Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM. Issue 21 (15th July 2016)

• Record Type: Journal Article
• Authors: Varshney, Dhaval; Petit, Alain-Pierre; Bueren-Calabuig, Juan A.; Jansen, Chimed; Fletcher, Dan A.; Peggie, Mark; Weidlich, Simone; Scullion, Paul; Pisliakov, Andrei V.; Cowling, Victoria H.
• Is Part Of: Nucleic acids research. Volume 44:Issue 21(2016)
• Page Start: 10423
• Page End: 10436
• Publication Date: 2016-07-15
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkw637
• Languages: English
• ISSNs: 0305-1048

Abstract

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.