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

Dhaval Varshney,Dan A Fletcher,Paul Scullion,Victoria H Cowling,Simone Weidlich,Alain-Pierre Petit,Juan A Bueren-Calabuig,Mark Peggie,Andrei V Pisliakov,Chimed Jansen

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.

Highlights

Eukaryotic mRNA is modified by the addition of the 5 cap structure; 7-methylguanosine linked to the first transcribed nucleotide by a 5 -5 triphosphate bridge [1,2]
The presence of RNMT-Activating Miniprotein (RAM) does not alter the canonical Class I methyltransferase fold in RNA guanine-7 methyltransferase (RNMT), it allows the refinement of a lobe structure, which was absent in 3BGV
The RNMT–RAM crystal structure in conjunction with biochemical analyses and molecular dynamics simulations has revealed that RAM activates RNMT by stabilising the RNMT lobe and ␣-helix hinge, positioning adjacent helix A in the active site in a position favourable for substrate binding and the methylation reaction

Summary

Introduction

Eukaryotic mRNA is modified by the addition of the 5 cap structure; 7-methylguanosine linked to the first transcribed nucleotide by a 5 -5 triphosphate bridge [1,2]. The mRNA cap is formed on the first transcribed nucleotide of transcripts by three sequential enzymatic activities; triphosphatase, guanylyltransferase and methyltransferase [4,9]. The 5 triphosphate of pre-mRNA is hydrolyzed to diphosphate by a 5 -triphosphatase, to which GMP is added by the RNA guanylyltransferase to create the cap intermediate, GpppN. The cap intermediate is methylated by the RNA guanine N-7 methyltransferase, utilising the methyl donor, AdoMet, to create the mature cap, m7GpppN, and byproduct, AdoHcy (S-adenosyl homocysteine). These activities have different configurations in different eukaryotic species and viruses, ranging from all being present on a single peptide to all being present on distinct peptides. RNA guanylyltransferase and 5 triphosphatase (RNGTT/CE) caps the nascent transcript and RNA guanine-7 methyltransferase (RNMT) methylates the cap

Methods

Results

Conclusion