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Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing

ABSTRACT: Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing

PROVIDER: PRJNA1068978 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR27723500_1.fastq.gz	Fastqsanger.gz
	SRR27723500_2.fastq.gz	Fastqsanger.gz
	SRR27723501_1.fastq.gz	Fastqsanger.gz
	SRR27723501_2.fastq.gz	Fastqsanger.gz
	SRR27723502_1.fastq.gz	Fastqsanger.gz

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Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing

Project description:5-Methylcytosine (m5C) is one of the major post-transcriptional modifications in mRNA and is highly involved in the pathogenesis of various diseases. However, the capacity of existing assays for accurately and comprehensively transcriptome-wide m5C mapping still needs improvement. Here, we developed a deaminase and reader protein assisted RNA methylation analysis, termed DRAM, in which deaminases (APOBEC1 and TadA-8e) are fused with m5C reader proteins (ALYREF and YBX1) to identify the m5C sites through deamination events neighboring the methylation sites. This antibody-free and bisulfite-free approach provided transcriptome-wide editing regions which were highly overlapped with the publicly available BS-seq datasets. Notably, DRAM-seq even discovered a new m5C methylation locus in KAT7, a previously demonstrated key aging regulator. In addition, DRAM system even supports ultra-low input RNA (10ng) and monitor the dynamic accumulation of cellular m5C. We anticipated that the DRAM system could pave the way for uncovering further biological functions of m5C modifications.

2025-03-14 | GSE254194 | GEO

Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via deaminase-assisted sequencing [RNA-Seq]

Project description:5gmC (5-glyceryl-methylcytosine), a vitamin C-derived hypermodified base, has been identified in the genome of Chlamydomonas reinhardtii. However, the global distribution of 5gmC and its role as an epigenetic mark remain poorly understood. In this study, we employed a DNA deaminase to distinguish 5gmC from 5mC (5-methylcytosine) and cytosines, enabling precise profiling of 5gmC across the genome. This deaminase-assisted sequencing (DEA-seq) demonstrates superior performance compared to the previously proposed TET-coupled bisulfite sequencing. Using both methods, we identified numerous confident 5gmC sites. Unlike 5mC, which predominantly occurs at CpG sites, 5gmC is preferentially located in CHH contexts. Remarkably, over half of 5gmC sites are mutually exclusive from 5mC, suggesting a role in active DNA demethylation. Additionally, 5gmC is enriched within introns, contrasting with the more extensive localization of 5mC in intergenic and promoter regions. Importantly, 5gmC levels are positively correlated with transcription, while 5mC typically exhibits an inverse relationship with gene expression, consistent with the enrichment of 5mC but loss of 5gmC at repressive H3K9me1 sites. Collectively, these findings suggest that 5gmC is not only an intermediate for active DNA demethylation but also functions as a stable epigenetic mark, potentially influencing transcriptional regulation independently of 5mC.

2025-09-05 | GSE301797 | GEO

Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via deaminase-assisted sequencing [Bisulfite-Seq]

2025-09-05 | GSE301796 | GEO

Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via deaminase-assisted sequencing [Bisulfite-Seq]

Project description:Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via deaminase-assisted sequencing [Bisulfite-Seq]

| PRJNA1287575 | ENA

Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via deaminase-assisted sequencing [RNA-Seq]

Project description:Base-resolution profiling of 5-glyceryl-methylcytosine in Chlamydomonas reinhardtii via deaminase-assisted sequencing [RNA-Seq]

| PRJNA1287576 | ENA

5-methylcytosine modification and the ALYREF reader protein promote retrovirus replication

Project description:The 5-methylcytosine (m5C) modification is present at appreciable amounts in the mammalian transcriptome, but its precise location and function is still poorly understood. Here, we identify high m5C levels and map their locations in a model retrovirus, the murine leukemia virus, and show that the ALYREF m5C reader protein regulates viral replication. Our results reveal a single-nucleotide m5C profile in a virus and its function in a eukaryotic mRNA.

2020-04-14 | GSE139606 | GEO

5-methylcytosine modification and the ALYREF reader protein promote retrovirus replication

Project description:5-methylcytosine modification and the ALYREF reader protein promote retrovirus replication

| PRJNA580484 | ENA

Identification of putative reader proteins of RNA 5-methylcytosine and its derivatives in Caenorhabditis elegans

Project description:Methylation of carbon-5 of cytosines (m5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. m5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. We recently showed that m5C is required for Caenorhabditis elegans development and fertility under heat stress. To contribute to the understanding of how m5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Our mass spectrometry analyses reveal unique binding proteins for each of the modifications. We validate our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m5C in vitro. The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m5C and its derivatives.

2022-09-29 | PXD035761 | Pride

Bisulfite-Free Whole-Genome Mapping of 5-Methylcytosine at Single-Base Resolution by NTD-seq

Project description:5-Methylcytosine (5mC) is a crucial epigenetic modification plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel NaegleriaTET-assisted deaminase sequencing (NTD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The TAD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, TAD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied NTD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. NTD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.

2025-04-02 | GSE263426 | GEO

Bisulfite-Free Whole-Genome Mapping of 5-Methylcytosine at Single-Base Resolution by TAD-seq

Project description:5-Methylcytosine (5mC) is a crucial epigenetic modification plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel nTET-assisted deaminase sequencing (TAD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The TAD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, TAD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied TAD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. TAD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.

2024-06-30 | GSE254203 | GEO

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