Functional characterization of the mammalian mRNA decapping enzyme hDcp2

Regulation of decapping is a critical determinant of mRNA stability. We recently identified hDcp2 as a human decapping enzyme with intrinsic decapping activity. This activity is specific to N(7)-methylated guanosine containing RNA. The hDcp2 enzyme does not function on the cap structure alone and is not sensitive to competition by cap analog, suggesting that hDcp2 requires the RNA for cap recognition. We now demonstrate that hDcp2 is an RNA-binding protein and its recognition and hydrolysis of the cap substrate is dependent on an initial interaction with the RNA moiety. A biochemical characterization of hDcp2 revealed that a 163 amino acid region containing two evolutionarily conserved regions, the Nudix fold hydrolase domain and the adjacent Box B region contained methyl-cap-specific hydrolysis activity. Maximum decapping activity for wild-type as well as truncation mutants of hDcp2 required Mn(2+) as a divalent cation. The demonstration that hDcp2 is an RNA-binding protein with an RNA-dependent decapping activity will now provide new approaches to identify specific mRNAs that are regulated by this decapping enzyme as well as provide novel avenues to control mRNA decapping and turnover by influencing the RNA-binding property of hDcp2.     

Multiple mRNA decapping enzymes in mammalian cells

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.     

Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn²⁺- or Mg²⁺-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.     

Differential utilization of decapping enzymes in mammalian mRNA decay pathways

mRNA decapping is a crucial step in the regulation of mRNA stability and gene expression. Dcp2 is an mRNA decapping enzyme that has been widely studied. We recently reported the presence of a second mammalian cytoplasmic decapping enzyme, Nudt16. Here we address the differential utilization of the two decapping enzymes in specified mRNA decay processes. Using mouse embryonic fibroblast (MEF) cell lines derived from a hypomorphic knockout of the Dcp2 gene with undetectable levels of Dcp2 or MEF cell lines harboring a Nudt16-directed shRNA to generate reduced levels of Nudt16, we demonstrate the distinct roles for Dcp2 and Nudt16 in nonsense-mediated mRNA decay (NMD), decay of ARE-containing mRNA and miRNA-mediated silencing. Our results indicated that NMD preferentially utilizes Dcp2 rather than Nudt16; Dcp2 and Nudt16 are redundant in miRNA-mediated silencing; and Dcp2 and Nudt16 are differentially utilized for ARE-mRNA decay. These data demonstrate that the two distinct decapping enzymes can uniquely function in specific mRNA decay processes in mammalian cells.     

Functional link between the mammalian exosome and mRNA decapping.

Mechanistic understanding of mammalian mRNA turnover remains incomplete. We demonstrate that the 3' to 5' exoribonuclease decay pathway is a major contributor to mRNA decay both in cells and in cell extract. An exoribonuclease-dependent scavenger decapping activity was identified that follows decay of the mRNA and hydrolyzes the residual cap. The decapping activity is associated with a subset of the exosome proteins in vivo, implying a higher-order degradation complex consisting of exoribonucleases and a decapping activity, which together coordinate the decay of an mRNA. These findings indicate that following deadenylation of mammal mRNA, degradation proceeds by a coupled 3' to 5' exoribonucleolytic activity and subsequent hydrolysis of the cap structure by a scavenger decapping activity.     

An mRNA decapping enzyme from ribosomes of Saccharomyces cerevisiae

By use of [³H]methyl-5′-capped [¹⁴C]mRNA from yeast as a substrate, a decapping enzyme activity has been detected in enzyme fractions derived from a high salt wash of ribosomes of $\text{Saccharomyces cerevisiae}$. The product of the decapping reaction is [³H]m⁷GDP. That the enzyme is not a non-specific pyrophosphatase is suggested by the finding that the diphosphate product, m⁷GpppA(G), and UDP-glucose are not hydrolyzed.     

Extensive homology between poly(A)-containing mRNA and purified nominal poly(A)-lacking mRNA in mouse kidney

To investigate poly(A)-lacking mRNA in mouse kidney, we studied a fraction of renal mRNA that does not bind to oligo(dT)-cellulose but can be purified by benzoylated cellulose chromatography. Nominal poly(A)-lacking mRNA and poly(A)-containing mRNA have complete nucleotide sequence homology, suggesting that kidney does not contain mRNAs that are not represented in the polyadenylated RNA fraction. Translation products directed by nominal poly(A)-lacking mRNA and poly(A)-containing mRNA are qualitatively and quantitatively similar in one-dimensional polyacrylamide gels. [3H]cDNA transcribed from poly(A)-containing mRNA hybridizes with its template and with nominal poly(A)-lacking mRNA to the same extent (95%) and with the same kinetics; reaction of [3H]cDNA to nominal poly(A)-lacking mRNA with the two mRNA populations gives the same result. The extensive homology these two mRNA populations share is important to the interpretation of mRNA lifetime and to the analysis of authentic poly(A)-lacking mRNAs.     

Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities

Nonsense-mediated mRNA decay (NMD) is a mechanism by which cells recognize and degrade mRNAs that prematurely terminate translation. To date, the polarity and enzymology of NMD in mammalian cells is unknown. We show here that downregulating the Dcp2 decapping protein or the PM/Scl100 component of the exosome (1) significantly increases the abundance of steady-state nonsense-containing but not nonsense-free mRNAs, and (2) significantly slows the decay rate of transiently induced nonsense-containing but not nonsense-free mRNA. Downregulating poly(A) ribonuclease (PARN) also increases the abundance of nonsense-containing mRNAs. Furthermore, NMD factors Upf1, Upf2, and Upf3X coimmunopurify with the decapping enzyme Dcp2, the putative 5'-->3' exonuclease Rat1, the proven 5'-->3' exonuclease Xrn1, exosomal components PM/Scl100, Rrp4, and Rrp41, and PARN. From these and other data, we conclude that NMD in mammalian cells degrades mRNAs from both 5' and 3' ends by recruiting decapping and 5'-->3' exonuclease activities as well as deadenylating and 3'-->5' exonuclease activities.     

Poly(A)-binding proteins regulate both mRNA deadenylation and decapping in yeast cytoplasmic extracts.

The pathway of mRNA degradation has been extensively studied in the yeast, Saccharomyces cerevisiae, and it is now clear that many mRNAs decay by a deadenylation-dependent mechanism. Although several of the factors required for mRNA decay have been identified, the regulation and precise roles of many of the proteins involved remains unclear. We have developed an in vitro system that recapitulates both the deadenylation and the decapping steps of mRNA decay. Furthermore, both deadenylation and decapping are inhibited by poly(A) binding proteins in our assay. Our system has allowed us to separate the decay process from translation and we have shown that the poly(A) tail is capable of inhibiting decapping in an eIF4E-independent manner. Our in vitro system should prove invaluable in dissecting the mechanisms of mRNA turnover.     

The poly(A)-binding protein facilitates in vitro translation of poly(A)-rich mRNA

To investigate the role of the 73-kDa poly(A)-binding protein in protein synthesis, the effect of the addition of homo-polyribonucleotides on the translation of polyadenylated and non-adenylated mRNA was studied in the rabbit reticulocyte lysate. Poly(A) was found to be the most effective polynucleotide in inhibiting duck-globin mRNA translation, whereas it had no effect on the translation of polyribosomal duck-globin mRNP, or on the endogenous synthesis of the rabbit reticulocyte lysate. The translation of poly(A)-free mRNA was not affected by the addition of poly(A). Furthermore, we found that the inhibiting effect of poly(A) can be reversed by addition of purified poly(A)-binding protein. It is thus likely that the 73-kDa poly(A)-binding protein is an essential factor necessary for poly(A)-rich mRNA translation.     

Energy requirement and kinetics of transport of poly(A)-free histone mRNA compared to poly(A)-rich mRNA from isolated L-cell nuclei

ATP-promoted efflux of poly(A)-rich RNA from isolated nuclei of prelabeled mouse lymphoma L5178y cells has an activation energy of 51.5 kJ/mol, similar to that found for the nuclear envelope nucleoside triphosphatase (48.1 kJ/mol) assumed to be involved in mediating nucleocytoplasmic transport of at least some RNA. Here we show that efflux of two specific poly(A)-rich mRNAs (actin and beta-tubulin) from isolated L-cell nuclei is almost totally dependent on the presence of ATP, while efflux of poly(A)-free histone mRNA (H4, H2B, and H1) also occurs to a marked extent in the absence of this nucleotide. Measurements of temperature dependence of transport rate revealed an activation energy of 56.1 kJ/mol for actin mRNA, while the activation energy for histone-H4-mRNA efflux was in the same range as that found for ATP-induced release of RNA from demembranated nuclei (about 15-20 kJ/mol). Addition of nonhydrolyzable nucleotide analogs of ATP to the in vitro system used for measurement of RNA transport did not result in release of nonhistone mRNA (actin), but enhanced the efflux of H4 mRNA to approximately the same extent as ATP. Although not absolutely required, addition of ATP stimulated the rate of export of histone mRNA about twofold. Only the poly(A)-rich RNA, but not the poly(A)-free RNA, released from isolated nuclei was found to compete with poly(A) for the nuclear envelope mRNA-binding site, indicating the mechanism of transport for both RNA classes to be distinct. Export of both nonhistone and histone mRNA was found to be inhibited by a monoclonal antibody against a p60 nuclear-pore-complex antigen. This antibody had no effect on the nucleoside triphosphatase, mediating transport of poly(A)-rich mRNA.     

An effective enzyme interacting with poly (dT-dA).poly (dT-dA): a dynamic enhancer-repressor action.

The Green function technique is used to study the open hydrogen bond probability of poly(dT-dA).poly(dT-dA) when an effective enzyme is attached to the helix. The DNA interstrand hydrogen bond mean motion and probability of fluctuating to an open state depends on the internal vibrational frequency of the enzyme. An enzyme with internal frequency of 80 cm-1 reduces hydrogen bond motion and the resulting probability of hydrogen bond fluctuational opening. An enzyme with internal frequency of 72 cm-1 increases hydrogen bond motion and the probability of hydrogen bond breaking.