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.

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

“…The arrest rate and mis-incorporation profiles are both strongly modulated under these altered conditions, with an increase in nucleotide skipping (deletions). Every RT polymerase shows an individual sensitivity to increased [Mn 2+ ] and it depends on the nature of the RNA modification tested, allowing for a more reliable detection [ 27 ].…”

General Principles for the Detection of Modified Nucleotides in RNA by Specific Reagents

“…In such cases, four general resolutions are conceivable on the molecular level, namely, i) continued reverse transcription with normal dNTP or ii) by misincorporation, iii) skipping of the modified nucleotide in the template, or iv) abortion of cDNA synthesis. [89,90] Many modifications, in particular those with alterations on the Watson-Crick face, display a mixture of (i)-(iv). The relative contributions of (i)-(iv) form a reverse transcription signature that is somewhat characteristic for a given modification but is sequence specific and may also vary according to the enzyme and reaction conditions employed for reverse transcription.…”

“…The relative contributions of (i)-(iv) form a reverse transcription signature that is somewhat characteristic for a given modification but is sequence specific and may also vary according to the enzyme and reaction conditions employed for reverse transcription. [89][90][91] A number of reagents with selectivity for modifications have been employed to trace modifications by comparing RNAseq data from treated and untreated samples. The alkylation of inosine [61b] or the acylation of Ψ with CMCT [33a,39b,92] was among the first to be carried to a transcriptome wide stage.…”

General Principles and Limitations for Detection of RNA Modifications by Sequencing

“…This is one example of many where the Watson–Crick edge is unaffected by the modification, causing a weak or invisible RT-signature. , In the case at hand, it becomes enhanced through modulation of the RT-conditions, namely lowered dNTP conditions which diminish the reverse transcriptase activity to the point where it stalls and produces abortive cDNA. Further sophistication included the combination of stalling and misincorporation as important contributors to the RT-signature, which was then shown to vary, e.g., among different enzymes such that the combined RT-signature of two RT-enzymes was more informative than one alone . This inspired the directed evolution of enzymes whose RT-signature was specifically sensitive to modifications such as m 6 A, Nm, Q and Ψ, which were otherwise considered to be RT-silent. − Another sophistication included jumps in the RT-signature, a feature that seems to be fraught with less background. − However, it became clear that the number of RNA modifications whose altered base pairing properties allowed comprehensive detection was limited …”