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Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome

ABSTRACT: Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome

PROVIDER: PRJNA882844 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR21659747.fastq.gz	Fastqsanger.gz
	SRR21659748.fastq.gz	Fastqsanger.gz
	SRR21659749.fastq.gz	Fastqsanger.gz
	SRR21659750.fastq.gz	Fastqsanger.gz
	SRR21659751.fastq.gz	Fastqsanger.gz

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Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome

Project description:While DNA N6-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method to measure 6mA at base-resolution with high sensitivity. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through successful mapping of the well-characterized G(6mA)TC motif in the E. coli DNA and identified 6mA sites in the mammalian mitochondrial DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is in general low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single-base resolution with high sensitivity for a comprehensive understanding of DNA 6mA in eukaryotes.

2023-12-28 | GSE213876 | GEO

High-precision mapping reveals rare N6-deoxyadenosine methylation in the mammalian genome

Project description:DNA modification has been characterized as a critical epigenetic regulator in gene expression. N6-deoxyadenosine methylation (6mA) is the most widespread type in prokaryotes and some unicellular eukaryotes. Nevertheless, the lack of a precise and comprehensive mapping method greatly hinders studying the presence and biological significance of 6mA in mammals. Here, we develop a new method MM-seq (Modification-induced Mismatch Sequencing) for genome-wide 6mA mapping in single-base resolution. This method balances accuracy and sensitivity based on a novel detection principle. We find modified DNA bases are likely to flip out of the DNA duplex, forming a mismatch-like structure that an antibody can capture. The mismatch-like structure can be recognized by endonuclease or exonuclease, which marks the exact modified site for readout via sequencing. Using this method, we examine the genomic positions of 6mA in bacteria, green algae, and mammalian cells. We find that human and mouse genomes contain rare and randomly distributed 6mA sites, with almost no overlap between different cell types. After knock-out of the RNA m6A methyltransferase Mettl3, limited 6mA sites in gDNA are diminished. Our results support the perspective that rare 6mA in the mammalian genome is caused by the misincorporation of N6-methyladenine via the nucleotide-salvage pathway.

2023-01-11 | GSE171698 | GEO

High-precision mapping reveals rare N6-deoxyadenosine methylation in the mammalian genome

Project description:High-precision mapping reveals rare N6-deoxyadenosine methylation in the mammalian genome

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Base Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome

Project description:The study of 5-hydroxylmethylcytosines (5hmC), the sixth base of the mammalian genome, as an epigenetic mark has been hampered by a lack of method to map it at single-base resolution. Previous affinity purification-based methods could not precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach for mapping 5hmC at base resolution. Application of this new method to the embryonic stem cells not only confirms widespread distribution of 5hmC in mammalian genome, but also reveals a strong sequence bias and strand asymmetry at sites of 5hmC. Additionally, the relative abundance of 5hmC varies significantly depending on the types of functional sequences, suggesting different mechanisms for 5hmC deposition and maintenance. Furthermore, we observe high levels of 5hmC and reciprocally low levels of 5mC at transcription factor binding sites, revealing a dynamic DNA methylation process at cis-regulatory elements. Base resolution sequencing of 5 hydroxymethylcytosine in human and mouse embryonic stem cells

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Identification and Characterization of the Mammalian Nuclear RNA N6-Adenosine Methyltransferase Core Complex

Project description:N6-methyladenosine (m6A) is the most prevalent internal modification found in mammalian messenger and non-coding RNAs. The discoveries of functionally significant demethylases that reverse this methylation as well as the recently revealed m6A distributions in mammalian transcriptomes strongly indicate regulatory functions of this modification. Here we report the identification and characterization of the mammalian nuclear RNA N6-adenosine methyltransferase core (RNMTC) complex. Besides METTL3, a methyltransferase which was the only known component of RNMTC in the past, we discovered that a previously uncharacterized methyltransferase, METTL14, exhibits a N6-adenosine methyltransferase activity higher than METTL3. Together with WTAP, the third component that dramatically affects the cellular m6A level, these three proteins form the core complex that orchestrates m6A deposition on mammalian nuclear RNA. Biochemistry assays, imaging experiments, as well as transcriptome-wide analyses of the binding sites and their effects on m6A methylation support methylation function and reveal new insights of RNMTC. PAR-CLIP and m6A-seq in HeLa cells

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2012-05-17 | GSE36173 | GEO

An atlas of single-base-resolution N6-methyl-adenine methylomes redefines RNA demethylase function as suppressors of disruptive RNA methylation

Project description:An atlas of single-base-resolution N6-methyl-adenine methylomes redefines RNA demethylase function as suppressors of disruptive RNA methylation

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Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes.

Project description:Various methyltransferases and demethylases catalyse methylation and demethylation of N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) but precise methylomes uniquely mediated by each methyltransferase/demethylase are still lacking. Here, we develop m6A-Crosslinking-Exonuclease-sequencing (m6ACE-seq) to map transcriptome-wide m6A and m6Am at quantitative single-base-resolution. This allows for the generation of a comprehensive atlas of distinct methylomes uniquely mediated by every individual known methyltransferase or demethylase. Our atlas reveals METTL16 to indirectly impact manifold methylation targets beyond its consensus target motif and highlights the importance of precision in mapping PCIF1-dependent m6Am. Rather than reverse RNA methylation, we find that both ALKBH5 and FTO instead maintain their regulated sites in an unmethylated steady-state. In FTO's absence, anomalous m6Am disrupts snRNA interaction with nuclear export machinery, potentially causing aberrant pre-mRNA splicing events.

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An atlas of single-base-resolution N6-methyl-adenine methylomes redefines RNA demethylase function as suppressors of disruptive RNA methylation

Project description:Various methylases and demethylases catalyze methylation and demethylation of N6-methyladenosine (m6A) and N6,2?-O-dimethyladenosine (m6Am) but precise methylomes uniquely mediated by each methylase/demethylase are still lacking. Here, we developed m6A-Crosslinking-Exonuclease-sequencing (m6ACE-seq) to map m6A and m6Am at transcriptome-wide single-base-resolution. m6ACE-seq's ability to quantify relative differences in methylation levels across samples enabled the generation of a comprehensive atlas of distinct methylomes uniquely mediated by every individual known methylase/demethylase. We determined METTL16 to indirectly impact manifold methylation targets beyond its consensus target motif, and highlighted the importance of precision in mapping PCIF1-dependent m6Am. Rather than reverse RNA methylation, we found that both ALKBH5 and FTO demethylases instead maintain their regulated sites in an unmethylated steady-state. In FTO's absence, anomalous m6Am disrupts snRNA interaction with nuclear export machinery, potentially causing aberrant pre-mRNA splicing events. We propose a model whereby RNA demethylases ensure normal RNA metabolism by suppressing disruptive RNA methylation in the nucleus.

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Single-base resolution methylome of 5-methylcytosine in mammalian transcriptome

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