ena-STUDY-Lorraine University-13-04-2018-13:46:14:680-57
AlkAniline-Seq: profiling of m7G and m3C RNA modifications at single nucleotide resolution.
We developed a novel chemical-based approach for 7-methylguanosine (m7G) mapping in RNA called AlkAniline-Seq which combines alkaline hydrolysis followed by aniline cleavage, library preparation and sequencing. AlkAniline-Seq provides a positive readout of intrinsically unstable abasic sites created in RNA by alkaline treatment and is therefore highly specific, sensitive, and accurate even for low abundant RNAs. AlkAniline-Seq detects not only fully modified m7G residues, but also sub-stoichiometric modifications down to 1-2% of molar amount. AlkAniline-Seq was successfully applied to bacterial and yeast rRNA and tRNAs to map all previously reported m7G sites with single nucleotide precision. Unexpectedly, in addition to m7G detection, AlkAniline-Seq revealed to be highly specific for 3-methylcytidine (m3C) residues with a comparable specificity and sensitivity. Therefore, AlkAniline-Seq was applied for S. cerevisiae transcriptome-wide mapping. No m7G/m3C modification sites were detected outside tRNAs and rRNAs, suggesting that potential sites in mRNAs and ncRNAs are modified at less than 1-2% or down-regulated under optimal growth conditions. AlkAniline-Seq is therefore a powerful method for the systematic profiling of diverse m7G and m3C transcriptomes, and for exploring their variations under various biological contexts (disease).
We developed a novel chemical-based approach for 7-methylguanosine (m7G) mapping in RNA called AlkAniline-Seq which combines alkaline hydrolysis followed by aniline cleavage, library preparation and sequencing. AlkAniline-Seq provides a positive readout of intrinsically unstable abasic sites created in RNA by alkaline treatment and is therefore highly specific, sensitive, and accurate even for low abundant RNAs. AlkAniline-Seq detects not only fully modified m7G residues, but also sub-stoichiometric modifications down to 1-2% of molar amount. AlkAniline-Seq was successfully applied to bacterial and yeast rRNA and tRNAs to map all previously reported m7G sites with single nucleotide precision. Unexpectedly, in addition to m7G detection, AlkAniline-Seq revealed to be highly specific for 3-methylcytidine (m3C) residues with a comparable specificity and sensitivity. Therefore, AlkAniline-Seq was applied for S. cerevisiae transcriptome-wide mapping. No m7G/m3C modification sites were detected outside tRNAs and rRNAs, suggesting that potential sites in mRNAs and ncRNAs are modified at less than 1-2% or down-regulated under optimal growth conditions. AlkAniline-Seq is therefore a powerful method for the systematic profiling of diverse m7G and m3C transcriptomes, and for exploring their variations under various biological contexts (disease).