Functional characterization of the mammalian mRNA decapping enzyme hDcp2

Piccirillo, Christopher; Khanna, Richie; Kiledjian, Megerditch

doi:10.1261/rna.5690503

Cited by 116 publications

(187 citation statements)

References 33 publications

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“…6). The dependence of the cap being linked to an RNA moiety and the selective release of m 7 GDP as the decapping product is analogous to Dcp2 (Piccirillo et al 2003) and suggests that RppH is likely an RNA-binding protein that binds the RNA at the 5 ′ end to hydrolyze within the triphosphate linkage, which would explain its requirement of the RNA body to be linked to the cap structure. The significance of a protein with decapping activity in prokaryotes is not evident.…”

Section: Discussionmentioning

confidence: 99%

Multiple Nudix family proteins possess mRNA decapping activity

Song

Bail

Kiledjian

2013

RNA

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120

139

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RNA decapping is an important contributor to gene expression and is a critical determinant of mRNA decay. The recent demonstration that mammalian cells harbor at least two distinct decapping enzymes that preferentially modulate a subset of mRNAs raises the intriguing possibility of whether additional decapping enzymes exist. Because both known decapping proteins, Dcp2 and Nudt16, are members of the Nudix hydrolase family, we set out to determine whether other members of this family of proteins also contain intrinsic RNA decapping activity. Here we demonstrate that six additional mouse Nudix proteins-Nudt2, Nudt3, Nudt12, Nudt15, Nudt17, and Nudt19-have varying degrees of decapping activity in vitro on both monomethylated and unmethylated capped RNAs. The decapping products from Nudt17 and Nudt19 were analogous to Dcp2 and predominantly generated m 7 GDP, while cleavage by Nudt2, Nudt3, Nudt12, and Nudt15 was more pleiotropic and generated both m 7 GMP and m 7 GDP. Interestingly, all six Nudix proteins as well as both Dcp2 and Nudt16 could hydrolyze the cap of an unmethylated capped RNA, indicating that decapping enzymes may be less constrained for the presence of the methyl moiety. Investigation of Saccharomyces cerevisiae Nudix proteins revealed that the yeast homolog of Nudt3, Ddp1p, also possesses decapping activity in vitro. Moreover, the bacterial Nudix pyrophosphohydrolase RppH displayed RNA decapping activity and released m 7 GDP product comparable to Dcp2, indicating that decapping is an evolutionarily conserved activity that preceded mammalian cap formation. These findings demonstrate that multiple Nudix family hydrolases may function in mRNA decapping and mRNA stability.

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Section: Discussionmentioning

confidence: 99%

Multiple Nudix family proteins possess mRNA decapping activity

Song

Bail

Kiledjian

2013

RNA

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120

139

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“…NUDT2, NUDT3, NUDT4, NUDT9, NUDT10, and NUDT11 encode enzymes active on a range of dinucleotides and the structurally unrelated diphosphoinositol polyphosphates (41 -46). Dcp2 is a nudix protein implicated in the control of mRNA decay pathways (47). The MutT-related nudix proteins MTH1/NUDT1 (the human MutT homologue) and NUDT5 degrade oxidized and potentially mutagenic purine nucleoside diphosphates and triphosphates to the corresponding monophosphates.…”

Section: Discussionmentioning

confidence: 99%

Basic Fibroblast Growth Factor (FGF-2) Overexpression Is a Risk Factor for Esophageal Cancer Recurrence and Reduced Survival, which Is Ameliorated by Coexpression of the FGF-2 Antisense Gene

Barclay¹,

Li²,

Geldenhuys³

et al. 2005

Clinical Cancer Research

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Purpose:The basic fibroblast growth factor (FGF-2) gene is bidirectionally transcribed to generate overlapping sense and antisense (FGF-AS) mRNAs. FGF-AS has been implicated in the posttranscriptional regulation of FGF-2 expression. The aim of this study was to characterize FGF-2 and FGF-AS in esophageal cancer and to correlate their expression with clinicopathologic findings and outcome. Experimental Design: Reverse transcription-PCR was used to study FGF-2 and FGF-AS mRNA expression (normalized to glyceraldehyde-3-phosphate dehydrogenase) in 48 esophageal cancers relative to matched histologically normal esophageal epithelia (internal control). We used Cox proportional hazards analysis to calculate hazard ratios for recurrence and survival of patients with underexpression relative to the overexpression of FGF-2 and/or FGF-AS. Results: Overexpression of FGF-2 mRNA, by comparison with tumors underexpressing FGF-2, was associated with significantly increased risk for tumor recurrence (hazard ratio, 3.80; 95% confidence interval, 1.64-8.76) and reduced overall survival (hazard ratio, 2.11; 95% confidence interval, 1.0-4.58). When the effects of FGF-2 and FGF-AS were considered simultaneously, the association of FGF-2 mRNA overexpression with recurrence and mortality was even more pronounced, whereas FGF-AS mRNA overexpression was associated with reduced risk for recurrence and improved survival. Conclusions: Overexpression of FGF-2 mRNA is associated with tumor recurrence and reduced survival after surgical resection of esophageal cancer and that these risks are reduced in tumors coexpressing the FGF-AS mRNA. These data support the hypothesis that FGF-AS is a novel tumor suppressor that modulates the effect of FGF-2 expression and may have potential clinical application to the development of novel therapeutic strategies.

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“…The purified RNA was labeled by using recombinant VACV mRNA guanylyltransferase (Ambion) in the presence of 0.132 M [␣- 32 RNA Decapping Assay. Reactions containing labeled capped RNA and purified recombinant MBP-D10 protein were carried out in 15 l of decapping buffer [100 mM K acetate/10 mM Tris⅐HCl (pH 7.5)/2 mM MgCl 2 /0.5 mM MnCl 2 /2 mM DTT) at 37°C for 30 min unless indicated otherwise (28). The products of the reaction were resolved on polyethyleneimine-cellulose TLC plates (Sigma, St. Louis, MO, or Alltech Biotechnology, Columbia, MD) developed in 0.75 M LiCl.…”

mentioning

confidence: 99%

Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression

Parrish

Resch

Moss

2007

Proc. Natl. Acad. Sci. U.S.A.

114

177

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Previous studies indicated that the vaccinia virus D10 protein, which is conserved in all sequenced poxviruses, participates in the rapid turnover of host and viral mRNAs. D10 contains a motif present in the family of Nudix/MutT enzymes, a subset of which has been shown to enhance mRNA turnover in eukaryotic cells through cleavage of the 5 cap (m 7 GpppNm-). Here, we demonstrate that a purified recombinant D10 fusion protein possesses an intrinsic activity that liberates m 7 GDP from capped RNA substrates. Furthermore, point mutations in the Nudix/MutT motif abolished decapping activity. D10 has a strong affinity for capped RNA substrates (K m Ϸ 3 nm). RNAs of 24 -309 nt were decapped to comparable extents, whereas the cap of a 12-nt RNA was uncleaved. At large molar ratios relative to capped RNA substrate, competitor m 7 GpppG, m 7 GTP, or m 7 GDP inhibited decapping, whereas even higher concentrations of unmethylated analogs did not. High concentrations of uncapped RNA were also inhibitory, suggesting that D10 recognizes its substrate through interaction with both cap and RNA moieties. Thus far, poxviruses represent the only virus family shown to encode a Nudix hydrolase-decapping enzyme. Although it may seem self-destructive for a virus to encode a decapping and a capping enzyme, accelerated mRNA turnover helps eliminate competing host mRNAs and allows stagespecific synthesis of viral proteins.mRNA metabolism ͉ MutT motif ͉ Nudix hydrolase ͉ poxvirus ͉ mRNA turnover T he steady-state concentration of an mRNA is determined by synthesis and decay, allowing cells to rapidly adapt their pattern of gene expression (1). Similarly, some viruses accelerate mRNA turnover to suppress synthesis of cellular proteins and regulate expression of their own genes (2). Studies of vaccinia virus (VACV), the laboratory prototype poxvirus, indicated that viral mRNAs are relatively unstable compared with those of uninfected cells (3, 4). Thus, rapid mRNA turnover coupled with robust and sequential transcription of viral early, intermediate, and late genes allow stage-specific protein synthesis (5). Likewise, VACV infection induces the destabilization of cellular transcripts, a process thought to contribute to the shutdown of host protein synthesis (6-9). The latter may enhance viral replication by alleviating competition from cellular mRNAs for the protein synthetic machinery and by diminishing host antiviral responses. Nevertheless, the mechanisms used by VACV to regulate mRNA turnover have not been elucidated.The VACV D9 and D10 proteins were identified as putative negative regulators of gene expression during a transfectionbased DNA library screen used to isolate activators of late VACV transcription (10, 11). Both proteins are highly conserved: D10 homologs are present in all sequenced poxviruses, and D9 homologs are in all members of the chordopoxvirus subfamily. Overexpression of D10 during infection significantly reduced the amount of VACV transcripts, and overexpression of D9 had a similar but lesser effect (11). Remarkabl...

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Functional characterization of the mammalian mRNA decapping enzyme hDcp2

Cited by 116 publications

References 33 publications

Multiple Nudix family proteins possess mRNA decapping activity

Multiple Nudix family proteins possess mRNA decapping activity

Basic Fibroblast Growth Factor (FGF-2) Overexpression Is a Risk Factor for Esophageal Cancer Recurrence and Reduced Survival, which Is Ameliorated by Coexpression of the FGF-2 Antisense Gene

Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression

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