Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
RNA
2018 May 01;245:633-642. doi: 10.1261/rna.065698.118.
Show Gene links
Show Anatomy links
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
.
???displayArticle.abstract???
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.
Arribas-Layton,
Structural and functional control of the eukaryotic mRNA decapping machinery.
2013, Pubmed
Arribas-Layton,
Structural and functional control of the eukaryotic mRNA decapping machinery.
2013,
Pubmed
Bessman,
The MutT proteins or "Nudix" hydrolases, a family of versatile, widely distributed, "housecleaning" enzymes.
1996,
Pubmed
Carreras-Puigvert,
A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family.
2017,
Pubmed
Cohen,
Dcp2 Decaps m2,2,7GpppN-capped RNAs, and its activity is sequence and context dependent.
2005,
Pubmed
Daniels,
Nudix hydrolases degrade protein-conjugated ADP-ribose.
2015,
Pubmed
Deshmukh,
mRNA decapping is promoted by an RNA-binding channel in Dcp2.
2008,
Pubmed
Franks,
The control of mRNA decapping and P-body formation.
2008,
Pubmed
Furuichi,
Viral and cellular mRNA capping: past and prospects.
2000,
Pubmed
Ghosh,
Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme.
2004,
Pubmed
,
Xenbase
Ghosh,
Enzymology of RNA cap synthesis.
2010,
Pubmed
Grudzien-Nogalska,
New insights into decapping enzymes and selective mRNA decay.
2017,
Pubmed
Iwasaki,
Characterization of Arabidopsis decapping proteins AtDCP1 and AtDCP2, which are essential for post-embryonic development.
2007,
Pubmed
Iyama,
NUDT16 is a (deoxy)inosine diphosphatase, and its deficiency induces accumulation of single-strand breaks in nuclear DNA and growth arrest.
2010,
Pubmed
Lange,
Nucleolar localization elements in U8 snoRNA differ from sequences required for rRNA processing.
1998,
Pubmed
,
Xenbase
Li,
Differential utilization of decapping enzymes in mammalian mRNA decay pathways.
2011,
Pubmed
Li,
Transcript-specific decapping and regulated stability by the human Dcp2 decapping protein.
2008,
Pubmed
Liu,
The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases.
2002,
Pubmed
Liu,
Functional analysis of mRNA scavenger decapping enzymes.
2004,
Pubmed
Lu,
hNUDT16: a universal decapping enzyme for small nucleolar RNA and cytoplasmic mRNA.
2011,
Pubmed
,
Xenbase
McLennan,
The Nudix hydrolase superfamily.
2006,
Pubmed
Meyer,
Messenger RNA turnover in eukaryotes: pathways and enzymes.
2004,
Pubmed
Mildvan,
Structures and mechanisms of Nudix hydrolases.
2005,
Pubmed
Milligan,
Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.
1987,
Pubmed
Niedzwiecka,
Biophysical approach to studies of cap-eIF4E interaction by synthetic cap analogs.
2007,
Pubmed
Palazzo,
Processing of protein ADP-ribosylation by Nudix hydrolases.
2015,
Pubmed
Peculis,
Metal determines efficiency and substrate specificity of the nuclear NUDIX decapping proteins X29 and H29K (Nudt16).
2007,
Pubmed
,
Xenbase
Piccirillo,
Functional characterization of the mammalian mRNA decapping enzyme hDcp2.
2003,
Pubmed
Scarsdale,
Crystal structures of U8 snoRNA decapping nudix hydrolase, X29, and its metal and cap complexes.
2006,
Pubmed
,
Xenbase
Schaeffer,
The exosome contains domains with specific endoribonuclease, exoribonuclease and cytoplasmic mRNA decay activities.
2009,
Pubmed
Song,
Multiple mRNA decapping enzymes in mammalian cells.
2010,
Pubmed
,
Xenbase
Steiger,
Analysis of recombinant yeast decapping enzyme.
2003,
Pubmed
Strenkowska,
Cap analogs modified with 1,2-dithiodiphosphate moiety protect mRNA from decapping and enhance its translational potential.
2016,
Pubmed
Taylor,
Evolutionary conservation supports ancient origin for Nudt16, a nuclear-localized, RNA-binding, RNA-decapping enzyme.
2008,
Pubmed
,
Xenbase
Trésaugues,
Structural Basis for the Specificity of Human NUDT16 and Its Regulation by Inosine Monophosphate.
2015,
Pubmed
Valkov,
Mille viae in eukaryotic mRNA decapping.
2017,
Pubmed
van Dijk,
Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures.
2002,
Pubmed
Wallace,
LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions.
1995,
Pubmed
