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15 Jul 2016

Maturation and translation of mRNA in eukaryotes requires the addition of the 7-​methylguanosine cap.  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 RAM binds 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 RAM and is required for RAM binding.  Structure-​guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises 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 RAM increases the recruitment of the methyl donor, AdoMet (S-​adenosyl methionine)​, to RNMT.  Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.