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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.

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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.

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