A Quantitative Sequencing Method for 5-Formylcytosine in RNA
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA659533 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12524830.fastq.gz | Fastqsanger.gz | |||
| SRR12524831.fastq.gz | Fastqsanger.gz | |||
| SRR12524832.fastq.gz | Fastqsanger.gz | |||
| SRR12524833.fastq.gz | Fastqsanger.gz | |||
| SRR12524834.fastq.gz | Fastqsanger.gz |
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Project description:5-Formylcytosine (f5C) modification is present in human mitochondrial methionine tRNA (mt-tRNAMet) and cytosolic leucine tRNA (ct-tRNALeu), with their formation mediated by NSUN3 and ALKBH1. f5C has also been detected in mRNA of yeast and human cells, but its transcriptome-wide distribution has not been studied. Here we report f5C-seq, a quantitative sequencing method to map f5C transcriptome-wide in HeLa and mouse embryonic stem cells (mESCs). We show that f5C in RNA can be reduced to dihydrouracil (DHU) by pico-brane, and DHU can be exclusively read as U during reverse transcription (RT) reaction, allowing the detection and quantification of f5C sites by a unique C-to-U mutation signature. We validated f5C-seq by identifying and quantifying the two known f5C sites in tRNA, in which the f5C modification fractions dropped significantly in ALKBH1-depleted cells. By applying f5C-seq to small RNA, we identified 13 and 11 new f5C sites in HeLa and mESC tRNA, respectively.
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Project description:Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Here, reduced bisulfite sequencing is developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells.
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Project description:Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Here, reduced bisulfite sequencing is developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells. 12 samples, reduced representation bisulphite treatment: 4 replicates each for bisulphite (BS), oxidative BS (oxBS) and reduced BS (redBS) for the detection of 5mC, 5hmC and 5fC. Mouse (strain B6C) embryonic stem cells.
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Project description:Epitranscriptomic RNA modifications can regulate biological processes, but there remains a major gap in our ability to identify and measure individual modifications at nucleotide resolution. Here we present Mal-Seq, a chemical method for sequencing 5-formylcytosine (f5C) modifications on RNA based on the selective and efficient malononitrile-mediated labeling of f5C residues to generate adducts that are read as C-to-T mutations upon reverse transcription and polymerase chain reaction amplification. We apply Mal-Seq to characterize the prevalence of f5C at the wobble position of mt-tRNA(Met) in different organisms and tissue types and find that high-level f5C modification is present in mammals but lacking in lower eukaryotes. Our work sheds light on mitochondrial tRNA modifications throughout eukaryotic evolution and provides a general platform for characterizing the f5C epitranscriptome.
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