Abstract
RNA bisulfite sequencing (RNA-BS-seq) represents a method for the detection of methylated cytosines in RNA. Developed originally for the analysis of DNA methylation, a modified version of this method can be used for the analysis of methylated cytosine in RNA. Treatment of nucleic acids with HSO3-ions under acidic conditions results in deamination of cytosine (C) to uracil, while 5-methylcytosine (m5C) or 5-hydroxymethylcytosine (hm5C) exhibit low reactivity in this reaction and remain unchanged. Subsequent PCR amplification and sequencing of specific targets allows for the assessment of the methylation status of single Cs in their native sequence context at nucleotide resolution. Here, we describe the application of this method for the analysis of cytosine methylation in low abundance poly(A)RNA using a combination of commercially available kits and standard lab methods to ensure reproducible results. Furthermore, useful information on optimizing the method, suitable controls for almost all steps, and general troubleshooting guides are provided.
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References
Grosjean H (2009) Nucleic acids are not boring long polymers of only four types of nucleotides: a guided tour. In: Grosjean H (ed) DNA and RNA modification enzymes: structure, mechanism, function and evolution. LandesBioscience, Austin
Motorin Y, Lyko F, Helm M (2010) 5-methylcytosine in RNA: detection, enzymatic formation and biological functions. Nucleic Acids Res 38:1415–1430. doi:10.1093/nar/gkp1117
Carell T, Brandmayr C, Hienzsch A et al (2012) Structure and function of noncanonical nucleobases. Angew Chem Int Ed Engl 51:7110–7131. doi:10.1002/anie.201201193
Cowling VH (2010) Regulation of mRNA cap methylation. Biochem J 425:295–302. doi:10.1042/BJ20091352
Dominissini D, Moshitch-Moshkovitz S, Schwartz S et al (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485:201–206. doi:10.1038/nature11112
Meyer KD, Saletore Y, Zumbo P 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
Squires JE, Patel HR, Nousch M et al (2012) Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res 40:5023–5033. doi:10.1093/nar/gks144
Khoddami V, Cairns BR (2013) Identification of direct targets and modified bases of RNA cytosine methyltransferases. Nat Biotechnol 31:458–464. doi:10.1038/nbt.2566
Hussain S, Sajini AA, Blanco S et al (2013) NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs. Cell Rep 4:255–261. doi:10.1016/j.celrep.2013.06.029
Carlile TM, Rojas-Duran MF, Zinshteyn B et al (2014) Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature. doi:10.1038/nature13802
Frommer M, McDonald LE, Millar DS (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Curr Issue 89(5):1827–1831
Schaefer M, Pollex T, Hanna K, Lyko F (2009) RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res 37:e12. doi:10.1093/nar/gkn954
Shapiro R, Servis RE, Welcher M (1970) Reactions of uracil and cytosine derivatives with sodium bisulfite. J Am Chem Soc 92(2):422–424
Hayatsu H, Wataya Y, Kai K, Iida S (1970) Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry 9:2858–2865
Amort T, Soulière MF, Wille A et al (2013) Long non-coding RNAs as targets for cytosine methylation. RNA Biol 10:1003–1008. doi:10.4161/rna.24454
Hayatsu H, Shiragami M (1979) Reaction of bisulfite with the 5-hydroxymethyl group in pyrimidines and in phage DNAs. Biochemistry 18:632–637
Schaefer M, Hagemann S, Hanna K, Lyko F (2009) Azacytidine inhibits RNA methylation at DNMT2 target sites in human cancer cell lines. Cancer Res 69:8127–8132. doi:10.1158/0008-5472.CAN-09-0458
Blanco S, Dietmann S, Flores JV et al (2014) Aberrant methylation of tRNAs links cellular stress to neuro-developmental disorders. EMBO J 33:2020–2039. doi:10.15252/embj.201489282
Acknowledgment
The research was funded by the Austrian Science Fund (FWF): P27024-BBL.
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Amort, T., Lusser, A. (2017). Detection of 5-Methylcytosine in Specific Poly(A) RNAs by Bisulfite Sequencing. In: Lusser, A. (eds) RNA Methylation. Methods in Molecular Biology, vol 1562. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6807-7_8
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DOI: https://doi.org/10.1007/978-1-4939-6807-7_8
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