Fig. 2: Identification of NUDT2 as PPP-RNA phosphatase.

a RNA was incubated for 3 h with the indicated Nudix hydrolase, and the integrity of the RNA was determined using a Bioanalyzer. One representative experiment of three is shown. b γ-³²P-radiolabeled in vitro transcribed 44-mer ssRNA substrate was left untreated or incubated for 90 min with 600 nM NUDT2, NUDT12, NUDT14 or NUDT17 (+) or was left untreated (−). Reaction mixtures were analyzed by TLC and autoradiography. γ-³²P: radiolabeled γ-phosphate; RNA: remaining input RNA after incubation. c As (b) but incubation for the time indicated at the top of the panel. 600 nM NUDT2 were used. d As (c) but 600 nM NUDT2 E58A or NUDT2 were used. e As (b) but γ-³²P-radiolabeled in vitro transcribed single-stranded (ss-) or double-stranded (ds-) RNA generated by annealing an antisense oligo, or a RIG-I activating hairpin RNA (IVT4) were incubated with 600 nM NUDT2 and 5 U RppH, respectively. f Phylogenetic analysis of all human Nudix hydrolases and bacterial RppH was performed using MAFFT. g Structural alignment of the crystal structures of RppH (brown) with an RNA substrate (red sticks) (PDB: 4S2Y) and NUDT2 (blue) (PDB: 3U53) using PYMOL⁵⁷. The green-colored magnesium ions spheres are essential for hydrolase activity. h Visualization of the electrostatic surface charges of RppH and NUDT2 displayed with the aligned RNA of the RppH structure using PYMOL. Acidic amino acids are depicted in red, and basic residues are shown in blue.