RiboMeth-seq: Profiling of 2′-O-Me in RNA

  • Nicolai Krogh
  • Ulf Birkedal
  • Henrik NielsenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)


RiboMeth-seq is a sequencing-based method for mapping and quantitation of one of the most abundant RNA modifications, ribose methylation. It is based on a simple chemical principle, namely the several orders of magnitude difference in nucleophilicity of a 2′-OH and a 2-O-Me. Thus, the method combines alkaline fragmentation and a specialized library construction protocol based on 5-OH and 2,3 cyclic phosphate ends to prepare RNA for sequencing. The read-ends of library fragments are used for mapping with nucleotide resolution and calculation of the fraction of molecules methylated at the 2-O-Me sites.

Key words

2-O-Me Ribose methylation Box C/D snoRNA Ribosome Ribosomal RNA RNA library RNA-seq 



The work was supported by the Danish Research Council for Independent Research and the Danish Cancer Research Foundation.


  1. 1.
    Motorin Y, Helm M (2011) RNA nucleotide methylation. Wiley interdisciplinary reviews. RNA 2:611–631CrossRefPubMedGoogle Scholar
  2. 2.
    Decatur WA, Fournier MJ (2002) rRNA modifications and ribosome function. Trends in biochemical sciences 27:344–351CrossRefPubMedGoogle Scholar
  3. 3.
    Watkins NJ, Bohnsack MT (2012) The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA. Wiley interdisciplinary reviews. RNA 3:397–414CrossRefPubMedGoogle Scholar
  4. 4.
    Helm M (2006) Post-transcriptional nucleotide modification and alternative folding of RNA. Nucleic acids research 34:721–733CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Decatur WA, Liang XH, Piekna-Przybylska D, Fournier MJ (2007) Identifying effects of snoRNA-guided modifications on the synthesis and function of the yeast ribosome. Methods in enzymology 425:283–316CrossRefPubMedGoogle Scholar
  6. 6.
    Noeske J, Wasserman MR, Terry DS, Altman RB, Blanchard SC, Cate JH (2015) High-resolution structure of the Escherichia coli ribosome. Nature structural & molecular biology 22:336–341CrossRefGoogle Scholar
  7. 7.
    Polikanov YS, Melnikov SV, Soll D, Steitz TA (2015) Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly. Nature structural & molecular biology 22:342–344CrossRefGoogle Scholar
  8. 8.
    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
  9. 9.
    Birkedal U, Christensen-Dalsgaard M, Krogh N, Sabarinathan R, Gorodkin J, Nielsen H (2015) Profiling of ribose methylations in RNA by high-throughput sequencing. Angewandte Chemie 54:451–455PubMedGoogle Scholar
  10. 10.
    Kiss-Laszlo Z, Henry Y, Bachellerie JP, Caizergues-Ferrer M, Kiss T (1996) Site-specific ribose methylation of preribosomal RNA: a novel function for small nucleolar RNAs. Cell 85:1077–1088CrossRefPubMedGoogle Scholar
  11. 11.
    Schutz K, Hesselberth JR, Fields S (2010) Capture and sequence analysis of RNAs with terminal 2',3'-cyclic phosphates. RNA 16:621–631CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Meyer M, Masquida B (2014) cis-Acting 5' hammerhead ribozyme optimization for in vitro transcription of highly structured RNAs. Methods Mol Biol 1086:21–40CrossRefPubMedGoogle Scholar
  13. 13.
    Maden BE (2001) Mapping 2'-O-methyl groups in ribosomal RNA. Methods 25:374–382CrossRefPubMedGoogle Scholar
  14. 14.
    Douthwaite S, Kirpekar F (2007) Identifying modifications in RNA by MALDI mass spectrometry. Methods in enzymology 425:3–20PubMedGoogle Scholar
  15. 15.
    Taoka M, Nobe Y, Hori M, Takeuchi A, Masaki S, Yamauchi Y, Nakayama H, Takahashi N, Isobe T (2015) A mass spectrometry-based method for comprehensive quantitative determination of post-transcriptional RNA modifications: the complete chemical structure of Schizosaccharomyces pombe ribosomal RNAs. Nucleic acids research 43:e115CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Buchhaupt M, Sharma S, Kellner S, Oswald S, Paetzold M, Peifer C, Watzinger P, Schrader J, Helm M, Entian KD (2014) Partial methylation at Am100 in 18S rRNA of baker's yeast reveals ribosome heterogeneity on the level of eukaryotic rRNA modification. PloS one 9:e89640CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.J 17(n. 1):10–12CrossRefGoogle Scholar
  18. 18.
    Langmead B, Salzberg S (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Ryvkin P, Leung YY, Silverman IM, Childress M, Valladares O, Dragomir I, Gregory BD, Wang LS (2013) HAMR: high-throughput annotation of modified ribonucleotides. RNA 19:1684–1692CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Cellular and Molecular MedicineThe Panum Institute, University of CopenhagenCopenhagenDenmark

Personalised recommendations