Illumina-based RiboMethSeq approach for mapping of 2'-O-Me residues in RNA

Nucleic Acids Res. 2016 Sep 19;44(16):e135. doi: 10.1093/nar/gkw547. Epub 2016 Jun 14.


RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from Bacteria, Archaea and Eukarya. RNAs bearing 2'-O-methylations show increased resistance to degradation and enhanced stability in helices. While the exact role of each 2'-O-Me residue remained elusive, the catalytic protein Fibrillarin (Nop1 in yeast) responsible for 2'-O-methylation in eukaryotes, is associated with human pathologies. Therefore, there is an urgent need to precisely map and quantify hundreds of 2'-O-Me residues in RNA using high-throughput technologies. Here, we develop a reliable protocol using alkaline fragmentation of total RNA coupled to a commonly used ligation approach, and Illumina sequencing. We describe a methodology to detect 2'-O-methylations with high sensitivity and reproducibility even with limited amount of starting material (1 ng of total RNA). The method provides a quantification of the 2'-O-methylation occupancy of a given site, allowing to detect relatively small changes (>10%) in 2'-O-methylation profiles. 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.

MeSH terms

  • Gene Deletion
  • Gene Library
  • Methylation
  • Nucleotides / metabolism
  • Oligonucleotides / metabolism
  • RNA, Fungal / metabolism*
  • RNA, Small Nucleolar / metabolism
  • Reproducibility of Results
  • Saccharomyces cerevisiae / metabolism*
  • Sequence Analysis, RNA / methods*


  • Nucleotides
  • Oligonucleotides
  • RNA, Fungal
  • RNA, Small Nucleolar