A cryptic pocket in METTL3-METTL14 regulates m(6)A 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 m(6)A 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.