Interactions, localization, and phosphorylation of the m6A generating METTL3-METTL14-WTAP complex.
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.
['Schöller|Eva|E|', 'Weichmann|Franziska|F|', 'Treiber|Thomas|T|', 'Ringle|Sam|S|', 'Treiber|Nora|N|', 'Flatley|Andrew|A|', 'Feederle|Regina|R|', 'Bruckmann|Astrid|A|', 'Meister|Gunter|G|']
D000241:Adenosine D000595:Amino Acid Sequence D020134:Catalytic Domain D018797:Cell Cycle Proteins D045744:Cell Line, Tumor D057809:HEK293 Cells D006367:HeLa Cells D006801:Humans D008780:Methyltransferases D046912:Multiprotein Complexes D009687:Nuclear Proteins D010766:Phosphorylation D025941:Protein Interaction Mapping D012323:RNA Processing, Post-Transcriptional D000072260:RNA Splicing Factors D012333:RNA, Messenger D016601:RNA-Binding Proteins D011994:Recombinant Proteins
m6A METTL3 METTL14 RNA modification methyltransferase