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Mol Cell. 2019 Aug 8;75(3):631-643.e8. doi: 10.1016/j.molcel.2019.06.006. Epub 2019 Jul 3.

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

Author information

1
Division of Newborn Medicine, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
2
Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY 10065, USA; Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, 02-109 Warsaw, Poland.
3
Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY 10065, USA.
4
IBMM, Université de Montpellier, CNRS, ENSCM, Montpellier, France.
5
NCBI, National Library of Medicine, NIH, Bethesda, MD 20894, USA.
6
Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY 10065, USA. Electronic address: srj2003@med.cornell.edu.
7
Division of Newborn Medicine, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Electronic address: eric.greer@childrens.harvard.edu.

Abstract

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.

KEYWORDS:

PCIF1; m(6)A; m(6)Am; mRNA methylation; mRNA stability; mRNA translation

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