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Open AccessArticle

Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides

1
Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg, University Mainz, Staudingerweg 5, 55128 Mainz, Germany
2
Epitranscriptomics and RNA Sequencing (EpiRNA-Seq) Core Facility, UMS2008 IBSLor CNRS, Université de Lorraine-INSERM, Biopôle, 9 Avenue de la Forêt de Haye, 54505 Vandœuvre-lès-Nancy, France
3
IMoPA, UMR7365 CNRS, Université de Lorraine, Biopôle, 9 Avenue de la Forêt de Haye, 54505 Vandœuvre-lès-Nancy, France
*
Author to whom correspondence should be addressed.
Genes 2020, 11(8), 950; https://doi.org/10.3390/genes11080950
Received: 16 July 2020 / Revised: 10 August 2020 / Accepted: 12 August 2020 / Published: 18 August 2020
(This article belongs to the Special Issue Functions and Dynamics of RNA Modifications)
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 Mg2+ with Mn2+ in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on m1A 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 Mn2+ on the reverse transcriptase, by promoting nucleotide skipping over synthesis abortion. While modifications such as m1A, m22G, m1G and m3C 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 Mn2+ 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. View Full-Text
Keywords: reverse transcription; RT signature; RNA modifications; m1A; manganese chloride reverse transcription; RT signature; RNA modifications; m1A; manganese chloride
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Kristen, M.; Plehn, J.; Marchand, V.; Friedland, K.; Motorin, Y.; Helm, M.; Werner, S. Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides. Genes 2020, 11, 950.

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