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 v.h.cowling@dundee.ac.uk.

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.

Name
Email *

	Sorry, but the email address you supplied was invalid.