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

  • Nucleic Acids Res. 2016 Dec 1;44(21):10423-10436. doi: 10.1093/nar/gkw637.
Dhaval Varshney  1  2 ,  Alain-Pierre Petit  3 ,  Juan A Bueren-Calabuig  4  5 ,  Chimed Jansen  3 ,  Dan A Fletcher  3 ,  Mark Peggie  6 ,  Simone Weidlich  6 ,  Paul Scullion  3 ,  Andrei V Pisliakov  4  5 ,  Victoria H Cowling  7  2
Affiliations
  • 1. Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
  • 2. MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
  • 3. Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
  • 4. Computational Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
  • 5. Physics, School of Science and Engineering, University of Dundee, Nethergate, Dundee DD1 5EH, UK.
  • 6. Division of Signal Transduction Therapies, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
  • 7. Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK [email protected].
PMID: 27422871 DOI: 10.1093/nar/gkw637
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.