Perturbation of m6A Writers Reveals Two Distinct Classes of mRNA Methylation at Internal and 5′ Sites

N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing “basal” degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification. [Display omitted] •METTL3, METTL14, WTAP, and KIAA1429 are required for mRNA methylation•Methylation maps, upon depletion of WTAP, reveal two classes of methylation•WTAP-dependent sites are mostly static and correlate with mRNA stability•Thousands of WTAP-independent sites are present at the first transcribed nucleotide N6-methyladenosine (m6A) is a highly abundant modification of mRNA. Schwartz et al. identify and validate a network of proteins required for mRNA methylation in mammalian cells. They define two distinct classes of methylation sites. The majority of sites depend on the identified proteins, are located at internal positions in transcripts, and inversely correlate with mRNA stability. Sites independent of these proteins form at the first transcribed base as part of the cap structure, forming a previously unappreciated layer of transcriptome complexity.