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Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution

ABSTRACT: Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution

PROVIDER: PRJNA506999 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8244730_1.fastq.gz	Fastqsanger.gz
	SRR8244730_2.fastq.gz	Fastqsanger.gz
	SRR8244740_1.fastq.gz	Fastqsanger.gz
	SRR8244740_2.fastq.gz	Fastqsanger.gz
	SRR8244750_1.fastq.gz	Fastqsanger.gz

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Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution [III]

Project description:Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution [III]

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Project description:Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution [II]

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Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution [III]

Project description:N6-methyladenosine (m6A) is the most abundant modification on mRNA, and is implicated in critical roles in development, physiology and disease. A major challenge in the field has been the inability to quantify m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MASTER-seq for systematic quantitative profiling of m6A at single nucleotide resolution, building on differential cleavage by an RNAse at methylated sites. MASTER-seq permitted validation and de novo discovery of m6A sites, calibration of the performance of antibody based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is ‘hard-coded’ in cis via a simple and predictable code. This code accounts for ~50% of the variability in methylation levels and allows accurate prediction of m6A loss/acquisition events across evolution. MASTER-seq will allow quantitative investigation of m6A regulation in diverse cell types and disease states.

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Single-nucleotide resolution mapping of m6A and m6Am throughout the transcriptome

Project description:N6-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate and function. Current m6A mapping approaches rely on immunoprecipitation of m6A-containing RNA fragments to identify regions of transcripts that contain m6A. This approach localizes m6A residues to 100-200 nt-long regions of transcripts. The precise position of m6A in mRNAs cannot be identified on a transcriptome-wide level because there are no chemical methods to distinguish between m6A and adenosine. Here we show that anti-m6A antibodies can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription. Similarly, we find these antibodies induce mutational signatures at N6, 2’-O-dimethyladenosine (m6Am), a nucleotide found at the first encoded position of certain mRNAs. Using these mutational signatures, we map m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identify snoRNAs as a novel class of m6A-containing ncRNAs. UV-crosslinking and immunoprecipitation with m6A-specific antibodies was used to map m6A and m6Am in cellular RNA with single nucleotide resolution.

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