High-Throughput Mapping of 2-O-Me Residues in RNA Using Next-Generation Sequencing (Illumina RiboMethSeq Protocol)

  • Virginie Marchand
  • Lilia Ayadi
  • Aseel El Hajj
  • Florence Blanloeil-Oillo
  • Mark Helm
  • Yuri MotorinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)


Detection of RNA modifications in native RNAs is a tedious and laborious task, since the global level of these residues is low and most of the suitable physico-chemical methods require purification of the RNA of interest almost to homogeneity. To overcome these limitations, methods based on RT-driven primer extension have been developed and successfully used, sometimes in combination with a specific chemical treatment. Nowadays, some of these approaches have been coupled to high-throughput sequencing technologies, allowing the access to transcriptome-wide data. RNA 2-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from bacteria, archaea, and eukarya. Here, we describe a reliable and optimized protocol based on alkaline fragmentation of total RNA coupled to a commonly used ligation approach followed by Illumina sequencing. We describe the methodology for detection and relative quantification of 2-O-methylations with a high sensitivity and reproducibility even with a limited amount of starting material (1 ng of total RNA). Altogether this technique unlocks a technological barrier since it will be applicable for routine parallel treatment of biological and clinical samples to decipher the functions of 2-O-methylations in pathologies.

Key words

2-O-Methylation High-throughput sequencing RNA modification Ribose methylation Alkaline fragmentation 



This work was supported by joint ANR-DFG grant HTRNAMod (ANR-13-ISV8-0001/HE 3397/8-1) to MH and YM, and AO Lorraine University-Lorraine Region “Aberrant RNA methylation in cancer” funding to YM.


  1. 1.
    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–D267CrossRefPubMedGoogle Scholar
  2. 2.
    Motorin Y, Helm M (2011) RNA nucleotide methylation. Wiley Interdiscip Rev RNA 2:611–631CrossRefPubMedGoogle Scholar
  3. 3.
    Ji L, Chen X (2012) Regulation of small RNA stability: methylation and beyond. Cell Res 22:624–636CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ohara T, Sakaguchi Y, Suzuki T, Ueda H, Miyauchi K, Suzuki T (2007) The 3′ termini of mouse Piwi-interacting RNAs are 2′-O-methylated. Nat Struct Mol Biol 14:349–350CrossRefPubMedGoogle Scholar
  5. 5.
    Tycowski KT, You ZH, Graham PJ, Steitz JA (1998) Modification of U6 spliceosomal RNA is guided by other small RNAs. Mol Cell 2:629–638CrossRefPubMedGoogle Scholar
  6. 6.
    Maden BE (2001) Mapping 2′-O-methyl groups in ribosomal RNA. Methods 25:374–382CrossRefPubMedGoogle Scholar
  7. 7.
    Maden BE, Corbett ME, Heeney PA, Pugh K, Ajuh PM (1995) Classical and novel approaches to the detection and localization of the numerous modified nucleotides in eukaryotic ribosomal RNA. Biochimie 77:22–29CrossRefPubMedGoogle Scholar
  8. 8.
    Yu YT, Shu MD, Steitz JA (1997) A new method for detecting sites of 2′-O-methylation in RNA molecules. RNA 3:324–331PubMedPubMedCentralGoogle Scholar
  9. 9.
    Huang C, Karijolich J, Yu Y-T (2016) Detection and quantification of RNA 2′-O-methylation and pseudouridylation. Methods 103:68–76. doi: 10.1016/j.ymeth.2016.02.003 CrossRefPubMedGoogle Scholar
  10. 10.
    Dong Z-W, Shao P, Diao L-T, Zhou H, Yu C-H, Qu L-H (2012) RTL-P: a sensitive approach for detecting sites of 2′-O-methylation in RNA molecules. Nucleic Acids Res 40:e157CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Aschenbrenner J, Marx A (2016) Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase. Nucleic Acids Res 44:3495–3502CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Dennis PP, Tripp V, Lui L, Lowe T, Randau L (2015) C/D box sRNA-guided 2′-O-methylation patterns of archaeal rRNA molecules. BMC Genomics 16:632CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Birkedal U, Christensen-Dalsgaard M, Krogh N, Sabarinathan R, Gorodkin J, Nielsen H (2015) Profiling of ribose methylations in RNA by high-throughput sequencing. Angew Chem Int Ed Engl 54:451–455PubMedGoogle Scholar
  14. 14.
    Wilfinger WW, Mackey K, Chomczynski P (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22(474–6):478–481Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Virginie Marchand
    • 1
    • 2
  • Lilia Ayadi
    • 1
  • Aseel El Hajj
    • 1
  • Florence Blanloeil-Oillo
    • 1
    • 2
  • Mark Helm
    • 3
  • Yuri Motorin
    • 1
    • 2
    Email author
  1. 1.IMoPA UMR7365 CNRS-ULBioPole Lorraine UniversityVandoeuvre-Les-NancyFrance
  2. 2.Next-Generation Sequencing Core Facility, FR3209 BMCT CNRS-ULBioPole Lorraine UniversityVandoeuvre-les-NancyFrance
  3. 3.Institute of Pharmacy and BiochemistryJohannes Gutenberg University MainzMainzGermany

Personalised recommendations