Anomalous reverse transcription through chemical modifications in polyadenosine stretches
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA627227 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11583837_1.fastq.gz | Fastqsanger.gz | |||
| SRR11583837_2.fastq.gz | Fastqsanger.gz | |||
| SRR11583838_1.fastq.gz | Fastqsanger.gz | |||
| SRR11583838_2.fastq.gz | Fastqsanger.gz | |||
| SRR11583839_1.fastq.gz | Fastqsanger.gz |
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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.
2020-04-22 | GSE149061 | GEO
Project description: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.
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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.
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Project description: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.
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Project description:Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA of Mtb, using tRNA sequencing (tRNA-seq). Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of 9 modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion of Mtb genes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species.
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