mRNA capping enzyme. Isolation and characterization of the gene encoding mRNA guanylytransferase subunit from Saccharomyces cerevisiae.

The highly purified yeast mRNA capping enzyme is composed of two separate chains of 52 (alpha) and 80 kDa (beta), responsible for the activities of mRNA guanylyltransferase and RNA 5'-triphosphatase, respectively (Itoh, N., Yamada, H., Kaziro, Y., and Mizumoto, K. (1987) J. Biol. Chem. 262, 1989-1995). The gene encoding the mRNA guanylyltransferase subunit (alpha subunit), CEG1, has been isolated by immunological screening of a yeast genomic expression library in lambda gt11 with polyclonal antibodies directed against purified yeast capping enzyme. The identity of CEG1 was confirmed by epitope selection and by expressing the gene in Escherichia coli to give a catalytically active mRNA guanylyltransferase. The gene is present in one copy per haploid genome, and encodes a polypeptide of 459 amino acid residues. From its primary structure as well as its mRNA size, it was concluded that the alpha and the beta subunits of yeast mRNA capping enzyme are encoded by two separate genes, not as a fused protein. CEG1 is located on the chromosome VII by a pulse-field gel electrophoresis. Gene disruption experiment indicated that CEG1 is essential for the growth of yeast. We have also found another open reading frame (ORF2) which lies in close proximity to CEG1 in our clones and encodes a 450 amino acid-polypeptide of yet unknown function.     

Messenger RNA guanylyltransferase from Saccharomyces cerevisiae. I. Purification and subunit structure

GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of the GMP moiety from GTP to the 5' end of the RNA to form a cap structure (G(5')pppN-), has been purified to an apparent homogeneity from Saccharomyces cerevisiae. The mRNA 5'-triphosphatase activity hydrolyzing the gamma-phosphoryl group from pppN-RNA was co-purified with mRNA guanylyltransferase activity through column chromatographies on CM-Sephadex and poly(U)-Sepharose, and centrifugation through glycerol gradients, suggesting that these two activities are physically associated. An 820,w value of 7.3, and Mr = 140,000 were estimated from the sedimentation behavior in glycerol gradients. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two major polypeptides, Mr = 45,000 (alpha) and 39,000 (beta), were detected with the purified enzyme preparation. Their molar ratios were close to unity when estimated by the relative density of silver staining. These results suggest that the yeast mRNA-capping enzyme is an oligomeric protein which may consist of two alpha and two beta chains (alpha 2 beta 2).     

Association of an RNA 5'-triphosphatase activity with RNA guanylyltransferase partially purified from rat liver nuclei.

An RNA 5'-triphosphatase activity hydrolyzing gamma-phosphate from pppN-RNA was found to be associated with mRNA guanylyltransferase partially purified from rat liver nuclei. The activity specifically removed 32P as inorganic phosphate from [gamma-32P]pppA(pA)n, but not from [beta-32P]pppA(pA)n or from [gamma-32P]ATP. Free SH group(s) were required for its activity, and the reaction was inhibited by N-ethylmaleimide. Divalent cations were not required, but were rather inhibitory for the reaction. The RNA 5'-triphosphatase activity could not be separated from the guanylyltransferase activity through successive chromatographies on Sephadex G-150, CM-Sephadex and blue dextran-Sepharose columns. Both activities remained physically associated during sedimentation in glycerol density gradients after high salt treatment. The heat stability of the RNA 5'-triphosphatase activity was almost identical with that of the guanylyltransferase activity. These results indicate that the 69000 mol. wt. protein purified from rat liver nuclei as guanylyltransferase possesses both mRNA capping and RNA 5'-triphosphatase activities.     

Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme)

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgCl2 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through successive chromatography steps on DEAE-cellulose, DNA-cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPi exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described. The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.     

Synthesis of Gp4N and Gp3N compounds by guanylyltransferase purified from yeast

Guanylyltransferase that catalyzes mRNA capping by the reaction, ppNpN + GTP----GpppNpN was purified from S. cerevisiae. The enzyme forms a nucleotidyl intermediate by phosphoamide linkage of GMP. Two guanylylated polypeptides of MR approximately 52,000 and 46,000 were obtained, the latter apparently by proteolysis of the larger component. Both forms transferred the covalently bound GMP to ppApG, yielding GpppApG. Dinucleoside tri- and tetraphosphates of the type Gp3N and Gp4N were also produced by using ribonucleoside 5'-di and triphosphates as acceptors. The purified yeast guanylyltransferase contained little or no RNA 5'-triphosphatase or methyltransferase.     

A Saccharomyces cerevisiae RNA 5'-triphosphatase related to mRNA capping enzyme

The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: the RNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Using computer homology searching, a S. cerevisiae gene was identified that encodes a protein resembling the C-terminal region of Cet1. Accordingly, we designated this gene CTL1 (capping enzyme RNAtriphosphatase-like 1). CTL1 is not essential for cell viability and no genetic or physical interactions with the capping enzyme genes were observed. The protein is found in both the nucleus and cytoplasm. Recombinant Ctl1 protein releases gamma-phosphate from the 5'-end of RNA to produce a diphosphate terminus. The enzyme is specific for polynucleotide RNA in the presence of magnesium, but becomes specific for nucleotide triphosphates in the presence of manganese. Ctl1 is the second member of the yeast RNA triphosphatase family, but is probably involved in an RNA processing event other than mRNA capping.     

The Helicase-Like Domain of Plant Potexvirus Replicase Participates in Formation of RNA 5′ Cap Structure by Exhibiting RNA 5′-Triphosphatase Activity

Open reading frame 1 (ORF1) of potexviruses encodes a viral replicase comprising three functional domains: a capping enzyme at the N terminus, a putative helicase in the middle, and a polymerase at the C terminus. To verify the enzymatic activities associated with the putative helicase domain, the corresponding cDNA fragment from bamboo mosaic virus (BaMV) was cloned into vector pET32 and the protein was expressed in Escherichia coli and purified by metal affinity chromatography. An activity assay confirmed that the putative helicase domain has nucleoside triphosphatase activity. We found that it also possesses an RNA 5'-triphosphatase activity that specifically removes the gamma phosphate from the 5' end of RNA. Both enzymatic activities were abolished by the mutation of the nucleoside triphosphate-binding motif (GKS), suggesting that they have a common catalytic site. A typical m(7)GpppG cap structure was formed at the 5' end of the RNA substrate when the substrate was treated sequentially with the putative helicase domain and the N-terminal capping enzyme, indicating that the putative helicase domain is truly involved in the process of cap formation by exhibiting its RNA 5'-triphosphatase activity.     

Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1

Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltransferase which forms GMP-enzyme complex. In this study, full length SINV nsP1 was expressed in a soluble form with an N-terminal histidine tag in Escherichia coli and purified to homogeneity. The purified protein is enzymatically active and contains both MTase and GTase activity indicating that SINV nsP1 does not require membrane association for its enzymatic function. Biochemical analysis shows that detergents abolish nsP1 GTase activity, whereas nonionic detergents do not affect MTase activity. Furthermore, SINV nsP1 contains the metal-ion dependent GTase, whereas MTase does not require a metal ion. Circular dichroism spectroscopic analysis of purified protein indicate that nsP1 has a mixed α/β structure and is in the folded native conformation.     

Cloning and characterization of mRNA capping enzyme and mRNA (Guanine-7-)-methyltransferase cDNAs from Xenopus laevis

The mRNA cap structure, which is synthesized by a series of reactions catalyzed by capping enzyme, mRNA (guanine-7-)-methyltransferase, and mRNA (ribose-2'-O-)-methyltransferase, has crucial roles for RNA processing and translation. Methylation of the cap structure is also implicated in polyadenylation-mediated translational activation during Xenopus oocyte maturation. Here we isolated two Xenopus laevis cDNAs, xCAP1a and xCAP1b, for mRNA capping enzyme and one cDNA for mRNA (guanine-7-)-methyltransferase, xCMT1, which encode 598, 511, and 402 amino acids, respectively. The deduced amino acid sequence of xCAP1a was highly homologous to that of human capping enzyme hCAP1a, having all the characteristic regions including N-terminal RNA 5'-triphosphatase as well as C-terminal mRNA guanylyltransferase domains which are conserved among animal mRNA guanylyltransferases, whereas in xCAP1b the most C-terminal motif was missing. The amino acid sequence of xCMT1 was also similar to human (guanine-7-)-methyltransferase, hCMT1a, with all the conserved motifs among cellular (guanine-7-)-methyltransferases, except for its N-terminal portion. The recombinant xCAP1a and xCMT1 exhibited cap formation and mRNA (guanine-7-)-methyltransferase activities, respectively. RT-PCR analysis showed that mRNA for xCAP1a and xCMT1 exist abundantly in fertilized eggs as maternal mRNAs, but xCMT1 mRNA gradually decreased in its amount in later stages of early development.     

Catalytic activity of vaccinia mRNA capping enzyme subunits coexpressed in Escherichia coli

RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase activities are associated with the vaccinia virus mRNA capping enzyme, a heterodimeric protein containing polypeptides of Mr 95,000 and Mr 31,000. The genes encoding the large and small subunits (corresponding to the D1 and the D12 ORFs, respectively, of the viral genome) were coexpressed in Escherichia coli BL21 (DE3) under the control of a bacteriophage T7 promoter. Guanylyltransferase activity (assayed as the formation of a covalent enzyme-guanylate complex) was detected in soluble lysates of these bacteria. A 1000-fold purification of the guanylyltransferase was achieved by ammonium sulfate precipitation and chromatography using phosphocellulose and SP5PW columns. Partially purified guanylytransferase synthesized GpppA caps when provided with 5'-triphosphate-terminated poly(A) as a cap acceptor. In the presence of AdoMet the enzyme catalyzed concomitant cap methylation with 99% efficiency. Inclusion of S-adenosyl methionine increased both the rate and extent of RNA capping, permitting quantitative modification of RNA 5' ends. Guanylyltransferase sedimented as a single component of 6.5 S during further purification in a glycerol gradient; this S value is identical with that of the heterodimeric capping enzyme from vaccinia virions. Electrophoretic analysis showed a major polypeptide of Mr 95,000 cosedimenting with the guanylyltransferase. RNA triphosphatase activity cosedimented exactly with guanylyltransferase. Methyltransferase activity was associated with guanylyltransferase and was also present in less rapidly sedimenting fractions. The methyltransferase activity profile correlated with the presence of a Mr 31,000 polypeptide. These results indicate that the D1 and D12 gene products are together sufficient to catalyze all three enzymatic steps in cap synthesis. A model for the domain structure of this enzyme is proposed.     

Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase

Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae. Open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that has long been postulated to be a replicase involved in the replication and formation of the cap structure at the 5' end of the viral genome. To identify and characterize the enzymatic activities associated with the N-terminal domain of the BaMV ORF1 protein, the intact replicase and two C-terminally truncated proteins were expressed in Saccharomyces cerevisiae. All three versions of BaMV ORF1 proteins could be radiolabeled by [alpha-(32)P]GTP, which is a characteristic of guanylyltransferase activity. The presence of S-adenosylmethionine (AdoMet) was essential for this enzymatic activity. Thin-layer chromatography analysis suggests that the radiolabeled moiety linked to the N-terminal domain of the BaMV ORF1 protein is m(7)GMP. The N-terminal domain also exhibited methyltransferase activity that catalyzes the transfer of the [(3)H]methyl group from AdoMet to GTP or guanylylimidodiphosphate. Therefore, during cap structure formation in BaMV, methylation of GTP may occur prior to transguanylation as for alphaviruses and brome mosaic virus. This study establishes the association of RNA capping activity with the N-terminal domain of the replicase of potexviruses and further supports the idea that the reaction sequence of RNA capping is conserved throughout the alphavirus-like superfamily of RNA viruses.     

Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme.

Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase activities. The enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The N-terminal 60-kDa of the D1 subunit (from residues 1 to 545) is an autonomous domain which catalyzes the triphosphatase and guanylyltransferase reactions. Mutations in the D1 subunit that specifically inactivate the guanylyltransferase without affecting the triphosphatase component have been described (P. Cong and S. Shuman, Mol. Cell. Biol. 15:6222-6231, 1995). In the present study, we identified two alanine-cluster mutations of D1(1-545), R77A-K79A and E192A-E194A, that selectively inactivated the triphosphatase, but not the guanylyltransferase. Concordant mutational inactivation of RNA triphosphatase and nucleoside triphosphatase functions (to approximately 1% of wild-type specific activity) suggests that both gamma-phosphate cleavage reactions occur at a single active site. The R77A-K79A and E192A-E194A mutant enzymes were less active than wild-type D1(1-545) in the capping of triphosphate-terminated poly(A) but could be complemented in vitro by D1(1-545)-K260A, which is inert in nucleotidyl transfer but active in gamma-phosphate cleavage. Whereas wild-type D1(1-545) formed only the standard GpppA cap, the R77A-K79A and E192A-E194A enzymes synthesized an additional dinucleotide, GppppA. This finding illuminates a novel property of the vaccinia virus capping enzyme, the use of triphosphate RNA ends as an acceptor for nucleotidyl transfer when gamma-phosphate cleavage is rate limiting.     

RNA capping by HeLa cell RNA guanylyltransferase. Characterization of a covalent protein-guanylate intermediate

RNA capping by partially purified HeLa cell GTP:RNA guanylyltransferase has been shown to occur in the following sequence of two partial reactions involving a covalent protein-guanylate intermediate: (i) E(P68) + GTP in equilibrium E(P68-GMP) + PPi (ii) E(P68-GMP) + ppRNA in equilibrium GpppRNA + E(P68) Initially, the enzyme reacts with GTP in the absence of an RNA cap acceptor to form a covalent protein-guanylate complex. This complex consists of a GMP residue linked via a phosphoamide bond to a Mr = 68,000 protein. The enzyme then transfers the guanylate residue from the Mr = 68,000 polypeptide to the 5' end of diphosphate-terminated poly(a) to yield the capped derivative GpppA(pA)n. Both partial reactions have been shown to be reversible. In the reverse of Reaction i, E(P68--GMP) reacts with PPi to regenerate GTP. In the reverse of Reaction ii, the enzyme catalyzes the transfer of the 5'-GMP from capped RNA to the Mr = 68,000 protein to form protein-guanylate complex. A divalent cation is required for both partial reactions. The Mr = 68,000 protein is presumed to be a subunit of the HeLa guanylyltransferase. This interpretation is consistent with the sedimentation coefficient of 4.2 S of the native enzyme. Preliminary studies of RNA guanylyltransferase from mouse myeloma tumors suggest a similar mechanism of transguanylylation involving a Mr = 68,000 protein-guanylate complex. These data, in conjunction with previous studies of vaccinia virus guanylyltransferase (Shuman, S., and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 187-191) suggests that covalent GMP-enzyme intermediates may be a general feature of the RNA capping reaction.     

Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus

Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5' cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an approximately 10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m(7)GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m(7)GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m(7)GTP sustained the formation of the m(7)GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m(7)GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m(7)GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m(7)GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m(7)GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.     

Genetic, Physical, and Functional Interactions between the Triphosphatase and Guanylyltransferase Components of the Yeast mRNA Capping Apparatus

We have characterized an essential Saccharomyces cerevisiae gene, CES5, that when present in high copy, suppresses the temperature-sensitive growth defect caused by the ceg1-25 mutation of the yeast mRNA guanylyltransferase (capping enzyme). CES5 is identical to CET1, which encodes the RNA triphosphatase component of the yeast capping apparatus. Purified recombinant Cet1 catalyzes hydrolysis of the γ phosphate of triphosphate-terminated RNA at a rate of 1 s⁻¹. Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to form a Cet1-Ceg1 heterodimer. The interaction of Cet1 with Ceg1 elicits >10-fold stimulation of the guanylyltransferase activity of Ceg1. This stimulation is the result of increased affinity for the GTP substrate. A truncated protein, Cet1(201-549), has RNA triphosphatase activity, heterodimerizes with and stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell growth in vivo. The more extensively truncated derivative Cet1(246-549) also has RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal when expressed in single copy in vivo. These data suggest that the Cet1-Ceg1 interaction is essential but do not resolve whether the triphosphatase activity is also necessary. The mammalian capping enzyme Mce1 (a bifunctional triphosphatase-guanylyltransferase) substitutes for Cet1 in vivo. A mutation of the triphosphatase active-site cysteine of Mce1 is lethal. Hence, an RNA triphosphatase activity is essential for eukaryotic cell growth. This work highlights the potential for regulating mRNA cap formation through protein-protein interactions.     

Purification and characterization of the messenger ribonucleic acid capping enzyme GTP:RNA guanylyltransferase from wheat germ

A GTP:RNA guanylyltransferase or capping enzyme has been purified approximately 2000-fold from wheat germ. The enzyme catalyzes the transfer of the GMP residue from GTP to the 5' end of RNA or synthetic polyribonucleotides. Diphosphate-ended polymers were capped more efficiently than molecules with triphosphate ends, and molecules with monophosphate ends were not capped at all. There appears to be little specificity since RNAs with purine or pyrimidine ends served as acceptors. Other features of the wheat germ RNA guanylyltransferase include relatively low Km values for GTP (2.7 microM) and ppA (pA)n (14.2 nM), a divalent cation requirement satisfied by low (0.5 mM) concentrations of MnCl2 or higher (5 mM) concentrations of MgCl2, and a pH optimum around neutrality.