N6-methyladenosine (m6A) is one of the most prevalent RNA modifications present within messenger RNAs (mRNAs), with an average of 3-5 modified adenosines per transcript. The methyltransferase-like protein 3 (METTL3) and methyltransferase-like protein 14 (METTL14) have been reported to form a sta- ble dimeric complex that is responsible for the formation of m6A within mRNAs. The METTL14 subunit of this methyltransferase complex has been proposed to be the catalytic subunit. This conclusion was, however, drawn from analyzing each subunit separately and not within the context of the assembled complex. Here, point mutations within METTL3 resulted in the complete loss of catalytic activity within the binary complex, whereas no such effect was observed when mutating METTL14. In addition to this, cross-linking experiments using tritiated S-adenosyl-methionine (SAM) and recombinantly purified METTL3/14 suggest that METTL3 is also the only subunit that can efficiently bind to the methyl donor. A third aspect that was analyzed in this work was if the METTL3/14 complex can specifically recognize its RNA substrate. Electrophoretic mobility shift assays (EMSAs) that were conducted suggest that the pro- tein complex mostly binds to polyanionic molecules in an unspecific manner and that target specificity is introduced by other means.
During the experimental phase of this work, not much was known about the architecture of the methyl- transferase. Here,experiments using truncation constructs of METTL14 provide evidence that the MT- A70 domain of this protein is essential for the dimerization with METTL3. An additional protein, namely the Wilms’ tumor 1-associating protein (WTAP), has been reported to stably associate with the methyl- transferase complex through a direct interaction with METTL3. This interaction between METTL3 and WTAP was also examined in this work. The data collected from immunoprecipitation experiments could demonstrate that the first N-terminal α-helix within METTL3 binds to a small protein region within WTAP’s N-terminus. Closer examination of these identified interaction surfaces led to the hypothesis that most likely coiled-coils promote the observed METTL3-WTAP interaction.
All of the above investigated proteins are localized within the cell nucleus. Many nuclear proteins rely on a nuclear localization signal (NLS) for them to be translocated across the nuclear envelope. Here, mutational analysis revealed that WTAP and METTL3 each possess a classical NLS sequence that is func- tional. For METTL14, this work provides evidence that METTL14 does not have a NLS embedded within its primary sequence and that its localization depends on the heterodimerization with METTL3.

N6-methyladenosine (m6A) is one of the most prevalent RNA modifications present within messenger RNAs (mRNAs), with an average of 3-5 modified adenosines per transcript. The methyltransferase-like protein 3 (METTL3) and methyltransferase-like protein 14 (METTL14) have been reported to form a sta- ble dimeric complex that is responsible for the formation of m6A within mRNAs. The METTL14 subunit of this methyltransferase complex has been proposed to be the catalytic subunit. This conclusion was, however, drawn from analyzing each subunit separately and not within the context of the assembled complex. Here, point mutations within METTL3 resulted in the complete loss of catalytic activity within the binary complex, whereas no such effect was observed when mutating METTL14. In addition to this, cross-linking experiments using tritiated S-adenosyl-methionine (SAM) and recombinantly purified METTL3/14 suggest that METTL3 is also the only subunit that can efficiently bind to the methyl donor. A third aspect that was analyzed in this work was if the METTL3/14 complex can specifically recognize its RNA substrate. Electrophoretic mobility shift assays (EMSAs) that were conducted suggest that the pro- tein complex mostly binds to polyanionic molecules in an unspecific manner and that target specificity is introduced by other means.
During the experimental phase of this work, not much was known about the architecture of the methyl- transferase. Here,experiments using truncation constructs of METTL14 provide evidence that the MT- A70 domain of this protein is essential for the dimerization with METTL3. An additional protein, namely the Wilms’ tumor 1-associating protein (WTAP), has been reported to stably associate with the methyl- transferase complex through a direct interaction with METTL3. This interaction between METTL3 and WTAP was also examined in this work. The data collected from immunoprecipitation experiments could demonstrate that the first N-terminal α-helix within METTL3 binds to a small protein region within WTAP’s N-terminus. Closer examination of these identified interaction surfaces led to the hypothesis that most likely coiled-coils promote the observed METTL3-WTAP interaction.
All of the above investigated proteins are localized within the cell nucleus. Many nuclear proteins rely on a nuclear localization signal (NLS) for them to be translocated across the nuclear envelope. Here, mutational analysis revealed that WTAP and METTL3 each possess a classical NLS sequence that is func- tional. For METTL14, this work provides evidence that METTL14 does not have a NLS embedded within its primary sequence and that its localization depends on the heterodimerization with METTL3.