Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides.
Fiche publication
Date publication
août 2020
Journal
Genes
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MOTORINE Iouri, Dr MARCHAND Virginie
Tous les auteurs :
Kristen M, Plehn J, Marchand V, Friedland K, Motorin Y, Helm M, Werner S
Lien Pubmed
Résumé
Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for the respective RNA modification and reverse transcriptase (RT). While known for DNA polymerases in so-called error-prone PCR, testing of four different RTs by replacing Mg with Mn in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on mA positions dropping from 82% down to 24%. Additionally, we observed a vast increase in nucleotide skipping events, with single positions rising from 4% to 49%, thus implying an enhanced read-through capability as an effect of Mn on the reverse transcriptase, by promoting nucleotide skipping over synthesis abortion. While modifications such as mA, mG, mG and mC showed a clear influence of manganese ions on their RT signature, this effect was individual to the polymerase used. In summary, the results imply a supporting effect of Mn on reverse transcription, thus overcoming blockades in the Watson-Crick face of modified ribonucleosides and improving both read-through rate and signal intensity in RT signature analysis.
Mots clés
RNA modifications, RT signature, m1A, manganese chloride, reverse transcription
Référence
Genes (Basel). 2020 Aug 18;11(8):