An experimental workflow for identifying RNA m6A alterations in cellular senescence by methylated RNA immunoprecipitation sequencing

Main Article Content

Yue Shi
Zeming Wu
Weiqi Zhang
Jing Qu
Weimin Ci
Guang-Hui Liu


m6A, cellular senescence, meRIP-seq, bioinformatic analysis, qPCR


N6-methyladenosine (m6A), the most prevalent mRNA modification in eukaryotic cells, is known to play regulatory roles in a wide array of biological processes, including aging and cellular senescence. To investigate such roles, the m6A modification can be identified across the entire transcriptome by immunoprecipitation of methylated RNA with an anti-m6A antibody, followed by high-throughput sequencing (meRIP-seq or m6A-seq). Presented here is a protocol for employing meRIP-seq to profile the RNA m6A landscape in senescent human cells. We described, in detail, sample preparation, mRNA isolation, immunoprecipitation, library preparation, sequencing, bioinformatic analysis and validation. We also provided tips and considerations for the optimization and interpretation of the results. Our protocol serves as a methodological resource for investigating transcriptomic m6A alterations in cellular senescence as well as a valuable paradigm for the validation of genes of interest.


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1. Di Micco R, Krizhanovsky V, Baker D, d'Adda di Fagagna F. Cellular senescence in ageing: from mechanisms to therapeutic opportunities. Nat Rev Mol Cell Biol. 2021;22(2):75-95. Epub 2020/12/18. PMID: 33328614
2. López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. Hallmarks of aging: An expanding universe. Cell. 2023;186(2):243-78. Epub 2023/01/05. PMID:36599349
3. Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O, Bishop C, et al. Cellular Senescence: Defining a Path Forward. Cell. 2019;179(4):813-27. Epub 2019/11/02. PMID:31675495
4. Liu B, Qu J, Zhang W, Izpisua Belmonte JC, Liu GH. A stem cell aging framework, from mechanisms to interventions. Cell Rep. 2022;41(3):111451. Epub 2022/10/20. PMID:36261013
5. Cai Y, Ji Z, Wang S, Zhang W, Qu J, Belmonte JC, et al. Genetic Enhancement: a New Avenue to Combat Aging-related Diseases. Life Med. 2022;1(3):307–18.
6. Sun Y, Li Q, Kirkland JL. Targeting senescent cells for a healthier longevity: the roadmap for an era of global aging. Life Med. 2022;1(2):103–19. PMID:36699942
7. Cai Y, Song W, Li J, Jing Y, Liang C, Zhang L, et al. The landscape of aging. Sci China Life Sci. 2022;65(12):2354-454. Epub 2022/09/07. PMID: 36066811
8. Yang Y, Li X, Zhang T, Xu D. RIP kinases and necroptosis in aging and aging-related diseases. LifeMed. 2022;1(1):2-20.
9. Wu Z, Wang S, Belmonte JC, Zhang W, Qu J, Liu GH. Emerging role of RNA m6A modification in aging regulation. Curr Med (Cham). 2022;1(1):8.
10. Wang K, Liu H, Hu Q, Wang L, Liu J, Zheng Z, et al. Epigenetic regulation of aging: implications for interventions of aging and diseases. Signal Transduct Target Ther. 2022;7(1):374. Epub 2022/11/07. PMID: 36336680
11. Roignant JY, Soller M. m(6)A in mRNA: An Ancient Mechanism for Fine-Tuning Gene Expression. Trends Genet. 2017;33(6):380-90. Epub 2017/05/14. PMID:28499622
12. Niu Y, Zhao X, Wu YS, Li MM, Wang XJ, Yang YG. N6-methyl-adenosine (m6A) in RNA: an old modification with a novel epigenetic function. Genomics Proteomics Bioinformatics. 2013;11(1):8-17. Epub 2013/03/05. 23453015
13. Deng X, Su R, Weng H, Huang H, Li Z, Chen J. RNA N(6)-methyladenosine modification in cancers: current status and perspectives. Cell Res. 2018;28(5):507-17. PMID: 29686311
14. Wu Z, Shi Y, Lu M, Song M, Yu Z, Wang J, et al. METTL3 counteracts premature aging via m6A-dependent stabilization of MIS12 mRNA. Nucleic Acids Res. 2020;48(19):11083-96. Epub 2020/10/10. 33035345
15. Wu Z, Lu M, Liu D, Shi Y, Ren J, Wang S, et al. m6A epitranscriptomic regulation of tissue homeostasis during primate aging. Nat Aging. 2023;3(6):705–21. PMID:37118553
16. Li Q, Li X, Tang H, Jiang B, Dou Y, Gorospe M, et al. NSUN2-Mediated m5C Methylation and METTL3/METTL14-Mediated m6A Methylation Cooperatively Enhance p21 Translation. J Cell Biochem. 2017;118(9):2587-98. Epub 2017/03/02. PMID: 28247949
17. Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012;485(7397):2016. Epub 2012/05/12. PMID:22575960.
18. Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell. 2012;149(7):1635-46. Epub 2012/05/23. PMID: 22608085
19. Li X, Xiong X, Yi C. Epitranscriptome sequencing technologies: decoding RNA modifications. Nat Methods. 2016;14(1):23–31. PMID:28032622
20. Song J, Yi C. Chemical Modifications to RNA: A New Layer of Gene Expression Regulation. ACS Chem Biol. 2017;12(2):316-25. Epub 2017/01/05. PMID:28051309
21. Min KW, Zealy RW, Davila S, Fomin M, Cummings JC, Makowsky D, et al. Profiling of m6A RNA modifications identified an age-associated regulation of AGO2 mRNA stability. Aging Cell. 2018;17(3):e12753. Epub 2018/03/25. PMID: 29573145
22. Zhang W, Qu J, Liu GH, Belmonte JC. The ageing epigenome and its rejuvenation. Nat Rev Mol Cell Biol. 2020;21(3):137–50. PMID:32020082
23. Liu X, Liu Z, Wu Z, Ren J, Fan Y, Sun L, et al. Resurrection of endogenous retroviruses during aging reinforces senescence. Cell. 2023;186(2):287-304.e26. Epub 2023/01/08. PMID:36610399
24. Zhao D, Chen S. Failures at every level: breakdown of the epigenetic machinery of aging. LifeMed. 2022;1(2):81-3.
25. Zhu J, An Y, Wang X, Huang L, Kong W, Gao M, et al. The natural product rotundic acid treats both aging and obesity by inhibiting PTP1B. LifeMed. 2022;1(3):372-86. PMID:36811113
26. Zhou S, Liu L, Lu X. Endogenous retroviruses make aging go viral. Life Med. 2023;2(1):lnad001. 27. Liu C, Sun H, Yi Y, Shen W, Li K, Xiao Y, et al. Absolute quantification of single-base m(6)A methylation in the mammalian transcriptome using GLORI. Nat Biotechnol. 2023;41(3):355-66. Epub 2022/10/28. PMID:36302990
28. Hu L, Liu S, Peng Y, Ge R, Su R, Senevirathne C, et al. m(6)A RNA modifications are measured at single-base resolution across the mammalian transcriptome. Nat Biotechnol. 2022;40(8):1210-9. Epub 2022/03/16. PMID: 35288668
29. Luo H, Liu W, Zhang Y, Yang Y, Jiang X, Wu S, et al. METTL3-mediated m(6)A modification regulates cell cycle progression of dental pulp stem cells. Stem Cell Res Ther. 2021;12(1):159. Epub 2021/03/03. PMID: 33648590
30. Zhang J, Ao Y, Zhang Z, Mo Y, Peng L, Jiang Y, et al. Lamin A safeguards the m(6) A methylase METTL14 nuclear speckle reservoir to prevent cellular senescence. Aging Cell. 2020;19(10):e13215. Epub 2020/08/20. PMID: 32813328
31. Zhang S, Wu Z, Shi Y, Wang S, Ren J, Yu Z, et al. FTO stabilizes MIS12 and counteracts senescence. Protein Cell. 2022; 13(12);954-60. PMID:35384602
32. Yankova E, Blackaby W, Albertella M, Rak J, De Braekeleer E, Tsagkogeorga G, et al. Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature. 2021;593(7860):597-601. Epub 2021/04/27. PMID: 33902106
33. Huang Y, Su R, Sheng Y, Dong L, Dong Z, Xu H, et al. Small-Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid Leukemia. Cancer Cell. 2019;35(4):677-91.e10. Epub 2019/04/17. PMID: 30991027
34. Wei X, Huo Y, Pi J, Gao Y, Rao S, He M, et al. METTL3 preferentially enhances non-m(6)A translation of epigenetic factors and promotes tumourigenesis. Nat Cell Biol. 2022;24(8):1278-90. Epub 2022/08/05. PMID:35927451
35. Huang H, Weng H, Sun W, Qin X, Shi H, Wu H, et al. Recognition of RNA N(6)-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat Cell Biol. 2018;20(3):285-95. PMID: 29476152
36. Bai Y, Li K, Peng J, Yi C. m 6A modification: a new avenue for anti-cancer therapy. Life Med. 2023;2(1):Inad008.