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

Abstract

-methyladenine (mA) is found on many eukaryotic RNAs including mRNAs. mA modification has been implicated in mRNA stability and turnover, localization, or translation efficiency. A heterodimeric enzyme complex composed of METTL3 and METTL14 generates mA 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.

RNA N6-methyladenosine: a promising molecular target in metabolic diseases

“…m 6 A modification is highly involved in RNA stability, localization, turnover and translation efficiency, which is crucial for the biological functions [128]. The mRNA m 6 A methylation has a wide range of effects on MDs.…”

Adenosine methylation as a molecular imprint defining the fate of RNA

“…Future studies should address how the m 6 A machinery is coupled to transcription. For example METTL3 has been shown to be post‐translationally modified, phosphorylated and SUMOylated at several sites . Such modification could impact catalytic activity, as has been shown when METTL3 is SUMOylated, as well as binding to interaction partners.…”

“…Identification of the proteins required for m 6 A installation enabled a subcellular interrogation of this process. Using microscopy, the METTL3/METTL14 dimer and associated factor WTAP were localized to nuclear speckles . Interestingly, WTAP knockdown in human cells resulted in loss of endogenous METTL3/METTL14 nuclear localization and not vice versa .…”

Enzymatischer oder In‐vivo‐Einbau von Propargylgruppen in Kombination mit Klick‐Chemie zur Anreicherung und Detektion von Methyltransferase‐Zielsequenzen in RNA

“…[15] Tatsächlich konnte mittels LC-MS-Analytik N 6 -Propargyladenosin in einer Substrat-RNAa us 27 Nukleotiden nachgewiesen werden, was bestätigt, dass SeAdoYn als Cosubstrat akzeptiert wird (Abbildung 1E,A bbildungen S7 und S8, Ta-belle S1). [15] Tatsächlich konnte mittels LC-MS-Analytik N 6 -Propargyladenosin in einer Substrat-RNAa us 27 Nukleotiden nachgewiesen werden, was bestätigt, dass SeAdoYn als Cosubstrat akzeptiert wird (Abbildung 1E,A bbildungen S7 und S8, Ta-belle S1).…”

“…Um herauszufinden, ob diese nichtnatürlichen Modifikationen auch enzymatisch eingebracht werden kçnnen, haben wir AdoMet-Analoga synthetisiert und getestet, ob diese von METTL3-METTL14 umgesetzt werden, das wir rekombinant in Sf21-Insektenzellen produziert haben (Abbildung S5). [15] Tatsächlich konnte mittels LC-MS-Analytik N 6 -Propargyladenosin in einer Substrat-RNAa us 27 Nukleotiden nachgewiesen werden, was bestätigt, dass SeAdoYn als Cosubstrat akzeptiert wird (Abbildung 1E,A bbildungen S7 und S8, Ta-belle S1). AdoPropen wurde ebenfalls umgesetzt, allerdings weniger effizient.…”