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

ABSTRACT: 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.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE124509 | GEO | 2019/09/20

REPOSITORIES: GEO

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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

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

m6A-seq data analysis of control and PCIF1 knockdown transcriptome

Project description:Purpose: To identify the in vivo RNA targets of PCIF1, we performed meRIP experiments for PCIF1 KD and control cells using an anti-m6A antibody. We envisioned that the cap-specific PCIF1 should selectively alter the m6Am modification at the 5-terminal region of transcripts. Indeed, we observed a reduction of modification peaks at the 5’-end but not the 3’-UTR regions of mRNA upon PCIF1 KD.

2018-12-20 | GSE120229 | GEO

HIV reprograms host m6Am RNA methylome by viral Vpr protein-mediated degradation of PCIF1

Project description:To identify m6Am genes that are responsible for HIV inhibition, m6Am-exo-seq was performed in control, PCIF1 KO T cells, and cells infected with HIV.

2021-08-09 | GSE171800 | GEO

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

FTO-mediated cytoplasmic m6Am demethylation adjusts stem-like properties in colorectal cancer cell

Project description:Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications dynamic in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its m6Am (N6,2'-O-dimethyladenosine) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a novel, reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.

2021-01-20 | GSE165115 | GEO

m6Am RNA methylation enzyme PCIF1 restricts HIV infection and is degraded by viral Vpr protein

Project description:To identify m6Am genes that are responsible for HIV inhibition, MeRIP-seq was performed in control and PCIF1 KO T cells infected with HIV.

2021-08-09 | GSE154035 | GEO

Effect of PCIF1 knockdown on gene exppression in HeLa S3 cells

Project description:We previously identified PCIF1 (Phosphorylated CTD Interacting Factor 1) as a novel phosphorylated C-terminal domain (CTD) of RNA polymerase II. We also recently identified PCIF1 as a new cap-specific adenine N6 methyltransferase (CAPAM) responsible for creating N6, 2’-O-dimethyladenosine (m6Am) at the 5’-end of mRNAs. However, it remains unclear how PCIF1 regulates gene expression. To identify genes whose expression levels are affected by PCIF1 knockdown in human cultured cells, we performed gene expression profiling by microarray analysis.

2023-09-30 | GSE156768 | GEO

Arabidopsis Messenger RNA N6-methyladenosine Demethylation Regulates Plant Development

Project description:N6-methyladenosine (m6A) is the most abundant internal modification in the messenger RNA (mRNA) of all higher eukaryotes. This modification has been shown to be reversible in mammals; it is installed by a methyltransferase heterodimer complex of METTL3 and METTL14 bound with WTAP, and reversed by iron(II)- and Î±-ketoglutarate-dependent demethylases FTO and ALKBH5. This modification exhibits significant functional roles in various biological processes. The m6A modification as a RNA mark is recognized by reader proteins, such as YTH domain family proteins and HNRNPA2B1; m6A can also act as a structure switch to affect RNA-protein interactions for biological regulation. In Arabidopsis thaliana, the methyltransferase subunit MTA (the plant orthologue of human METTL3, encoded by At4g10760) was well characterized and FIP37 (the plant orthologue of human WTAP) was first identified as the interacting partner of MTA. Here we report the discovery and characterization of reversible m6A methylation mediated by AtALKBH10B (encoded by At4g02940) in A. thaliana, and noticeable roles of this RNA demethylase in affecting plant development and floral transition. Our findings reveal potential broad functions of reversible mRNA methylation in plants. m6A peaks were identified from wild type Columbia-0 and atalkbh10b-1 mutant in two biological replicates

2016-03-24 | E-GEOD-79523 | biostudies-arrayexpress

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