C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system

Jaïs, Philippe H; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila; Verdier, Fréderique; Canard, Bruno; Goncalves, Sergio; Chiron, Stéphane; Le Gall, Maude; Mayeux, Patrick; Shmulevitz, Maya

NP868R capping enzyme was previously shown to form a covalent link with GTP, an activity required for GTase activity (48) , but RTPase and N7-MTase activities were only inferred from sequence annotation (59) . To test if NP868R has all bona fide enzymatic activities, we expressed and purified histidine-tagged NP868R by baculovirus expression system and Ni 2+ -affinity chromatography, respectively ( Figure 5A ). The NTPase activity of NP868R was evidenced by in vitro conversion assay of [␣-32 P]-GTP or ATP into GDP or ATP. Figure 5B shows that NP868R displayed NTPase activity similar to that of the VVD1R subunit, which hydrolyses [␣-32 P]-GTP into GDP, or ATP into ADP (not shown). The GTase activity results from two steps reaction process in which the GTase first form a covalent link with GMP which is subsequently transferred on the 5 end of pRNA. The first step of this process (GTP-covalent binding) was demonstrated through incubation of NP868R with [␣-32 P]-GTP and MgCl 2 . A radiolabeled 99 kDa protein was observed under denaturing electrophoresis conditions, suggesting that NP868R forms a covalent adduct with the guanosine nucleotide similar to VVD1R and VVD1R-MTase ( Figure 5C ). The subsequent transfer of GMP to the 5 -end of RNAs was next monitored by incubating NP868R with synthetic 3 -end radiolabeled RNAs (pppAC 4 Cp), followed by urea-PAGE RNA product analysis. In presence of MgCl 2, NP868R induced a band-shift of RNA products. Migration patterns were similar to those observed when using VVD1R and VVD1R-MTase as positive controls, suggesting similar modification at the 5 -end of RNA blocked at their 3 -end by pCp labeling ( Figure 5D ). The nature of the cap structure was further analyzed by thin layer chromatography after RNA hydrolysis by nuclease P1. In the presence of the methyl donor S-Adenosyl-Methionine (SAM) and MgCl 2 , the positive control VVD1R, as well as NP868R could synthesize a m7 GpppA cap structure ( Figure 5E ), indicating that NP868R carries both N7-MTase and GTase activities. NP868R methylated GpppAC 4 , whereas a blocked N7-atom in the synthetic capped RNA ( m7 GpppAC 4 substrate) abolished 3 H-methyl transfer. NP868R thus carries N7-MTase but not ribose-2 -O-methyltransferase activities on these short substrates ( Figure 5F ). Altogether, these assays demonstrate that NP868R is a fully active capping enzyme.