Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches

Thermostable reverse
transcriptases are workhorse enzymes underlying
nearly all modern techniques for RNA structure mapping and for the
transcriptome-wide discovery of RNA chemical modifications. Despite
their wide use, these enzymes’ behaviors at chemical modified
nucleotides remain poorly understood. Wellington-Oguri et al. recently
reported an apparent loss of chemical modification within putatively
unstructured polyadenosine stretches modified by dimethyl sulfate
or 2′ hydroxyl acylation, as probed by reverse transcription.
Here, reanalysis of these and other publicly available data, capillary
electrophoresis experiments on chemically modified RNAs, and nuclear
magnetic resonance spectroscopy on (A)12 and variants show
that this effect is unlikely to arise from an unusual structure of
polyadenosine. Instead, tests of different reverse transcriptases
on chemically modified RNAs and molecules synthesized with single
1-methyladenosines implicate a previously uncharacterized reverse
transcriptase behavior: near-quantitative bypass through chemical
modifications within polyadenosine stretches. All tested natural and
engineered reverse transcriptases (MMLV; SuperScript II, III, and
IV; TGIRT-III; and MarathonRT) exhibit this anomalous bypass behavior.
Accurate DMS-guided structure modeling of the polyadenylated HIV-1
3′ untranslated region requires taking into account this anomaly.
Our results suggest that poly­(rA-dT) hybrid duplexes can trigger an
unexpectedly effective reverse transcriptase bypass and that chemical
modifications in mRNA poly­(A) tails may be generally undercounted.