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Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides

Authors
  • Kristen, Marco1
  • Plehn, Johanna1
  • Marchand, Virginie2
  • Friedland, Kristina1
  • Motorin, Yuri2, 3
  • Helm, Mark1
  • Werner, Stephan1
  • 1 (M.H.)
  • 2 (Y.M.)
  • 3 IMoPA, UMR7365 CNRS, Université de Lorraine, Biopôle, 9 Avenue de la Forêt de Haye, 54505 Vandœuvre-lès-Nancy, France
Type
Published Article
Journal
Genes
Publisher
MDPI AG
Publication Date
Aug 18, 2020
Volume
11
Issue
8
Identifiers
DOI: 10.3390/genes11080950
PMID: 32824672
PMCID: PMC7466121
Source
PubMed Central
Keywords
License
Green

Abstract

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.

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