Interpreting reverse transcriptase termination and mutation events for greater insight into the chemical probing of RNA
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ABSTRACT: Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely non-overlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context, and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.
Project description: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 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 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.
Project description:Chemical modifications on mRNA are increasingly recognized as a critical regulatory layer of the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile directed evolution platform that rapidly selects for reverse transcriptases that install mutations during reverse transcription at sites of a given type of RNA modification, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N1-methyladenosine (m1A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m1A sites. We apply the evolved reverse transcriptase to identify thousands of statistically confident m1A sites in human mRNA, some of which can be detected in antibody-free RNA-seq libraries. Together, this work develops and validates the reverse transcriptase evolution platform and provides new tools, analysis methods, and datasets to study m1A biology.
Project description:SHAPE-MaP structure probing experiment was performed on SARS-CoV-2 infected Vero cells at 4 days post infection with two biological replicates. For each replciate, SHAPE-MaP includes a sample treated with 2-methylnicotinic acid imidazolide acid (modified) or a minue reagent (unmodified). NAI preferentially reacts with unpaired bases in RNA, forming acylated bases. These modifications are encoded as mutation during reverse transcripatse and library preparation. After sequencing and alignment, the reactivity profiles of 'modified' and 'unmodified' samples are used to calculate SHAPE reactivity of each base
Project description:SHAPE-MaP structure probing experiment was performed on SARS-CoV-2 infected Vero or C6/36 cells at 4 days post infection with two biological replicates. For each replciate, SHAPE-MaP includes a sample treated with 2-methylnicotinic acid imidazolide acid (modified) or a minuse reagent (unmodified). NAI preferentially reacts with unpaired bases in RNA, forming acylated bases. These modifications are encoded as mutation during reverse transcripatse and library preparation. After sequencing and alignment, the reactivity profiles of 'modified' and 'unmodified' samples are used to calculate SHAPE reactivity of each base
Project description:Given the interest in the COVID mRNA vaccines, we sought to investigate how the RNA modification N1-methylpseudouridine (and its related modification, pseudouridine) is read by ribosomes and reverse transcriptases. By looking at reverse transcriptase data, we can gain information on how the modification affects duplex stability, which may have important consequences for the tRNA-mRNA interactions found in the ribosome.
Project description:We present a novel N-nitrosation strategy for deamination capable of tolerating DNA/RNA biological macromolecules under mild conditions. A cooperative catalysis combining a carbonyl organocatalyst with a Lewis acid catalyst facilitates the formation of a C-nitro intermediate from a primary amine, which, upon rearrangement into N-nitrosamine, leads to selective deamination of unsubstituted canonical DNA/RNA bases under mild conditions. We employed this new approach to deamination of adenine into hypoxanthine, read as guanine by reverse transcriptases or DNA polymerases, while N6-methyladenosine (m6A) sites resist deamination and remain identified as adenine. We therefore report a chemically mild, low-input detection method for adenosine methylation sequencing at base resolution, named Chemical cooperative catalysis-Assisted for m6A sequencing (CAM-seq).
Project description:We present a novel N-nitrosation strategy for deamination capable of tolerating DNA/RNA biological macromolecules under mild conditions. A cooperative catalysis combining a carbonyl organocatalyst with a Lewis acid catalyst facilitates the formation of a C-nitro intermediate from a primary amine, which, upon rearrangement into N-nitrosamine, leads to selective deamination of unsubstituted canonical DNA/RNA bases under mild conditions. We employed this new approach to deamination of adenine into hypoxanthine, read as guanine by reverse transcriptases or DNA polymerases, while N6-methyladenosine (m6A) sites resist deamination and remain identified as adenine. We therefore report a chemically mild, low-input detection method for adenosine methylation sequencing at base resolution, named Chemical cooperative catalysis-Assisted for m6A sequencing (CAM-seq).
Project description:We present a novel N-nitrosation strategy for deamination capable of tolerating DNA/RNA biological macromolecules under mild conditions. A cooperative catalysis combining a carbonyl organocatalyst with a Lewis acid catalyst facilitates the formation of a C-nitro intermediate from a primary amine, which, upon rearrangement into N-nitrosamine, leads to selective deamination of unsubstituted canonical DNA/RNA bases under mild conditions. We employed this new approach to deamination of adenine into hypoxanthine, read as guanine by reverse transcriptases or DNA polymerases, while N6-methyladenosine (m6A) sites resist deamination and remain identified as adenine. We therefore report a chemically mild, low-input detection method for adenosine methylation sequencing at base resolution, named Chemical cooperative catalysis-Assisted for m6A sequencing (CAM-seq).
Project description:Structure probing coupled with high-throughput sequencing holds the potential to revolutionize our understanding of the role of RNA structure in regulation of gene expression. Despite major technological advances, intrinsic noise and high coverage requirements greatly limit the applicability of these techniques. Here we describe a probabilistic modeling pipeline which accounts for biological variability and biases in the data, yielding statistically interpretable scores for the probability of nucleotide modification transcriptome-wide. We demonstrate on two yeast data sets that our method has greatly increased sensitivity, enabling the identification of modified regions on many more transcripts compared with existing pipelines. It also provides confident predictions at much lower coverage levels than previously reported. Our results show that statistical modeling greatly extends the scope and potential of transcriptome-wide structure probing experiments.