Research Article

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

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Science  11 Jan 2019:
Vol. 363, Issue 6423, eaav0080
DOI: 10.1126/science.aav0080

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A cap-specific m6A writer

N6,2′-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. Akichika et al. quantified the abundance of this modification in the transcriptome and identified the writer protein, cap-specific adenosine methyltransferase (CAPAM), needed for this modification. CAPAM contains a unique structure that recognizes cap-specific N6-methyladenosine (m6A) as the substrate. The protein interacts with RNA polymerase II, suggesting that the modification occurs cotranscriptionally. The m6Am promotes the translation of capped mRNAs in a eIF4E-independent fashion.

Science, this issue p. eaav0080

Structured Abstract

INTRODUCTION

N6-methyladenosine (m6A), an abundant modification in eukaryotic mRNAs and long-noncoding RNAs, has been recognized as a major epitranscriptome mark that 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 site of capped mRNAs in vertebrates. Previous studies reported that an eraser protein, FTO, demethylates N6-methyl group of m6Am and destabilizes a subset of mRNAs, suggesting a possible function of m6Am in stabilizing A-starting capped mRNAs. However, the biogenesis and functional role of m6Am remain elusive.

RATIONALE

To reveal the functional and physiological roles of m6Am, it is necessary to identify a writer protein for N6-methylation of m6Am. We first established a highly sensitive method to analyze 5′-terminal chemical structures of the capped mRNAs using mass spectrometry (RNA-MS), and then measured m6Am methylation status accurately. We employed RNA-MS to identify the writer gene by a reverse genetic approach. We chose several candidates of uncharacterized methyltransferases (MTases) that are conserved in vertebrates, but not in yeast, which does not have m6Am. Each of the candidates was knocked out in human cells. If the target gene is disrupted, RNA-MS can detect the absence of m6Am in mRNAs prepared from the knockout cells.

RESULTS

RNA-MS analysis revealed that m6Am modification in human mRNAs is more abundant (92%) than previously estimated. We identified human PCIF1 as cap-specific adenosine-N6-MTase (CAPAM) responsible for N6-methylation of m6Am. Indeed, m6Am disappeared completely and converted to Am modification in mRNAs prepared from the CAPAM knockout (KO) cells. The CAPAM KO cells were viable, but sensitive to oxidative stress, implying the physiological importance of m6Am. We showed that CAPAM catalyzes N6-methylation of m6Am in the capped mRNAs in an S-adenosylmethionine (SAM)–dependent manner. A series of biochemical studies revealed that CAPAM specifically recognizes the 7-methylguanosine (m7G) cap structure and preferentially N6-methylates m7GpppAm rather than m7GpppA, indicating the importance of the 2′-O-methyl group of the target site for efficient m6Am formation. CAPAM has a N-terminal WW domain that specifically interacts with the Ser5-phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), suggesting that the CAPAM is recruited to the early elongation complex of RNAPII and introduces m6Am in a nascent mRNA chain cotranscriptionally. We also solved the crystal structure of CAPAM complexed with the cap analog and SAM analog. The core region of CAPAM is composed of MTase and helical domains. The m7G cap is bound to a pocket formed by these two domains. The SAM analog is recognized by an active site with the characteristic NPPF motif in the MTase domain. This structure reveals the molecular basis of cap-specific m6A formation. RNA-sequencing analysis of the CAPAM KO cells revealed that loss of m6Am does not affect transcriptome alteration. This result does not support the proposed function of m6Am in stabilizing A-starting capped mRNAs. Instead, ribosome profiling of the CAPAM KO cells showed that N6-methylation of m6Am promotes the translation of capped mRNAs.

CONCLUSION

We identified PCIF1/CAPAM as a cap-specific m6A writer for vertebrate mRNAs. Structural analysis revealed the molecular basis of cap-specific m6A formation by CAPAM. The ribosome profiling experiment revealed that CAPAM-mediated m6Am formation promotes translation of A-starting mRNAs, rather than stabilization of mRNAs.

Sequential and cotranscriptional m6Am formation mediated by CAPAM.

CAPAM is recruited to the early elongation stage of RNAPII through specific interaction between the WW domain and Ser5-phosphorylated CTD. The m7G cap MTase (RNMT) complexed with the capping enzyme (RNGTT) and 2′-O-MTase (CMTR1) are also recruited to this complex, indicating a hierarchical formation of m7Gpppm6Am—pppA, GpppA, m7GpppA, m7GpppAm, and m7Gpppm6Am.

Abstract

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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