Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA382421 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5442560_1.fastq.gz | Fastqsanger.gz | |||
| SRR5442560_2.fastq.gz | Fastqsanger.gz | |||
| SRR5442570_1.fastq.gz | Fastqsanger.gz | |||
| SRR5442570_2.fastq.gz | Fastqsanger.gz | |||
| SRR5442561_1.fastq.gz | Fastqsanger.gz |
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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: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:Pseudouridine (Y) is an abundant chemical modification that plays critical roles in cellular RNA metabolism and functions, and in RNA therapeutic applications. Transcriptome-wide mapping technologies present major advantages in discovering the functional contexts and crucial effector proteins of Y. Current mapping strategies for Ys are limited in identifying Ys at base-resolution within consecutive uridine sequence contexts where Y occurs frequently. Here we report “Mut-Y-seq” that utilities a recently evolved reverse transcriptase (“RT-1306”) coupled with the selective labeling of Y by the CMC treatment to identify Ys in mRNAs and non-coding RNAs from human cells. We showed that RT-1306 showed significantly promoted read-through and mutation signatures against the CMC-Y adduct; the mutation signatures generated by RT-1306 can discern the position of Y in consecutive uridine sequence contexts. We quantitatively characterized the Y-identification efficiencies by Mut-Y-seq under variable chemical treatment conditions by the receiver operating characteristic curve analysis and revealed the presence of potential false positive discoveries generated by side-reactions on unmodified uridines. Finally, the intersection of sites identified by orthogonal chemical treatments (i.e. CMC and bisulfite) produced a high-confidence list of Y sites in mRNAs and non-ribosomal long non-coding RNAs from HEK-293T cells. This report presents datasets for biological functional studies of endogenous Y sites, and sheds light on the combination of the reverse transcriptase engineering and selective chemical reactions for further expanding the toolkit for studying RNA chemical modifications.
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