Development and validation of monoclonal antibodies against N6-methyladenosine for the detection of RNA modifications

Autoři: Shun Matsuzawa aff001;  Yuka Wakata aff001;  Fumiya Ebi aff003;  Masaharu Isobe aff004;  Nobuyuki Kurosawa aff004
Působiště autorů: Graduate School of Innovative Life Science, University of Toyama, Toyama-shi, Toyama, Japan aff001;  Medical & Biological Laboratories Co., Ltd., Akaho, Komagane, Nagano, Japan aff002;  Graduate School of Science and Engineering for Education, University of Toyama, Gofuku, Toyama-shi, Toyama, Japan aff003;  Laboratory of Molecular and Cellular Biology, Faculty of Science and Engineering, Graduate School, University of Toyama, Gofuku, Toyama-shi, Toyama, Japan aff004
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223197


RNA contains various chemical modifications, among which N6-methyladenosine (m6A) is the most prevalent modified nucleotide in eukaryotic mRNA. Emerging evidence suggests that m6A plays an important role in regulating a variety of cellular functions by controlling mRNA processing, translation and degradation. Because m6A is not detectable by standard chemical modification-based approaches, immunological methods, such as ELISA, immunoblotting, immunohistochemistry, m6A RNA immunoprecipitation sequencing and m6A individual-nucleotide resolution cross-linking and immunoprecipitation, have been employed to detect m6A in RNA. Although the most important factor determining the success of these methods is the integrity of highly specific antibodies against m6A, the development of m6A-specific monoclonal antibodies has been challenging. We developed anti-m6A monoclonal antibodies using our recently developed single cell-based monoclonal antibody production system. The binding of one selected antibody, #B1-3, to RNA oligoribonucleotide containing a single m6A had an equilibrium dissociation constant of 6.5 nM, and this antibody exhibited negligible binding to oligoribonucleotides containing a single N1-methyladenosine and unmodified adenosine. The binding was competed by the addition of increasing concentrations of N6-methyl-ATP but not N1-methyl-ATP or ATP. Furthermore, this mAb specifically crosslinked m6A-containing oligoribonucleotide by ultraviolet light, resulting in the induction of cDNA truncation at m6A position. These results show the feasibility of using the validated m6A monoclonal antibody for the specific detection of m6A in RNA.

Klíčová slova:

Antibodies – Cell staining – Enzyme-linked immunoassays – Guinea pigs – Immunohistochemistry techniques – Immunoprecipitation – Monoclonal antibodies – RNA sequencing


1. Limbach PA, Crain PF, McCloskey JA (1994) Summary: the modified nucleosides of RNA. Nucleic Acids Res 22: 2183–2196. doi: 10.1093/nar/22.12.2183 7518580

2. Machnicka MA, Milanowska K, Osman Oglou O, Purta E, Kurkowska M, et al. (2013) MODOMICS: a database of RNA modification pathways—2013 update. Nucleic Acids Res 41: D262–267. doi: 10.1093/nar/gks1007 23118484

3. Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, et al. (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell 149: 1635–1646. doi: 10.1016/j.cell.2012.05.003 22608085

4. Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, et al. (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485: 201–206. doi: 10.1038/nature11112 22575960

5. Perry RP, Kelley DE, Friderici K, Rottman F (1975) The methylated constituents of L cell messenger RNA: evidence for an unusual cluster at the 5' terminus. Cell 4: 387–394. doi: 10.1016/0092-8674(75)90159-2 1168101

6. Desrosiers R, Friderici K, Rottman F (1974) Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc Natl Acad Sci U S A 71: 3971–3975. doi: 10.1073/pnas.71.10.3971 4372599

7. Adams JM, Cory S (1975) Modified nucleosides and bizarre 5'-termini in mouse myeloma mRNA. Nature 255: 28–33. doi: 10.1038/255028a0 1128665

8. Wei CM, Gershowitz A, Moss B (1975) Methylated nucleotides block 5' terminus of HeLa cell messenger RNA. Cell 4: 379–386. doi: 10.1016/0092-8674(75)90158-0 164293

9. Wang RY, Gehrke CW, Ehrlich M (1980) Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res 8: 4777–4790. doi: 10.1093/nar/8.20.4777 7443525

10. Wei CM, Moss B (1977) Nucleotide sequences at the N6-methyladenosine sites of HeLa cell messenger ribonucleic acid. Biochemistry 16: 1672–1676. doi: 10.1021/bi00627a023 856255

11. Yang Y, Hsu PJ, Chen YS, Yang YG (2018) Dynamic transcriptomic m(6)A decoration: writers, erasers, readers and functions in RNA metabolism. Cell Res 28: 616–624. doi: 10.1038/s41422-018-0040-8 29789545

12. Louloupi A, Ntini E, Conrad T, Orom UAV (2018) Transient N-6-Methyladenosine Transcriptome Sequencing Reveals a Regulatory Role of m6A in Splicing Efficiency. Cell Rep 23: 3429–3437. doi: 10.1016/j.celrep.2018.05.077 29924987

13. Slobodin B, Han R, Calderone V, Vrielink J, Loayza-Puch F, et al. (2017) Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation. Cell 169: 326–337 e312. doi: 10.1016/j.cell.2017.03.031 28388414

14. Xiao W, Adhikari S, Dahal U, Chen YS, Hao YJ, et al. (2016) Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Mol Cell 61: 507–519. doi: 10.1016/j.molcel.2016.01.012 26876937

15. Wang X, Lu Z, Gomez A, Hon GC, Yue Y, et al. (2014) N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505: 117–120. doi: 10.1038/nature12730 24284625

16. Feederle R, Schepers A (2017) Antibodies specific for nucleic acid modifications. RNA Biol 14: 1089–1098. doi: 10.1080/15476286.2017.1295905 28277931

17. Bringmann P, Luhrmann R (1987) Antibodies specific for N6-methyladenosine react with intact snRNPs U2 and U4/U6. FEBS Lett 213: 309–315. doi: 10.1016/0014-5793(87)81512-0 2951275

18. Munns TW, Liszewski MK, Sims HF (1977) Characterization of antibodies specific for N6-methyladenosine and for 7-methylguanosine. Biochemistry 16: 2163–2168. doi: 10.1021/bi00629a019 861202

19. Kurosawa N, Wakata Y, Inobe T, Kitamura H, Yoshioka M, et al. (2016) Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies. Sci Rep 6: 25174. doi: 10.1038/srep25174 27125496

20. Kurosawa N, Yoshioka M, Fujimoto R, Yamagishi F, Isobe M (2012) Rapid production of antigen-specific monoclonal antibodies from a variety of animals. BMC Biol 10: 80. doi: 10.1186/1741-7007-10-80 23017270

21. Kurosawa N, Yoshioka M, Isobe M (2011) Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells. BMC Biotechnol 11: 39. doi: 10.1186/1472-6750-11-39 21486488

22. Yoshioka M, Kurosawa N, Isobe M (2011) Target-selective joint polymerase chain reaction: a robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells. BMC Biotechnol 11: 75. doi: 10.1186/1472-6750-11-75 21774833

23. Potapov V, Fu X, Dai N, Correa IR, Jr., Tanner NA, et al. (2018) Base modifications affecting RNA polymerase and reverse transcriptase fidelity. Nucleic Acids Res 46: 5753–5763. doi: 10.1093/nar/gky341 29750267

24. Picelli S, Bjorklund AK, Faridani OR, Sagasser S, Winberg G, et al. (2013) Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10: 1096–1098. doi: 10.1038/nmeth.2639 24056875

25. Zhao BS, He C (2015) Pseudouridine in a new era of RNA modifications. Cell Res 25: 153–154. doi: 10.1038/cr.2014.143 25367125

26. Ge J, Yu YT (2013) RNA pseudouridylation: new insights into an old modification. Trends Biochem Sci 38: 210–218. doi: 10.1016/j.tibs.2013.01.002 23391857

27. Linder B, Grozhik AV, Olarerin-George AO, Meydan C, Mason CE, et al. (2015) Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 12: 767–772. doi: 10.1038/nmeth.3453 26121403

Článek vyšel v časopise


2019 Číslo 10