Mapping of 7-methylguanosine (mG), 3-methylcytidine (mC), dihydrouridine (D) and 5-hydroxycytidine (hoC) RNA modifications by AlkAniline-Seq.
Fiche publication
Date publication
janvier 2021
Journal
Methods in enzymology
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MOTORINE Iouri, Dr MARCHAND Virginie
Tous les auteurs :
Marchand V, Bourguignon-Igel V, Helm M, Motorin Y
Lien Pubmed
Résumé
Precise and reliable mapping of modified nucleotides in RNA is a challenging task in epitranscriptomics analysis. Only deep sequencing-based methods are able to provide both, a single-nucleotide resolution and sufficient selectivity and sensitivity. A number of protocols employing specific chemical reagents to distinguish modified RNA nucleotides from canonical parental residues have already proven their performance. We developed a deep-sequencing analytical pipeline for simultaneous detection of several modified nucleotides of different nature (methylation, hydroxylation, reduction) in RNA. The AlkAniline-Seq protocol uses intrinsic fragility of the N-glycosidic bond present in certain modified residues (7-methylguanosine (mG), 3-methylcytidine (mC), dihydrouridine (D) and 5-hydroxycytidine (hoC)) to induce cleavage under heat combined with alkaline conditions. The resulting RNA abasic site is decomposed by aniline-driven β-elimination and creates a 5'-phosphate (5'-P) at the adjacent N+1 residue. This 5'-P is the crucial entry point for a highly selective ligation of sequencing adapters during the subsequent Illumina library preparation protocol. AlkAniline-Seq protocol has a very low background, and is both highly sensitive and specific. Applications of AlkAniline-Seq include mapping of mG, mC, D, and hoC in variety of cellular RNAs, including in particular rRNAs and tRNAs.
Mots clés
Deep sequencing, Epitranscriptome, Mapping, Selective ligation, Single-nucleotide resolution, rRNA, tRNA
Référence
Methods Enzymol. 2021 ;658:25-47