Anomalous reverse transcription through chemical modifications in polyadenosine stretches

Abstract

Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for 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, re-analysis 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 1methyladenosines implicate a previously uncharacterized reverse transcriptase behavior: nearquantitative 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 RNA requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger unexpectedly effective reverse transcriptase bypass and that chemical modifications in poly(A) mRNA tails may be generally undercounted.

RNA:RNA interaction in ternary complexes resolved by chemical probing

“…The reactivity profiles from two independent experimental replicates (each with three technical replicates) were used to predict the secondary structure formation of the main loop containing the target RNA insert, using both RNAprobing and MC-Fold (Parisien and Major, 2008;Washietl et al, 2012). No stable secondary structure of the main loops was observed for either TS#1 or TS#2 (Figure S1), although some nucleotides show reduced reactivity (Figure S1b), namely tC4 to tC6, tA14, and tC16, which could be caused by a transient fold of the target sequence (Kladwang and Das, 2010;Bindewald et al, 2011;Mlýnský and Bussi, 2018;Strobel, Yu and Lucks, 2018), sequence-specific biases by the chemical modification (Weeks and Mauger, 2011) or the reverse transcription step (Kladwang et al, 2020). Generally, base-paired nucleotides are supposed to show low (near zero) reactivity values (Weidmann et al, 2021).…”

RNA secondary structure packages evaluated and improved by high-throughput experiments

“…Adenosine nucleotides preceded by 6 or more As were also removed due to evidence of anomalous transcription effects in such stretches 66 , though this removal was not shown to alter package correlations to data (data not included). External chemical mapping datasets were obtained from the supplementary information from the papers and processed similarly (outliers, nucleotides in poly-A stretches removed).…”

RNA secondary structure packages evaluated and improved by high-throughput experiments

“…Cloud Lab round 2 was filtered to exclude experiments that had FMN present, which pertained to Eterna Cloud Lab challenges to design riboswitches. Adenosine nucleotides preceded by six or more As were also removed due to evidence of anomalous reverse transcription effects in such stretches 73 . External chemical mapping datasets were obtained from the supplementary information from the papers and processed similarly (outliers, nucleotides in poly-A stretches removed).…”