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

Abstract

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.