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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.
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Submitted on : Wednesday, February 1, 2017 - 5:56:17 PM
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Virginie Marchand, Florence Blanloeil-Oillo, Mark Helm, Yuri Motorin. Illumina-based RiboMethSeq approach for mapping of 2'-O-Me residues in RNA. Nucleic Acids Research, Oxford University Press, 2016, 44 (16), ⟨10.1093/nar/gkw547⟩. ⟨hal-01452312⟩



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