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Target-Specific Profiling of RNA m5C Methylation Level Using Amplicon Sequencing

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Post-Transcriptional Gene Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2404))

Abstract

Mapping the position and quantifying the level of 5-methylcytosine (m5C) as a modification in different types of cellular RNA is an important objective in the field of epitranscriptomics. Bisulfite conversion has long been the gold standard for the detection of m5C in DNA, but it can also be applied to RNA. Here, we detail methods for bisulfite treatment of RNA, locus-specific PCR amplification, and detection of candidate sites by sequencing on the Illumina MiSeq platform.

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References

  1. Cantara WA, Crain PF, Rozenski J, McCloskey JA, Harris KA, Zhang X, Vendeix FA, Fabris D, Agris PF (2011) The RNA modification database, RNAMDB: 2011 update. Nucleic Acids Res 39(Database issue):D195–D201. https://doi.org/10.1093/nar/gkq1028

    Article  CAS  PubMed  Google Scholar 

  2. Machnicka MA, Milanowska K, Osman Oglou O, Purta E, Kurkowska M, Olchowik A, Januszewski W, Kalinowski S, Dunin-Horkawicz S, Rother KM, Helm M, Bujnicki JM, Grosjean H (2013) MODOMICS: a database of RNA modification pathways--2013 update. Nucleic Acids Res 41(Database issue):D262–D267. https://doi.org/10.1093/nar/gks1007

    Article  CAS  PubMed  Google Scholar 

  3. Milanowska K, Mikolajczak K, Lukasik A, Skorupski M, Balcer Z, Machnicka MA, Nowacka M, Rother KM, Bujnicki JM (2013) RNApathwaysDB--a database of RNA maturation and decay pathways. Nucleic Acids Res 41(Database issue):D268–D272. https://doi.org/10.1093/nar/gks1052

    Article  CAS  PubMed  Google Scholar 

  4. Fu Y, He C (2012) Nucleic acid modifications with epigenetic significance. Curr Opin Chem Biol 16(5–6):516–524. https://doi.org/10.1016/j.cbpa.2012.10.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Saletore Y, Meyer K, Korlach J, Vilfan ID, Jaffrey S, Mason CE (2012) The birth of the Epitranscriptome: deciphering the function of RNA modifications. Genome Biol 13(10):175. https://doi.org/10.1186/gb-2012-13-10-175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sibbritt T, Patel HR, Preiss T (2013) Mapping and significance of the mRNA methylome. Wiley Interdiscip Rev RNA 4(4):397–422. https://doi.org/10.1002/wrna.1166

    Article  CAS  PubMed  Google Scholar 

  7. Amort T, Rieder D, Wille A, Khokhlova-Cubberley D, Riml C, Trixl L, Jia XY, Micura R, Lusser A (2017) Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain. Genome Biol 18(1):1. https://doi.org/10.1186/s13059-016-1139-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T (2012) Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res 40(11):5023–5033. https://doi.org/10.1093/nar/gks144

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Fu L, Guerrero CR, Zhong N, Amato NJ, Liu Y, Liu S, Cai Q, Ji D, Jin SG, Niedernhofer LJ, Pfeifer GP, Xu GL, Wang Y (2014) Tet-mediated formation of 5-hydroxymethylcytosine in RNA. J Am Chem Soc 136(33):11582–11585. https://doi.org/10.1021/ja505305z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Huber SM, van Delft P, Mendil L, Bachman M, Smollett K, Werner F, Miska EA, Balasubramanian S (2015) Formation and abundance of 5-hydroxymethylcytosine in RNA. Chembiochem 16(5):752–755. https://doi.org/10.1002/cbic.201500013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, Sorek R, Rechavi G (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485(7397):201–206. https://doi.org/10.1038/nature11112

    Article  CAS  PubMed  Google Scholar 

  12. Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell 149(7):1635–1646. https://doi.org/10.1016/j.cell.2012.05.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Schwartz S, Mumbach MR, Jovanovic M, Wang T, Maciag K, Bushkin GG, Mertins P, Ter-Ovanesyan D, Habib N, Cacchiarelli D, Sanjana NE, Freinkman E, Pacold ME, Satija R, Mikkelsen TS, Hacohen N, Zhang F, Carr SA, Lander ES, Regev A (2014) Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites. Cell Rep 8(1):284–296. https://doi.org/10.1016/j.celrep.2014.05.048

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bar-Yaacov D, Frumkin I, Yashiro Y, Chujo T, Ishigami Y, Chemla Y, Blumberg A, Schlesinger O, Bieri P, Greber B, Ban N, Zarivach R, Alfonta L, Pilpel Y, Suzuki T, Mishmar D (2016) Mitochondrial 16S rRNA is methylated by tRNA methyltransferase TRMT61B in all vertebrates. PLoS Biol 14(9):e1002557. https://doi.org/10.1371/journal.pbio.1002557

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dominissini D, Nachtergaele S, Moshitch-Moshkovitz S, Peer E, Kol N, Ben-Haim MS, Dai Q, Di Segni A, Salmon-Divon M, Clark WC, Zheng G, Pan T, Solomon O, Eyal E, Hershkovitz V, Han D, Dore LC, Amariglio N, Rechavi G, He C (2016) The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA. Nature 530(7591):441–446. https://doi.org/10.1038/nature16998

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hauenschild R, Tserovski L, Schmid K, Thuring K, Winz ML, Sharma S, Entian KD, Wacheul L, Lafontaine DL, Anderson J, Alfonzo J, Hildebrandt A, Jaschke A, Motorin Y, Helm M (2015) The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent. Nucleic Acids Res 43(20):9950–9964. https://doi.org/10.1093/nar/gkv895

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Carlile TM, Rojas-Duran MF, Zinshteyn B, Shin H, Bartoli KM, Gilbert WV (2014) Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature 515(7525):143–146. https://doi.org/10.1038/nature13802

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lovejoy AF, Riordan DP, Brown PO (2014) Transcriptome-wide mapping of pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae. PLoS One 9(10):e110799. https://doi.org/10.1371/journal.pone.0110799

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Schwartz S, Bernstein DA, Mumbach MR, Jovanovic M, Herbst RH, Leon-Ricardo BX, Engreitz JM, Guttman M, Satija R, Lander ES, Fink G, Regev A (2014) Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159(1):148–162. https://doi.org/10.1016/j.cell.2014.08.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Edelheit S, Schwartz S, Mumbach MR, Wurtzel O, Sorek R (2013) Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs. PLoS Genet 9(6):e1003602. https://doi.org/10.1371/journal.pgen.1003602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Khoddami V, Cairns BR (2013) Identification of direct targets and modified bases of RNA cytosine methyltransferases. Nat Biotechnol 31(5):458–464. https://doi.org/10.1038/nbt.2566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hussain S, Sajini AA, Blanco S, Dietmann S, Lombard P, Sugimoto Y, Paramor M, Gleeson JG, Odom DT, Ule J, Frye M (2013) NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs. Cell Rep 4(2):255–261. https://doi.org/10.1016/j.celrep.2013.06.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gu W, Hurto RL, Hopper AK, Grayhack EJ, Phizicky EM (2005) Depletion of Saccharomyces cerevisiae tRNA(His) guanylyltransferase Thg1p leads to uncharged tRNAHis with additional m(5)C. Mol Cell Biol 25(18):8191–8201. https://doi.org/10.1128/MCB.25.18.8191-8201.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pollex T, Hanna K, Schaefer M (2010) Detection of cytosine methylation in RNA using bisulfite sequencing. Cold Spring Harb Protoc 2010(10):pdb prot5505. https://doi.org/10.1101/pdb.prot5505

    Article  PubMed  Google Scholar 

  25. Schaefer M, Pollex T, Hanna K, Lyko F (2009) RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res 37(2):e12. https://doi.org/10.1093/nar/gkn954

    Article  CAS  PubMed  Google Scholar 

  26. Legrand C, Tuorto F, Hartmann M, Liebers R, Jacob D, Helm M, Lyko F (2017) Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs. Genome Res 27(9):1589–1596. https://doi.org/10.1101/gr.210666.116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Huang T, Chen W, Liu J, Gu N, Zhang R (2019) Genome-wide identification of mRNA 5-methylcytosine in mammals. Nat Struct Mol Biol 26(5):380–388. https://doi.org/10.1038/s41594-019-0218-x

    Article  CAS  PubMed  Google Scholar 

  28. Yang Y, Wang L, Han X, Yang WL, Zhang M, Ma HL, Sun BF, Li A, Xia J, Chen J, Heng J, Wu B, Chen YS, Xu JW, Yang X, Yao H, Sun J, Lyu C, Wang HL, Huang Y, Sun YP, Zhao YL, Meng A, Ma J, Liu F, Yang YG (2019) RNA 5-methylcytosine facilitates the maternal-to-zygotic transition by preventing maternal mRNA decay. Mol Cell 75(6):1188–1202 e1111. https://doi.org/10.1016/j.molcel.2019.06.033

    Article  CAS  PubMed  Google Scholar 

  29. Schumann U, Zhang HN, Sibbritt T, Pan A, Horvath A, Gross S, Clark SJ, Yang L, Preiss T (2020) Multiple links between 5-methylcytosine content of mRNA and translation. BMC Biol 18(1):40. https://doi.org/10.1186/s12915-020-00769-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Tusnady GE, Simon I, Varadi A, Aranyi T (2005) BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes. Nucleic Acids Res 33(1):e9. https://doi.org/10.1093/nar/gni012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17(1):3. https://doi.org/10.14806/ej.17.1.200

    Article  Google Scholar 

  32. Rieder D, Amort T, Kugler E, Lusser A, Trajanoski Z (2016) meRanTK: methylated RNA analysis ToolKit. Bioinformatics 32(5):782–785. https://doi.org/10.1093/bioinformatics/btv647

    Article  CAS  PubMed  Google Scholar 

  33. Krueger F, Andrews SR (2011) Bismark: a flexible aligner and methylation caller for bisulfite-Seq applications. Bioinformatics 27(11):1571–1572. https://doi.org/10.1093/bioinformatics/btr167

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We thank Wenjia Qu for helpful suggestions for the MiSeq library preparation protocol. This work was supported by an NHMRC grant (APP1061551) and a senior research fellowship (514904) awarded to TP.

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Author notes

  1. Tennille Sibbritt and Ulrike Schumann contributed equally to this work.

Authors and Affiliations

  1. Department of Genome Sciences, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia

    Tennille Sibbritt, Ulrike Schumann, Andrew Shafik, Marco Guarnacci & Thomas Preiss

  2. Epigenetics Research Laboratory, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, NSW, Australia

    Susan J. Clark

  3. St. Vincent Clinical School, University of New South Wales, Sydney, NSW, Australia

    Susan J. Clark

  4. Victor Chang Cardiac Research Institute, Sydney, NSW, Australia

    Thomas Preiss

Authors
  1. Tennille Sibbritt
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  2. Ulrike Schumann
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  3. Andrew Shafik
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  4. Marco Guarnacci
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  5. Susan J. Clark
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  6. Thomas Preiss
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Corresponding author

Correspondence to Thomas Preiss .

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Editors and Affiliations

  1. Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Trento, Italy

    Erik Dassi

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Sibbritt, T., Schumann, U., Shafik, A., Guarnacci, M., Clark, S.J., Preiss, T. (2022). Target-Specific Profiling of RNA m5C Methylation Level Using Amplicon Sequencing. In: Dassi, E. (eds) Post-Transcriptional Gene Regulation. Methods in Molecular Biology, vol 2404. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1851-6_21

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  • DOI: https://doi.org/10.1007/978-1-0716-1851-6_21

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1850-9

  • Online ISBN: 978-1-0716-1851-6

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