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• W2047479529 abstract "Regulation of RNA degradation plays an important role in the control of gene expression. One mechanism of eukaryotic mRNA decay proceeds through an initial deadenylation followed by 5′ end decapping and exonucleolytic decay. Dcp2 is currently believed to be the only cytoplasmic decapping enzyme responsible for decapping of all mRNAs. Here we report that Dcp2 protein modestly contributes to bulk mRNA decay and surprisingly is not detectable in a subset of mouse and human tissues. Consistent with these findings, a hypomorphic knockout of Dcp2 had no adverse consequences in mice. In contrast, the previously reported Xenopus nucleolar decapping enzyme, Nudt16, is an ubiquitous cytoplasmic decapping enzyme in mammalian cells. Like Dcp2, Nudt16 also regulates the stability of a subset of mRNAs including a member of the motin family of proteins involved in angiogenesis, Angiomotin-like 2. These data demonstrate mammalian cells possess multiple mRNA decapping enzymes, including Nudt16 to regulate mRNA turnover. Regulation of RNA degradation plays an important role in the control of gene expression. One mechanism of eukaryotic mRNA decay proceeds through an initial deadenylation followed by 5′ end decapping and exonucleolytic decay. Dcp2 is currently believed to be the only cytoplasmic decapping enzyme responsible for decapping of all mRNAs. Here we report that Dcp2 protein modestly contributes to bulk mRNA decay and surprisingly is not detectable in a subset of mouse and human tissues. Consistent with these findings, a hypomorphic knockout of Dcp2 had no adverse consequences in mice. In contrast, the previously reported Xenopus nucleolar decapping enzyme, Nudt16, is an ubiquitous cytoplasmic decapping enzyme in mammalian cells. Like Dcp2, Nudt16 also regulates the stability of a subset of mRNAs including a member of the motin family of proteins involved in angiogenesis, Angiomotin-like 2. These data demonstrate mammalian cells possess multiple mRNA decapping enzymes, including Nudt16 to regulate mRNA turnover. Dcp2 decapping enzyme in differentially expressed and not detectable in a subset of mammalian tissues Mammalian cells contain multiple decapping enzymes Nudt16 is a second cytoplasmic mRNA decapping enzyme in mammalian cells Regulation of RNA degradation plays an important role in the control of gene expression. In eukaryotic cells, most mRNAs have a 5′ monomethyl guanosine cap structure (Shatkin, 1976Shatkin A.J. Capping of eucaryotic mRNAs.Cell. 1976; 9: 645-653Abstract Full Text PDF PubMed Scopus (692) Google Scholar) and a 3′ poly(A) tail (Sachs, 1993Sachs A.B. Messenger RNA degradation in eukaryotes.Cell. 1993; 74: 413-421Abstract Full Text PDF PubMed Scopus (758) Google Scholar) which are important for mRNA translation and stability. Bulk mRNA decay usually initiates with the removal of the 3′ poly(A) tail (Decker and Parker, 1993Decker C.J. Parker R. A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation.Genes Dev. 1993; 7: 1632-1643Crossref PubMed Scopus (503) Google Scholar). The resulting deadenylated mRNAs can be degraded by two exonucleolytic pathways involving either 5′ end or 3′ end decay (Badis et al., 2004Badis G. Saveanu C. Fromont-Racine M. Jacquier A. Targeted mRNA degradation by deadenylation-independent decapping.Mol. Cell. 2004; 15: 5-15Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, Cougot et al., 2004Cougot N. Babajko S. Seraphin B. Cytoplasmic foci are sites of mRNA decay in human cells.J. Cell Biol. 2004; 165: 31-40Crossref PubMed Scopus (474) Google Scholar). The 3′ end decay is carried out by a cytoplasmic multisubunit exosome complex (Anderson and Parker, 1998Anderson J.S.J. Parker R.P. The 3′ to 5′ degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3′ to 5′ exonucleases of the exosome complex.EMBO J. 1998; 17: 1497-1506Crossref PubMed Scopus (502) Google Scholar, Mitchell et al., 1997Mitchell P. Petfalski E. Shevchenko A. Mann M. Tollervey D. The exosome: a conserved eukaryotic RNA processing complex containing multiple 3′→5′ exoribonucleases.Cell. 1997; 91: 457-466Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar) and the resulting cap dinucleotide subsequently hydrolyzed by the scavenger decapping enzyme, DcpS (Liu et al., 2002Liu H. Rodgers N.D. Jiao X. Kiledjian M. The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases.EMBO J. 2002; 21: 4699-4708Crossref PubMed Scopus (194) Google Scholar, Wang and Kiledjian, 2001Wang Z. Kiledjian M. Functional link between the mammalian exosome and mRNA decapping.Cell. 2001; 107: 751-762Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Alternatively, following deadenylation, the mRNA can be decapped to remove the m7GDP of the cap to expose a monophosphorylated 5′ end, which is subsequently degraded by the 5′ monophospate-dependent 5′ to 3′ exoribonuclease, Xrn1 (Decker and Parker, 1993Decker C.J. Parker R. A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation.Genes Dev. 1993; 7: 1632-1643Crossref PubMed Scopus (503) Google Scholar, Hsu and Stevens, 1993Hsu C.L. Stevens A. Yeast cells lacking 5′→3′ exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5′ cap structure.Mol. Cell. Biol. 1993; 13: 4826-4835Crossref PubMed Scopus (312) Google Scholar). Removal of the 5′ cap structure (decapping) is therefore an important prerequisite for decay of the mRNA body from the 5′ end. The Dcp2 protein has been identified as the major mRNA decapping enzyme in cells (Dunckley and Parker, 1999Dunckley T. Parker R. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif.EMBO J. 1999; 18: 5411-5422Crossref PubMed Scopus (262) Google Scholar, Lykke-Andersen, 2002Lykke-Andersen J. Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay.Mol. Cell. Biol. 2002; 22: 8114-8121Crossref PubMed Scopus (288) Google Scholar, van Dijk et al., 2002van Dijk E. Cougot N. Meyer S. Babajko S. Wahle E. Seraphin B. Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures.EMBO J. 2002; 21: 6915-6924Crossref PubMed Scopus (363) Google Scholar, Wang et al., 2002Wang Z. Jiao X. Carr-Schmid A. Kiledjian M. The hDcp2 protein is a mammalian mRNA decapping enzyme.Proc. Natl. Acad. Sci. USA. 2002; 99: 12663-12668Crossref PubMed Scopus (254) Google Scholar). Decapping is a highly regulated process and involves both positive and negative regulators. In yeast, Dcp1p interacts with Dcp2p and enhances the decapping activity of Dcp2p (She et al., 2004She M. Decker C.J. Sundramurthy K. Liu Y. Chen N. Parker R. Song H. Crystal structure of Dcp1p and its functional implications in mRNA decapping.Nat. Struct. Mol. Biol. 2004; 11: 249-256Crossref PubMed Scopus (69) Google Scholar, Steiger et al., 2003Steiger M. Carr-Schmid A. Schwartz D.C. Kiledjian M. Parker R. Analysis of recombinant yeast decapping enzyme.RNA. 2003; 9: 231-238Crossref PubMed Scopus (138) Google Scholar). Dcp2p decapping is also stimulated by decapping enhancers Edc1p, Edc2p and Edc3p proteins, as well as Dhh1p and Lsm1–7 protein complex (Coller and Parker, 2004Coller J. Parker R. Eukarryotic mRNA Decapping.Annu. Rev. Biochem. 2004; 73: 861-890Crossref PubMed Scopus (386) Google Scholar). In mammalian cells, Hedls (also known as EDC4 or Ge-1) is also a positive effector of Dcp2 decapping (Fenger-Gron et al., 2005Fenger-Gron M. Fillman C. Norrild B. Lykke-Andersen J. Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping.Mol. Cell. 2005; 20: 905-915Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). In addition to protein trans factors that augment decapping, RNA cis elements can also stimulate decapping. The best characterized is the AU-rich element (ARE) present in yeast (Vasudevan and Peltz, 2001Vasudevan S. Peltz S.W. Regulated ARE-mediated mRNA decay in Saccharomyces cerevisiae.Mol. Cell. 2001; 7: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) and mammals (Fenger-Gron et al., 2005Fenger-Gron M. Fillman C. Norrild B. Lykke-Andersen J. Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping.Mol. Cell. 2005; 20: 905-915Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, Gao et al., 2001Gao M. Wilusz C.J. Peltz S.W. Wilusz J. A novel mRNA-decapping activity in HeLa cytoplasmic extracts is regulated by AU-rich elements.EMBO J. 2001; 20: 1134-1143Crossref PubMed Scopus (135) Google Scholar, Lykke-Andersen and Wagner, 2005Lykke-Andersen J. Wagner E. Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1.Genes Dev. 2005; 19: 351-361Crossref PubMed Scopus (342) Google Scholar). A more recently described element consists of a uracil (U)-tract added to the 3′ end of mRNAs or mRNA decay intermediates to enhance decapping by the recruitment of decapping stimulatory proteins (Mullen and Marzluff, 2008Mullen T.E. Marzluff W.F. Degradation of histone mRNA requires oligouridylation followed by decapping and simultaneous degradation of the mRNA both 5′ to 3′ and 3′ to 5′.Genes Dev. 2008; 22: 50-65Crossref PubMed Scopus (255) Google Scholar, Rissland et al., 2007Rissland O.S. Mikulasova A. Norbury C.J. Efficient RNA polyuridylation by noncanonical poly(a) polymerases.Mol. Cell. Biol. 2007; 27: 3612-3624Crossref PubMed Scopus (123) Google Scholar, Shen and Goodman, 2004Shen B. Goodman H.M. Uridine addition after microRNA-directed cleavage.Science. 2004; 306: 997Crossref PubMed Scopus (160) Google Scholar, Song and Kiledjian, 2007Song M.G. Kiledjian M. 3′ Terminal oligo U-tract-mediated stimulation of decapping.RNA. 2007; 13: 2356-2365Crossref PubMed Scopus (96) Google Scholar). Conversely, the Dcp2 decapping activity is also negatively regulated. In yeast, the cap binding protein eIF4E negatively affects decapping (Ramirez et al., 2002Ramirez C.V. Vilela C. Berthelot K. McCarthy J.E. Modulation of eukaryotic mRNA stability via the cap-binding translation complex eIF4F.J. Mol. Biol. 2002; 318: 951-962Crossref PubMed Scopus (42) Google Scholar, Schwartz and Parker, 1999Schwartz D.C. Parker R. Mutations in translation initiation factors lead to increased rates of deadenylation and decapping of mRNAs in Saccharomyces cerevisiae.Mol. Cell. Biol. 1999; 19: 5247-5256Crossref PubMed Scopus (186) Google Scholar, Schwartz and Parker, 2000Schwartz D.C. Parker R. mRNA decapping in yeast requires dissociation of the cap binding protein, eukaryotic translation initiation factor 4E.Mol. Cell. Biol. 2000; 20: 7933-7942Crossref PubMed Scopus (127) Google Scholar). In mammals, eIF4E inhibits decapping in vitro (Khanna and Kiledjian, 2004Khanna R. Kiledjian M. Poly(A)-binding-protein-mediated regulation of hDcp2 decapping in vitro.EMBO J. 2004; 23: 1968-1976Crossref PubMed Scopus (50) Google Scholar) and RNAs with synthetic cap structures that bind eIF4E with higher affinity are more stable in vivo (Grudzien et al., 2006Grudzien E. Kalek M. Jemielity J. Darzynkiewicz E. Rhoads R.E. Differential inhibition of mRNA degradation pathways by novel cap analogs.J. Biol. Chem. 2006; 281: 1857-1867Crossref PubMed Scopus (64) Google Scholar), suggesting eIF4E protects the cap by preventing access of the decapping enzyme. The poly(A) tail can also negatively influence decapping by both an indirect or direct association of the poly(A)-binding protein (PABP) with the cap (Khanna and Kiledjian, 2004Khanna R. Kiledjian M. Poly(A)-binding-protein-mediated regulation of hDcp2 decapping in vitro.EMBO J. 2004; 23: 1968-1976Crossref PubMed Scopus (50) Google Scholar). Similarly, an RNA binding protein that can preferentially associate with the 5′ cap implicated in X-linked mental retardation, VCX-A, can also inhibit decapping to stabilize mRNAs and silence translation (Jiao et al., 2006Jiao X. Wang Z. Kiledjian M. Identification of an mRNA-decapping regulator implicated in X-linked mental retardation.Mol. Cell. 2006; 24: 713-722Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, Jiao et al., 2009Jiao X. Chen H. Chen J. Herrup K. Firestein B.L. Kiledjian M. Modulation of neuritogenesis by a protein implicated in X-linked mental retardation.J. Neurosci. 2009; 29: 12419-12427Crossref PubMed Scopus (26) Google Scholar). Dcp2 is a member of the Nudix hydrolases superfamily of proteins that predominantly catalyze the hydrolysis of a wide range of small nucleotide substrates composed of a nucleoside diphosphate linked to another moiety X (Bessman et al., 1996Bessman M.J. Frick D.N. O'Handley S.F. The MutT proteins or “Nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes.J. Biol. Chem. 1996; 271: 25059-25062Crossref PubMed Scopus (562) Google Scholar). The Nudix family of proteins are evolutionarily conserved and present in viruses, bacteria, archaea, and eukaryotes (McLennan, 2006McLennan A.G. The Nudix hydrolase superfamily.Cell. Mol. Life Sci. 2006; 63: 123-143Crossref PubMed Scopus (413) Google Scholar). They contain a conserved Nudix motif consisting of the consensus sequence Gx5Ex7REUXEEXGU (where U represents a hydrophobic residue, and X represents any amino acid), which forms part of the versatile catalytic site for diphosphate hydrolysis (Bessman et al., 1996Bessman M.J. Frick D.N. O'Handley S.F. The MutT proteins or “Nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes.J. Biol. Chem. 1996; 271: 25059-25062Crossref PubMed Scopus (562) Google Scholar). To date, 22 Nudix hydrolase genes and at least five pseudogenes have been identified in mammals. Dcp2 and Nudt16 are the only mammalian Nudix proteins that have been reported to decap RNA. Dcp2 can bind RNA and cleave only cap structure that is linked to an RNA moiety. The decapping activity can be efficiently inhibited by uncapped RNA, but not cap analog, suggesting Dcp2 contains a prerequisite RNA binding requirement to recognize and hydrolyze the cap (Piccirillo et al., 2003Piccirillo C. Khanna R. Kiledjian M. Functional characterization of the mammalian mRNA decapping enzyme hDcp2.RNA. 2003; 9: 1138-1147Crossref PubMed Scopus (93) Google Scholar, Steiger et al., 2003Steiger M. Carr-Schmid A. Schwartz D.C. Kiledjian M. Parker R. Analysis of recombinant yeast decapping enzyme.RNA. 2003; 9: 231-238Crossref PubMed Scopus (138) Google Scholar, Wang et al., 2002Wang Z. Jiao X. Carr-Schmid A. Kiledjian M. The hDcp2 protein is a mammalian mRNA decapping enzyme.Proc. Natl. Acad. Sci. USA. 2002; 99: 12663-12668Crossref PubMed Scopus (254) Google Scholar). Interestingly, the RNA binding property of Dcp2 preferentially targets it to a subset of mRNAs containing a distinct stem-loop structure located within the first ten nucleotides of an mRNA which leads to enhanced decapping (Li et al., 2008Li Y. Song M.G. Kiledjian M. Transcript-specific decapping and regulated stability by the human Dcp2 decapping protein.Mol. Cell. Biol. 2008; 28: 939-948Crossref PubMed Scopus (48) Google Scholar, Li et al., 2009Li Y. Ho E.S. Gunderson S.I. Kiledjian M. Mutational analysis of a Dcp2-binding element reveals general enhancement of decapping by 5′-end stem-loop structures.Nucleic Acids Res. 2009; 37: 2227-2237Crossref PubMed Scopus (21) Google Scholar). Nudt16 was initially identified in Xenopus as a U8 snoRNA binding protein, termed X29, and shown to possess decapping activity (Ghosh et al., 2004Ghosh T. Peterson B. Tomasevic N. Peculis B.A. Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme.Mol. Cell. 2004; 13: 817-828Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). X29 is a nucleolar protein capable of specifically binding and decapping the U8 snoRNA in vitro in the presence of Mg2+ although interestingly possessed a more pleiotropic decapping activity when Mn2+ was the cation source (Ghosh et al., 2004Ghosh T. Peterson B. Tomasevic N. Peculis B.A. Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme.Mol. Cell. 2004; 13: 817-828Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Although X29 has been implicated in nucleolar decapping, a direct role for this protein in cellular U8 snoRNA stability has yet to be addressed. The Nudt16, mammalian ortholog of X29, also possesses decapping activity (Taylor and Peculis, 2008Taylor M.J. Peculis B.A. Evolutionary conservation supports ancient origin for Nudt16, a nuclear-localized, RNA-binding, RNA-decapping enzyme.Nucleic Acids Res. 2008; 36: 6021-6034Crossref PubMed Scopus (26) Google Scholar) and has been proposed as a nucleolar decapping enzyme. Interestingly although conserved in metazoans, an obvious ortholog of Nudt16 is lacking in S. cerevisiae, C. elegans, and Drosophila (Taylor and Peculis, 2008Taylor M.J. Peculis B.A. Evolutionary conservation supports ancient origin for Nudt16, a nuclear-localized, RNA-binding, RNA-decapping enzyme.Nucleic Acids Res. 2008; 36: 6021-6034Crossref PubMed Scopus (26) Google Scholar). In contrast to current perceptions, here we demonstrate that the Dcp2 protein is differentially expressed in mouse tissues with a subset of organs lacking detectable levels of Dcp2. Surprisingly, modest alterations in mRNA half-lives were detected by global analysis of Dcp2 dependent changes in mRNA stability, suggesting the presence of other decapping enzyme(s) in mammalian cells. Importantly, we demonstrate Nudt16 is a cytoplasmic protein capable of regulating the stability of a subset of mRNAs and propose Nudt16 is a second cytoplasmic mRNA decapping enzyme present in mammalian cells. Since its isolation, Dcp2 has been postulated to be the major decapping enzyme in eukaryotic cells. This is mainly based on the observation that disruption of Dcp2 in yeast oblates decapping in this single cell fungi (Dunckley and Parker, 1999Dunckley T. Parker R. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif.EMBO J. 1999; 18: 5411-5422Crossref PubMed Scopus (262) Google Scholar). Our recent demonstration that Dcp2 can selectively regulate a subset of mRNAs possessing a Dcp2 binding and decapping element (DBDE) at their 5′ end (Li et al., 2008Li Y. Song M.G. Kiledjian M. Transcript-specific decapping and regulated stability by the human Dcp2 decapping protein.Mol. Cell. Biol. 2008; 28: 939-948Crossref PubMed Scopus (48) Google Scholar, Li et al., 2009Li Y. Ho E.S. Gunderson S.I. Kiledjian M. Mutational analysis of a Dcp2-binding element reveals general enhancement of decapping by 5′-end stem-loop structures.Nucleic Acids Res. 2009; 37: 2227-2237Crossref PubMed Scopus (21) Google Scholar) indicates that this decapping enzyme can preferentially function on a selected population of mRNAs. These findings raise an intriguing question of whether Dcp2 is necessarily the only decapping enzyme in multicellular organisms and whether it is the major decapping enzyme responsible for hydrolyzing bulk mRNA in cells. To begin addressing these questions, we first asked whether Dcp2 was equivalently expressed in all tissues as would be expected of a decapping enzyme that functions on all mRNAs and all tissues in mammals. Tissue samples from 4-week-old C57BL/6 mice were probed for the presence of Dcp2 protein. Surprisingly, a broad range of expression levels were evident for full-length Dcp2 protein, with the highest levels detected in testis and brain. However, the most striking observation was the undetectable level of full-length Dcp2 protein in half of the tissues tested: heart, liver, kidney, and muscle (Figure 1A ). At present, it is not clear whether the small bands detected by the anti-Dcp2 antibody are degradation products or simply nonspecific cross-reactivity. The observed drastic differential expression of Dcp2 is not restricted to mice as shown with three different examples of human tissue tested for Dcp2. Similar to mice, Dcp2 protein is detected in human brain and testis extract but not liver (Figure 1B). The surprising finding that Dcp2 was at undetectable levels in certain adult tissues prompted us to test whether it could be developmentally regulated in these tissues. Extract from mouse embryonic, newborn, and adult organs were isolated and tested for the presence of Dcp2. As shown in Figure 1C, Dcp2 is present in embryonic brain, heart, liver, and kidney. However, a substantial decrease of Dcp2 is evident in heart, liver, and kidney at birth and a continual decrease to undetectable levels in the adult. In contrast, Dcp2 can be detected in the brain at all the developmental stages tested. These data demonstrate Dcp2 is developmentally regulated and its levels dramatically decrease in a subset of tissues in adult mice. Moreover, they illustrate that Dcp2-directed mRNA decapping is likely differentially utilized and not uniform in all tissues and may minimally contribute to mRNA decapping in a subset of adult tissues. In an effort to begin addressing the function of Dcp2 at the organismal level, we initiated generation of a Dcp2 knockout mouse using the available mouse embryonic stem cell (ESC) lines containing a gene trap vector (pGT2Lxf) insertion into the Dcp2 locus (RRE061, BayGenomics; San Francisco, CA) (see Figure S1A available online). We were able to obtain viable mice containing a homozygous insertion of the β-galactosidase and neomycin phosphotransferase fusion gene (β-geo) (Stanford et al., 2001Stanford W.L. Cohn J.B. Cordes S.P. Gene-trap mutagenesis: past, present and beyond.Nat. Rev. Genet. 2001; 2: 756-768Crossref PubMed Scopus (302) Google Scholar) inserted within the 25 kb Dcp2 intron 1 on mouse chromosome 18. To confirm the homozygous insertion of the β-geo gene, we initially mapped the precise insertion site within the RRE061 ESC line to nucleotide 12535 within intron 1 by a PCR strategy (Figure S1B) followed by sequencing of the insertion junction. PCR based screening of offspring obtained from heterozygote matings yielded a Mendelian 1:2:1 genotype with viable mice containing a homozygous insertion of the β-geo gene in Dcp2 intron 1. A representative example of the PCR based screen is shown in Figure S1C. We will refer to these mice containing a homozygous insertion of the β-geo gene as Dcp2β/β. Western analysis of several organs from 4-week-old wild-type (WT) and Dcp2β/β littermates revealed the mice containing a homozygous insertion of the β-geo gene were not completely devoid of Dcp2 expression and were instead phenotypically hypomorphic with reduced protein expression in the different tissues that Dcp2 can normally be detected (Figure 2A ). Although the gene trap knockout system is generally efficient, “leaky” expression of the WT mRNA can occasionally occur (Galy et al., 2004Galy B. Ferring D. Benesova M. Benes V. Hentze M.W. Targeted mutagenesis of the murine IRP1 and IRP2 genes reveals context-dependent RNA processing differences in vivo.RNA. 2004; 10: 1019-1025Crossref PubMed Scopus (42) Google Scholar) and is thought to be due to alternative splicing that bypasses the β-geo gene and splices the normal mRNA (Figure S1). That appears to be the case with the Dcp2 animals since WT Dcp2 protein can be detected with varying penetrance in the tissues tested. However, we have thus far not detected any obvious phenotypic difference between the WT animals and Dcp2β/β mice or differences in fertility, life span, or litter size. To begin investigating the functional consequence of the Dcp2 protein in cells, a representative cell line with no detectable Dcp2 protein was generated. Stably transformed mouse embryonic fibroblast (MEF) cell lines derived from an E12-E14 Dcp2β/β embryo were generated and clonal cell lines lacking detectable Dcp2 protein were isolated. Dcp2 protein and mRNA levels in a representative Dcp2β/β cell line are shown in Figures 2B and 2C, respectively. To test the extent of any potential differential decapping in these cells, electroporation of cap-labeled RNA that we have shown can be degraded by the 5′ decay pathway (Wang and Kiledjian, 2001Wang Z. Kiledjian M. Functional link between the mammalian exosome and mRNA decapping.Cell. 2001; 107: 751-762Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) was used. 32P-cap-labeled RNAs containing a G-tract at their 3′ end to minimize 3′ end decay were transfected into either WT or Dcp2β/β MEF cells and the extent of RNA remaining was followed over time. Surprisingly, despite the dramatic difference in Dcp2 protein levels in the two different MEF cell lines, decay of the capped RNA was indistinguishable over the time course of the experiment (Figure 3). These data demonstrate that the presence or absence of detectable Dcp2 did not appreciably alter stability of the transfected RNA and suggests Dcp2 may not be involved in bulk mRNA decapping in cells. The global influence of Dcp2 on mRNA stability was next addressed. We initially analyzed total mRNA poly(A) tail length distribution. Unlike the situation in yeast where disruption of the Dcp2 gene results in the accumulation of deadenylated but capped mRNA decay intermediates (Dunckley and Parker, 1999Dunckley T. Parker R. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif.EMBO J. 1999; 18: 5411-5422Crossref PubMed Scopus (262) Google Scholar), the poly(A) tail distribution appeared similar in both the WT and Dcp2β/β cells (Figure S2) suggesting bulk mRNA decapping was not hampered in the Dcp2β/β cells. In a second approach, we employed microarray analyses using the Illumina Sentrix Mouse 24K Array to follow overall mRNA turnover. WT and Dcp2β/β MEF cells were treated with Actinomycin D for 1 or 4 hr and mRNA levels before and after treatment were compared in the two different cell lines. The two time points were chosen to enable detection of more labile mRNAs at the 1 hr time point and relatively more stable mRNAs at the 4 hr time point. Surprisingly, the stability of only a small subset of mRNAs increased in the Dcp2β/β cells relative to the WT cells. Following 1 hr of transcriptional arrest, 90 mRNAs were at least 1.5-fold more stable and 55 mRNAs appeared more stable after 4 hr of transcriptional inhibition (Table S1) relative to the same analysis in WT MEF cells. Unexpectedly, the stability of 10 and 11 transcripts decreased 1.5-fold or greater at 1 and 4 hr transcriptional arrest, respectively. Of note, a disproportionate number of Histone mRNAs are represented in the less stable mRNAs and might indicate Dcp2 controls the expression of a labile histone mRNA decay factor. Collectively, consistent with the results obtained with the exogenous chimeric RNA transfection experiments above, Dcp2 appears to be a minor contributor to mRNA stability and raises the intriguing possibility for the presence of a Dcp2-independent decapping activity in mammalian cells. To begin addressing whether mammalian cells contain an mRNA decapping activity distinct from Dcp2, we focused on other Nudix proteins. We rationalized that proteins with a Nudix hydrolase domain possessing pyrophosphatase activity would be potential candidates. A total of 22 Nudix proteins have been identified in the human genome and termed Nudt1 through 22 (where Nudt20 is Dcp2 and Nudt16 is the X29 nucleolar decapping protein ortholog). The remaining 20 Nudix proteins were grouped based on their known catalytic activity (e.g., hydrolyze dinucleotide oligophosphates) as well as conservation with the Dcp2 BoxB region, which is a critical component for Dcp2 RNA binding and essential for decapping (Deshmukh et al., 2008Deshmukh M.V. Jones B.N. Quang-Dang D.U. Flinders J. Floor S.N. Kim C. Jemielity J. Kalek M. Darzynkiewicz E. Gross J.D. mRNA decapping is promoted by an RNA-binding channel in Dcp2.Mol. Cell. 2008; 29: 324-336Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Piccirillo et al., 2003Piccirillo C. Khanna R. Kiledjian M. Functional characterization of the mammalian mRNA decapping enzyme hDcp2.RNA. 2003; 9: 1138-1147Crossref PubMed Scopus (93) Google Scholar). Both Nudt1 and Nudt15 contain 8-OH-dGTPase activity. Nudt3, 4, 10, and 11 are members of the DIPP family and can hydrolyse DIPs and ApnA dinucleotide, while Nudt2 functions on NpnN (n ≥ 4). Nudt5, 6, 9, and 14 have ADP-ribose hydrolase activity and Nudt7, 8, and 19 are confirmed or putative peroxisomal CoA pyrophosphohydrolases. Last, Nudt12 and Nudt13 preferentially hydrolyse NAD(P)H (McLennan, 2006McLennan A.G. The Nudix hydrolase superfamily.Cell. Mol. Life Sci. 2006; 63: 123-143Crossref PubMed Scopus (413) Google Scholar). Four representative proteins were chosen (Nudt1, 4, 5, and 7) in addition to Nudt16, and their ability to decap mRNA was tested. His-tagged recombinant proteins were generated and the extent of decapping detected by thin layer chromatography (TLC). As expected, Dcp2 and Nudt16 both contain decapping activity, while cap hydrolysis activity was not evident with the Nudt1, 4, 5, and 7 proteins under our assay conditions (Figure 4A ). Decapping was also not detected with the Nudt16-like 1 (Nudt16L1) protein negative control, which lacks the critical cation binding residues and is devoid of decapping activity (Taylor and Peculis, 2008Taylor M.J. Peculis B.A. Evolutionary conservation supports ancient origin for Nudt16, a nuclear-localized, RNA-binding, RNA-decapping enzyme.Nucleic Acids Res. 2008; 36: 6021-6034Crossref PubMed Scopus (26) Google Scholar). Similar to Dcp2, Nudt16 can only function on capped RNA but not N7-methyl cap structure under these conditions (Figure S3). Although th" @default.
• W2047479529 created "2016-06-24" @default.
• W2047479529 creator A5006775091 @default.
• W2047479529 creator A5022950257 @default.
• W2047479529 creator A5085856839 @default.
• W2047479529 date "2010-11-01" @default.
• W2047479529 modified "2023-10-11" @default.
• W2047479529 title "Multiple mRNA Decapping Enzymes in Mammalian Cells" @default.
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