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

ABSTRACT: 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.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE63753 | GEO | 2015/06/29

SECONDARY ACCESSION(S): PRJNA268974

REPOSITORIES: GEO

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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.

2015-06-29 | E-GEOD-63753 | biostudies-arrayexpress

m6A in 5’ untranslated regions promotes cap-independent translation of mRNA

Project description:N6-methyladenosine (m6A) is a widespread internal RNA modification whose function is poorly understood. Here we report that m6A residues within the 5'UTR promote a novel form of cap-independent translation which is mediated through an interaction between m6A residues and the translation initiation factor, eIF3. We performed m6A profiling in cells subjected to heat shock stress and observed increased 5'UTR methylation after heat shock. For these studies, we used single-nucleotide resolution m6A mapping (miCLIP), which enables the detection of m6A residues separately from m6Am residues. Thus, the goal of these experiments was to identify m6A residues which are increased in the 5'UTR after heat shock stress.

2015-10-26 | GSE73405 | GEO

Identification of the m6Am Methyltransferase PCIF1 Reveals the Location and Functions of m6Am in the Transcriptome

Project description:mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2′-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to “internal” sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am’s function.

2019-07-03 | GSE122948 | GEO

Cap-specific terminal N6-methylation of RNA by an RNA polymerase II-associated methyltransferase.

Project description:N6-methyladenosine (m6A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6, 2'-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. However, its biogenesis and functional role remain elusive. Using a reverse genetics approach, we identified PCIF1, a factor that interacts with the serine-5-phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) responsible for N6-methylation of m6Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m6A formation. A transcriptome-wide analysis revealed that N6-methylation of m6Am promotes the translation of capped mRNAs. Thus, a cap-specific m6A writer promotes translation of mRNAs starting from m6Am.

2018-11-02 | GSE122071 | GEO

The mammalian cap-specific m6Am RNA methyltransferase PCIF1 regulates transcript levels in mouse tissues

Project description:The 5' end of eukaryotic mRNAs is protected by the m7G-cap structure. The transcription start site nucleotide is ribose methylated (Nm) in many eukaryotes, while an adenosine at this position is further methylated at the N6 position (m6A) by the mammalian Phosphorylated CTD-interacting Factor 1 (PCIF1) to generate m6Am. Here we show that while loss of cap-specific m6Am in mice does not affect viability or fertility, the Pcif1 mutants display reduced body-weight. Transcriptome analyses of mutant mouse tissues support a role for the cap-specific m6Am modification in stabilizing transcripts. In contrast, the Drosophila Pcif1 is catalytically-dead, but like its mammalian counterpart it retains the ability to associate with the Ser5-phosphorylated CTD of RNA pol II. Finally, we show that the Trypanosoma Pcif1 is an m6Am methylase that contributes to the N6,N6,2?-O-trimethyladenosine (m62Am) in the hypermethylated cap4 structure of Trypanosomatids. Thus, PCIF1 has evolved to function in catalytic and non-catalytic roles.

2020-08-28 | GSE151229 | GEO

Reversible methylation of m6Am in the 5′ cap controls mRNA stability

Project description:Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

2016-12-21 | GSE78040 | GEO

Recruitment of the m6A/Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48 [iCLIP-seq]

Project description:Fat mass and obesity-associated protein (FTO) can remove both the N6-methyladenosine (m6A) and N6, 2′-O-dimethyladenosine (m6Am) methylation marks that function in multiple aspects of posttranscriptional regulation. Here, we demonstrate that Zbtb48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of Zbtb48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. Zbtb48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO-dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2. Our findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which Zbtb48 co-ordinates RNA-binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.

2024-08-26 | GSE228607 | GEO

Recruitment of the m6A/Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48 [RNA-seq]

2024-08-26 | GSE228606 | GEO

Recruitment of the m6A/Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48 [ChIP-seq]

2024-08-26 | GSE228605 | GEO

m6Am-seq reveals the dynamic m6Am methylation in the human transcriptome

Project description:We report m6Am-seq, based on selective in vitro demethylation and RNA immunoprecipitation. m6Am-seq directly distinguishes m6Am and 5’-UTR N6-methyladenosine (m6A).

2021-07-19 | GSE180253 | GEO

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