Structure of METTL3-METTL14 with an m6A nucleotide reveals insights into m6A conversion and sensing.

The nuclear METTL3-METTL14 transfers a methyl group from SAM to convert the N ⁶ of adenosine (A) in RNA to m⁶A and in ssDNA to 6mA. m⁶A marks are prevalent in eukaryotic mRNAs and lncRNAs and modulate their stability and fate in a context-dependent manner. The cytoplasmic METTL3 can act as a m⁶A reader. However, the precise mechanism during m6A writing, reading, or sensing is unclear. Here, we present a ~2.5 Å structure of the methyltransferase core of human METTL3-METTL14 in complex with the reaction product mimic, N ⁶ -methyladenosine monophosphate (m⁶A), representing a state post-catalysis but before the release of m⁶A. m⁶A occupies an evolutionarily conserved RNA-binding pocket ~16 Å away from the SAM pocket that also frequently mutates in cancer. We propose a two-step model of swiveling of target A upon conversion to m⁶A and sensing its methylation status by this pocket, enabling it to actuate enzymes' switch from writer to an m⁶A-sensor. Cancer-associated mutations show impaired RNA binding dynamics, de-stacking, and defective m⁶A writing and sensing.