Abstract
The RNA guanine-N7 methyltransferase (RNMT) in complex with RNMT-activating miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5′ end of nascent RNA polymerase II transcripts. The mRNA cap protects the primary transcript from exonucleases and recruits cap-binding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the network community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM. Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme’s active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.
Highlights
Enzymatic modification of the 5 -end of eukaryotic messenger RNA by the addition of a cap structure is a key process that provides protection against 5 -exonucleases and facilitates the export and translation of mRNA [1,2,3,4]
By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNA guanine-N7 methyltransferase (RNMT) by RNMT-activating miniprotein (RAM)
RNMT–RAM structure and conformational dynamics in the presence of ligands. accelerated molecular dynamics (aMD) simulations of the RNMT– RAM complex in the presence of AdoMet and cap showed a high degree of stability of the key RNMT structural elements (Figure 2B)
Summary
Enzymatic modification of the 5 -end of eukaryotic messenger RNA by the addition of a cap structure is a key process that provides protection against 5 -exonucleases and facilitates the export and translation of mRNA [1,2,3,4]. The cap is formed on the first transcribed nucleotide of the premRNA as a result of three consecutive enzymatic reactions: (i) the 5 end triphosphate of pre-mRNA is hydrolysed to diphosphate by a 5 -triphosphatase; (ii) GMP is added by the RNA guanylyltransferase to create the cap intermediate GpppN, with a 5 →5 triphosphate linkage and (iii) the RNA guanine-N7 methyltransferase (RNMT) catalyses the transfer of a methyl group from S-adenosylmethionine (AdoMet) to GpppN to create the mature cap, m7GpppN, and byproduct, AdoHcy (S-adenosyl homocysteine) (Figure 1A) [2] This capping mechanism targets the nascent RNA polymerase II transcripts (i.e. pre-mRNAs) and is conserved amongst eukaryotes and viruses, structural organization of enzymes varies between organisms, e.g. the capping process can be carried out by a single complex or multiple enzymes [2,5]. RAM is highly expressed in embryonic stem cells; during neural differentiation, RAM is repressed while high RNMT levels are maintained contributing to down regulation of pluripotency-
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