Interactions, localization, and phosphorylation of the m6A generating METTL3–METTL14–WTAP complex

• DOI: 10.1261/rna.064063.117
• Publication Date: 2018-01-19T01:35:14Z
• Authors: Eva Schöller; Franziska Weichmann; Thomas Treiber; Sam Ringle; Nora Treiber; Andrew Flatley; Regina Feederle; Astrid Bruckmann; Gunter Meister
• Keywords: 0301 basic medicine; Adenosine; Nuclear Proteins; RNA-Binding Proteins; Cell Cycle Proteins; Methyltransferases; Article; Recombinant Proteins; 03 medical and health sciences; M6a ; Methyltransferase ; Mettl14 ; Mettl3 ; Rna Modification; HEK293 Cells; Catalytic Domain; Cell Line, Tumor; Multiprotein Complexes; Protein Interaction Mapping; Humans; Amino Acid Sequence; RNA Splicing Factors; RNA, Messenger; Phosphorylation; RNA Processing, Post-Transcriptional; HeLa Cells

N6-methyladenine (m6A) is found on many eukaryotic RNAs including mRNAs. m6A modification has been implicated in mRNA stability and turnover, localization, or translation efficiency. A heterodimeric enzyme complex composed of METTL3 and METTL14 generates m6A on mRNAs. METTL3/14 is found in the nucleus where it is localized to nuclear speckles and the splicing regulator WTAP is required for this distinct nuclear localization pattern. Although recent crystal structures revealed how the catalytic MT-A70 domains of METTL3 and METTL14 interact with each other, a more global architecture including WTAP and RNA interactions has not been reported so far. Here, we used recombinant proteins and mapped binding surfaces within the METTL3/14-WTAP complex. Furthermore, we identify nuclear localization signals and identify phosphorylation sites on the endogenous proteins. Using an in vitro methylation assay, we confirm that monomeric METTL3 is soluble and inactive while the catalytic center of METTL14 is degenerated and thus also inactive. In addition, we show that the C-terminal RGG repeats of METTL14 are required for METTL3/14 activity by contributing to RNA substrate binding. Our biochemical work identifies characteristic features of METTL3/14-WTAP and reveals novel insight into the overall architecture of this important enzyme complex.