Illumina-based RiboMethSeq approach for mapping of 2'-O-Me residues in RNA.
Fiche publication
Date publication
septembre 2016
Journal
Nucleic acids research
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MOTORINE Iouri, Dr MARCHAND Virginie
Tous les auteurs :
Marchand V, Blanloeil-Oillo F, Helm M, Motorin Y
Lien Pubmed
Résumé
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.
Mots clés
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
Référence
Nucleic Acids Res.. 2016 Sep;44(16):e135