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RNA
2018 May 01;245:633-642. doi: 10.1261/rna.065698.118.
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Hydrolytic activity of human Nudt16 enzyme on dinucleotide cap analogs and short capped oligonucleotides.
Grzela R, Nasilowska K, Lukaszewicz M, Tyras M, Stepinski J, Jankowska-Anyszka M, Bojarska E, Darzynkiewicz E.
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Human Nudt16 (hNudt16) is a member of the Nudix family of hydrolases, comprising enzymes catabolizing various substrates including canonical (d)NTPs, oxidized (d)NTPs, nonnucleoside polyphosphates, and capped mRNAs. Decapping activity of the Xenopus laevis (X29) Nudt16 homolog was observed in the nucleolus, with a high specificity toward U8 snoRNA. Subsequent studies have reported cytoplasmic localization of mammalian Nudt16 with cap hydrolysis activity initiating RNA turnover, similar to Dcp2. The present study focuses on hNudt16 and its hydrolytic activity toward dinucleotide cap analogs and short capped oligonucleotides. We performed a screening assay for potential dinucleotide and oligonucleotide substrates for hNudt16. Our data indicate that dinucleotide cap analogs and capped oligonucleotides containing guanine base in the first transcribed nucleotide are more susceptible to enzymatic digestion by hNudt16 than their counterparts containing adenine. Furthermore, unmethylated dinucleotides (GpppG and ApppG) and respective oligonucleotides (GpppG-16nt and GpppA-16nt) were hydrolyzed by hNudt16 with greater efficiency than were m7GpppG and m7GpppG-16nt. In conclusion, we found that hNudt16 hydrolysis of dinucleotide cap analogs and short capped oligonucleotides displayed a broader spectrum specificity than is currently known.
Figure 1. Structures of the dinucleotides used in the present study.
Figure 2. HPLC profiles of m7Gpppm2′-OG hydrolyzed with recombinant hNudt16: (A) before hydrolysis, (B) after 60 min of hydrolysis, (C) after 100 min of hydrolysis. Reaction was performed at 37°C, in 40 mM Tris buffer (pH 7.9) containing 100 mM NaCl, 6 mM MgCl2, and 2 mM DTT.
Figure 3. Hydrolysis of capped oligonucleotides by hNudt16. (A) Gel electrophoretic analysis of the progress of the hydrolysis of GpppG-16nt and GpppA-16nt. For comparison, reaction with Dcp1/2 was performed for m7GpppG-16nt. (B) Gel electrophoretic analysis of 16-nt oligonucleotides bearing different cap structures. (C) Comparison of decapping yield for capped and uncapped 16-nt oligonucleotides. (D) Gel electrophoretic analysis of decapping for methylated and unmethylated 34-nt oligonucleotides. All oligonucleotides were treated with recombinant hNudt16 at 30°C in 50 mM Tris buffer (pH 7.9) containing 10 mM NaCl, 6 mM MgCl2, 10 mM DTT, and 1 mM spermidine. The decapping percentage was calculated as the percent loss in the capped band, normalized by total quantity in the capped and decapped bands.
Figure 4. Interactions between Nudt16 and its ligands. (A) (Upper panel) Visualization of the GTP-bound XL Nudt16 active site (PDB 2A8S). Color codes for the protein, ligand, and metal ions are indicated. For clarity, only interactions with the base and selected residues are shown. (Lower panel) Ligplot (Wallace et al. 1995) representation of the structure shown in the upper panel. (B) (Upper panel) Visualization of the XL Nudt16 active site with m7GpppA. Color codes are the same as those in A. (C) (Upper panel) Visualization of the IMP-bound hNudt16 active site (PDB: 2XSQ). (Lower panel) Ligplot representation of the structure shown in the upper panel.
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