title: Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM.
abstract: 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.
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.|MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Dhaval
authors.initials: D
authors.lastname: Varshney
authors.affiliation: Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Alain-Pierre
authors.initials: AP
authors.lastname: Petit
authors.affiliation: Computational Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.|Physics, School of Science and Engineering, University of Dundee, Nethergate, Dundee DD1 5EH, UK.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Bueren-Calabuig
authors.affiliation: Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Chimed
authors.initials: C
authors.lastname: Jansen
authors.affiliation: Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Dan A
authors.initials: DA
authors.lastname: Fletcher
authors.affiliation: Division of Signal Transduction Therapies, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Peggie
authors.affiliation: Division of Signal Transduction Therapies, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Simone
authors.initials: S
authors.lastname: Weidlich
authors.affiliation: Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Scullion
authors.affiliation: Computational Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.|Physics, School of Science and Engineering, University of Dundee, Nethergate, Dundee DD1 5EH, UK.
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Pisliakov
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK v.h.cowling@dundee.ac.uk.|MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
authors.forename: Victoria H
authors.initials: VH
authors.lastname: Cowling
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D004789:Enzyme Activation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D016601:RNA-Binding Proteins; D008780:Methyltransferases; C569288:RAMAC protein, human; C055097:mRNA (guanine(N7))-methyltransferase