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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.
Highlights
Research on RNA modifications has experienced an impressive renaissance in the last few years, driven by a rapid growth in the field of epitranscriptomic research [1] and based on ever improving in vitro and in silico methods
To investigate how manganese ions influence cDNA synthesis in reverse transcription, we processed total tRNA from S. cerevisiae with different reverse transcriptase (RT), using various concentrations of MnCl2
We examined the influence of manganese ions on reverse transcription and derived RNA modification analysis by performing reverse transcription with four different RTs, therefrom derived RNA modification analysis by performing reverse transcription with four thereby replacing MgCl2 with various concentrations of MnCl2
Summary
Research on RNA modifications has experienced an impressive renaissance in the last few years, driven by a rapid growth in the field of epitranscriptomic research [1] and based on ever improving in vitro and in silico methods. A wide range of alterations, from single methylations on adenosine as in m1 A or m6 A, up to complex modifications comprising multiple steps of formation and rare biological structures such as a thioacetal [8] has been identified. From this variety, several modifications are considered key factors in cancer research, emphasizing the importance of m1 A, m6 A, m5 C, inosine and pseudouridine [9]. One approach uses the altered transcriptional behavior of modifications such as m1 A, an adenosine bearing a methyl group in the Watson-Crick face, impeding conventional base pairing. According to the necessity of converting RNA into DNA for current
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