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. Epub 2016 Jul 15.

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.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Binding Sites
  • Catalysis
  • Catalytic Domain
  • Enzyme Activation
  • Humans
  • Magnetic Resonance Spectroscopy
  • Methyltransferases / chemistry*
  • Methyltransferases / genetics
  • Methyltransferases / metabolism*
  • Models, Molecular
  • Mutation
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Stability
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Structure-Activity Relationship

Substances

  • RNA-Binding Proteins
  • Methyltransferases
  • RAMAC protein, human
  • mRNA (guanine(N7))-methyltransferase