A cryptic pocket in METTL3-METTL14 regulates m6A conversion and sensing.

The nuclear METTL3-METTL14 enzyme complex transfers a methyl group from S-adenosyl-L-methionine (SAM) to the N 6 amino group of an adenosine (A) base in RNA to convert it to m 6 A and in ssDNA to 6mA. m 6 A 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 to regulate mRNA translation. However, the precise mechanism that actuates the switch from m 6 A writer to reader/sensor is unclear. Here, we present a ~2.5Å crystal structure of the methyltransferase core of human METTL3-METTL14 in complex with the reaction product, N 6 -methyladenosine monophosphate (m 6 A), representing a state post-catalysis but before the release of m 6 A. m 6 A occupies a novel evolutionarily conserved cryptic pocket in METTL3-METTL14 located ~16Å away from the SAM pocket that frequently mutates in cancer. We propose a two-step model of swiveling of target A upon conversion to m 6 A and sensing its methylation status by the cryptic pocket, enabling it to actuate enzymes' switch from writer to an m 6 A-sensor. Cancer-associated mutations cannot distinguish methylated from unmethylated adenine and show impaired RNA binding, de-stacking, and defective m 6 A writing and sensing.