Illumina-based RiboMethSeq approach for mapping of 2'-O-Me residues in RNA.
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.
IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France Next-Generation Sequencing Core Facility, FR3209 BMCT, Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France.
Virginie
V
Marchand
IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France Next-Generation Sequencing Core Facility, FR3209 BMCT, Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France.
Florence
F
Blanloeil-Oillo
Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany.
Mark
M
Helm
IMoPA UMR7365 CNRS-UL, BioPole Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France Next-Generation Sequencing Core Facility, FR3209 BMCT, Lorraine University, 9 avenue de la Foret de Haye, 54505 Vandoeuvre-les-Nancy, France iouri.motorine@univ-lorraine.fr.
Yuri
0000-0002-8018-334X
Y
Motorin
D017353:Gene Deletion; D015723:Gene Library; D008745:Methylation; D009711:Nucleotides / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000378:metabolism; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017423:Sequence Analysis, RNA / Q000379:methods*
D009711:Nucleotides; D009841:Oligonucleotides; D012331:RNA, Fungal; D020537:RNA, Small Nucleolar