. G. et al. EditR: a method to quantify base editing from Sanger sequencing. CRISPR J. 1, 239–250 (2018).
Schaefer, M., Pollex, T., Hanna, K. & Lyko, F. RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res. 37, e12 (2009).
Liu, N. et al. N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature 518, 560–564 (2015).
Spitale, R. C. et al. Structural imprints in vivo decode RNA regulatory mechanisms. Nature 519, 486–490 (2015).
Liu, J. et al. N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science 367, 580–586 (2020).
Hagemann-Jensen, M. et al. Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nat. Biotechnol. 38, 708–714 (2020).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 3 (2011).
Smith, T., Heger, A. & Sudbery, I. UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy. Genome Res. 27, 491–499 (2017).
Zhang, Y., Park, C., Bennett, C., Thornton, M. & Kim, D. Rapid and accurate alignment of nucleotide conversion sequencing reads with HISAT-3N. Genome Res. 31, 1290–1295 (2021).
Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Ramaswami, G. et al. Accurate identification of human Alu and non-Alu RNA editing sites. Nat. Methods 9, 579–581 (2012).
Lo Giudice, C., Tangaro, M. A., Pesole, G. & Picardi, E. Investigating RNA editing in deep transcriptome datasets with REDItools and REDIportal. Nat. Protoc. 15, 1098–1131 (2020).
Cuddleston, W. H. et al. Cellular and genetic drivers of RNA editing variation in the human brain. Nat. Commun. 13, 2997 (2022).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013).
Chen, C. Y., Ezzeddine, N. & Shyu, A. B. Messenger RNA half-life measurements in mammalian cells. Methods Enzymol. 448, 335–357 (2008).
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.
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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Supplementary Notes 1–9, Tables 1–10 and Figs. 1–24.
TadA8.20-enabled A-to-I conversion in different sequence contexts as reported by nonmethylated RNA probes.
The sequencing and processing statistics of eTAM-seq libraries.
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 (2023). https://doi.org/10.1038/s41587-022-01587-6