Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides

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.

Keywords: RNA modifications; RT signature; m1A; manganese chloride; reverse transcription.

Figure 1

Overview of the sample preparation, comprising individual reaction mixtures for every transcriptase and addition of either MgCl₂ (reference) or four concentrations of MnCl₂, thereby resulting in a total of 20 distinct reaction conditions.

Figure 2

(A) Boxplot showing the RT signature of all non-modified sites in tRNA of S. cerevisiae at differing manganese concentrations, using EpiScript RT. The triangled color palette represents increasing concentrations of Mn²⁺, with black dots accounting for single data points. The 95% confidence intervals are displayed in light blue (median) and red (average), with colored asterisk (*) indicating significant differences (p-value < 0.05). (B) Violin plot based on the same dataset and color code as (A), displaying the jump rate. The width of each plot differs by amount of data at the given axis intercept.

Figure 3

(A) Boxplot showing the RT signatures of all m¹A and m²₂G positions in tRNA from Saccharomyces cerevisiae at differing manganese concentrations, using EpiScript RT. The triangled color palette represents increasing concentrations of Mn²⁺. The 95% confidence intervals are displayed in light blue (median) and red (average), with colored asterisk (*) indicating significant differences (p-value < 0.05). (B) Normalized bar plot based on the same dataset and color code as (A), displaying the difference in jump rate between normalized reference and manganese treated samples.

Figure 4

Comparative plot of all used polymerases breaking down their average profile for m¹A positions in S. cerevisiae tRNA. The 95% confidence interval is displayed in red, with colored asterisk (*) indicating significant differences (p-value < 0.05) between Mg²⁺ and Mn²⁺ conditions. Enzymes are displayed in different shapes with colors signaling Mg²⁺ (pale) or Mn²⁺ (intense) treatment.