Identification Of RNA N~6-methyladenosine (m~6A) Methyltransferase Complex

M6A (N6-methyladenosine) is the most abundant internal modification on mRNA in eukaryotes, accounting for about 80% of all the methylation forms. The identification of m6A demethylases FTO (Fat mass and obesity associated) and ALKBH5 (a-ketoglutarate-dependent dioxygenase alkB homolog 5) indicates that RNA methylation is reversible and represents a novel epitranscriptomic mechanism just like DNA methylation, and it may function as a novel epitranscriptomic marker of profound biological significance. M6 A occurs in highly conserved regions with the consensus sequence RRm6ACH([G/A][G> A]m6AC[U>A> C]), while only a fraction of these consensus sequences are methylated, indicating that some new mechanisms responsible for its site specificity exist, whereby, the indentification of the compoments of m A methyltransferase complex could help to understand the site specificity of m6A.In eukyraotes, the formation of m6A has been reported to be catalyzed by a huge methyltransferase complex (about 1000 kDa), the nature of which is still not well-characterized. The mammalian m6A methyltransferase complex contains at least two key subcomplexes designated as MT-A (200 kDa) and MT-B (800 kDa). So far, METTL3 (methyltransferase like 3) (about 70 kDa) is the only component that has been identified in MT-A subcomplex. Our purpose is to dissect new components of m6A methyltransferase complex and the mechanisms how they modulate mRNA processing and metabolism.In this study, we discovered two new components of the human m6A methyltransferase complex, WTAP (Wilms'tumor 1-associating protein) and METTL14 (methyltransferase like 14) by using immunoprecipation combined with mass spectrum analysis. The METTL14 protein, also containing the SAM binding domain and the catalytic motif, shares about 43% identity with METTL3. Furthermore, immonofluoresence analysis revealed that WTAP is required for the subcellular localization of METTL3 and METTL14 into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo, which was further confirmed by PAR-CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) assay, in which the binding affinity of METTL3 to RNA decreases obviously in WTAP-deficient cells, suggesting that WTAP may function as a regulative subunit to recruit m A methyltransferase complex to mRNA targets.We also found that the majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m A motif by PAR-CLIP-seq. Furthermore, transcriptomic analyses in combination with PAR-CLIP illustrated that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. The phenotypes as a result of WTAP deletion can only be reversed by the N-terminus and the full-length of ectopic expressed WTAP, but not C-terminus.These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.