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. Author manuscript; available in PMC: 2018 Mar 9.
Published in final edited form as: Cell. 2017 Mar 9;168(6):1015–1027.e10. doi: 10.1016/j.cell.2017.02.019

Figure 2. Mammalian DXO possesses robust deNADding activity.

Figure 2

(A) In vitro decapping assays with 10nM mouse recombinant DXO protein and indicated 32P-cap-labeled RNA substrates. Reaction products were resolved by polyethyleneimine (PEI)-cellulose thin-layer chromatography (TLC) developed in 0.45 M (NH4)2SO4. (B) Schematic of NAD+-capped RNA and sites of DXO cleavage. The red “P” denotes the position of the 32P. (C) Quantitation from (A) carried out with ImageQuant software and plotted from three independent experiments with the error bars representing +/−SD. (D) Catalytically inactive mutants E234A and K256Q abolish DXO deNADding activity. (E) Mammalian Dcp2 does not possess intrinsic deNADding activity. Recombinant (50nM) human Dcp2, mouse DXO or bacteria NudC proteins were incubated with 32P-cap-labeled NAD-RNA at 37°C for 30 minutes. The reaction products were either untreated (−) or treated with 1U Nuclease P1 (+), resolved by PEI-TLC developed in 0.45 M (NH4)2SO4. Product standards were denoted on the right. The asterisks represent the position of the 32P-labeling. (F) Similar to B, except sites of NudC and Nuclease P1 (Nuc. P1) hydrolysis are denoted.