Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM.

Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosinecap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase(RNMT), has an activating subunit, RNMT-Activating Miniprotein ( RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAMbinds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAMand is required for RAMbinding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAMstabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAMincreases the recruitment of the methyl donor, AdoMet ( S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAMallosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.