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PCIF1 Catalyzes m6Am mRNA Methylation to Regulate Gene Expression
Erdem Sendinc1, David Valle-Garcia1, Abhinav Dhall1
1Division of Newborn Medicine and Epigenetics Program, Department of Medicine, Boston Children's Hospital, Boston, MA 02115, USA; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
mRNA modifications play important roles in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5' ends of mRNAs. Furthermore, PCIF1 catalyzes only 5' m6Am methylation of capped mRNAs but not internal m6A methylation in vitro and in vivo. To study the biological role of m6Am, we developed a robust methodology (m6Am-Exo-Seq) to map its transcriptome-wide distribution, which revealed no global crosstalk between m6Am and m6A under assayed conditions, suggesting that m6Am is functionally distinct from m6A. Importantly, we find that m6Am does not alter mRNA transcription or stability but negatively impacts cap-dependent translation of methylated mRNAs. Together, we identify the only human mRNA m6Am methyltransferase and demonstrate a mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation.
Insights
Phosphorylated CTD Interacting Factor 1 (PCIF1) mediates N6,2-O-dimethyladenosine (m6Am) mRNA methylation. This conserved modification specifically impacts cap-dependent translation, offering a new gene expression regulatory mechanism.
Area of Science:
- Molecular Biology
- Epigenetics
- Gene Expression Regulation
Background:
- mRNA modifications are crucial for gene expression control.
- N6,2-O-dimethyladenosine (m6Am) is a prevalent mRNA modification.
- The enzymes and biological roles of m6Am remain largely uncharacterized.
Purpose of the Study:
- To identify the enzyme responsible for m6Am methylation.
- To map the transcriptome-wide distribution of m6Am.
- To elucidate the functional impact of m6Am on gene expression.
Main Methods:
- In vitro and in vivo enzymatic assays to characterize PCIF1 activity.
- Development of m6Am-Exo-Seq for transcriptome-wide m6Am mapping.
- Analysis of mRNA transcription, stability, and translation following m6Am modification.
Main Results:
- PCIF1 identified as the sole human mRNA m6Am methyltransferase.
- PCIF1 specifically catalyzes 5' m6Am methylation of capped mRNAs.
- m6Am distribution is distinct from m6A, with no global crosstalk observed.
- m6Am negatively regulates cap-dependent translation without affecting mRNA transcription or stability.
Conclusions:
- PCIF1 is an evolutionarily conserved methyltransferase for 5' m6Am.
- m6Am acts as a distinct epigenetic mark regulating translation.
- PCIF1-mediated m6Am methylation provides a novel mechanism for gene expression control.
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