Thioguanosine conversion enables mRNA life-time evaluation by RNA sequencing via double metabolic labeling.
Fiche publication
Date publication
janvier 2020
Journal
Angewandte Chemie (International ed. in English)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr ENNIFAR Eric
Tous les auteurs :
Gasser C, Delazer I, Neuner E, Pascher K, Brillet K, Klotz S, Trixl L, Himmelstoß M, Ennifar E, Rieder D, Lusser A, Micura R
Lien Pubmed
Résumé
Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed hydrazine/NH 4 Cl/OsO 4 -based conversion of 6-thioguanosine (6sG)- into A'-containing RNA, where A' constitutes a 6-hydrazino purine derivative. The A' nucleoside retains the Watson-Crick base pair mode as shown by crystal structure analysis of a short palindromic duplex and thermodynamic analysis of UV-melting profiles. We further demonstrate that A' is efficiently decoded as adenosine in primer extension assays and in RNA sequencing experiments. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the novel conversion chemistry is fully compatible with the conversion of the frequently applied metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual labeling set-ups enables high accuracy measurements of RNA decay. Our novel approach that we name TUC-seq DUAL uses the two modified nucleotides in subsequent pulses and their simultaneous detection. Thus, synthesis and decay of mRNA can be clearly distinguished based on the differential presence of G-to-A and U-to-C mutations. This enables mRNA life-time evaluation with unprecedented precision.
Mots clés
Nucleoside modifications, oligonucleotides, osmium tetroxide, RNA structures, RNA sequencing
Référence
Angew. Chem. Int. Ed. Engl.. 2020 Jan 30;: