Structure-function analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase

The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit and a stimulatory subunit. Structure-function analysis of the catalytic subunit by alanine scanning and conservative substitutions (49 mutations at 25 amino acids) identified 12 functional groups essential for methyltransferase activity in vivo, most of which were essential for cap methylation in vitro. Defects in cap binding were demonstrated for a subset of lethal mutants that displayed residual activity in vitro. We discuss our findings in light of a model of the Michaelis complex derived from crystal structures of AdoHcy-bound vaccinia cap methyltransferase and GTP-bound cellular cap methyltransferase. The structure-function data yield a coherent picture of the vaccinia cap methyltransferase active site and the determinants of substrate specificity and affinity.     

Structure and mechanism of mRNA cap (guanine-N7) methyltransferase

A suite of crystal structures is reported for a cellular mRNA cap (guanine-N7) methyltransferase in complex with AdoMet, AdoHcy, and the cap guanylate. Superposition of ligand complexes suggests an in-line mechanism of methyl transfer, albeit without direct contacts between the enzyme and either the N7 atom of guanine (the attacking nucleophile), the methyl carbon of AdoMet, or the sulfur of AdoMet/AdoHcy (the leaving group). The structures indicate that catalysis of cap N7 methylation is accomplished by optimizing proximity and orientation of the substrates, assisted by a favorable electrostatic environment. The enzyme-ligand structures, together with new mutational data, fully account for the biochemical specificity of the cap guanine-N7 methylation reaction, an essential and defining step of eukaryotic mRNA synthesis.     

Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs

The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2′O-methyladenosine (A_m), generating N6, 2′O-dimethyladenosine (m⁶A_m) when A_m is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and A_m), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2′O-methylated A_m over unmodified A nucleosides is due mainly to increased binding affinity for A_m. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5′-A_m oligonucleotide with approximately the same affinity as that of a cap analog (K_M = 0.4 versus 0.3 μM). In addition, PCIF1 methylates the uncapped 5′-A_m with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs.     

Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin's antifungal activity

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo,14 reduced activity in vitro to < or = 2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.     

Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene.

RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.     

Ribavirin is not a functional mimic of the 7-methyl guanosine mRNA cap

Ribavirin is a guanosine ribonucleoside analog that displays broad-spectrum anti-viral activity and is currently used for the treatment of some viral infections. Ribavirin has recently been proposed to also be a mimic of the 7-methyl guanosine cap found at the 5' end of mRNAs. To obtain supporting functional data for this hypothesis, we assessed the ability of ribavirin triphosphate to interfere with the interaction between eIF4E and 7-methyl guanosine capped mRNA. In chemical cross-linking assays, cap-affinity chromatography, and cap-dependent translation assays, ribavirin was unable to function as a cap analog.     

Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity

During mRNA synthesis, the polymerase of vesicular stomatitis virus (VSV) copies the genomic RNA to produce five capped and polyadenylated mRNAs with the 5'-terminal structure 7mGpppA(m)pApCpApGpNpNpApUpCp. The 5' mRNA processing events are poorly understood but presumably require triphosphatase, guanylyltransferase, [guanine-N-7]- and [ribose-2'-O]-methyltransferase (MTase) activities. Consistent with a role in mRNA methylation, conserved domain VI of the 241-kDa large (L) polymerase protein shares sequence homology with a bacterial [ribose-2'-O]-MTase, FtsJ/RrmJ. In this report, we generated six L gene mutations to test this homology. Individual substitutions to the predicted MTase active-site residues K1651, D1762, K1795, and E1833 yielded viruses with pinpoint plaque morphologies and 10- to 1,000-fold replication defects in single-step growth assays. Consistent with these defects, viral RNA and protein synthesis was diminished. In contrast, alteration of residue G1674 predicted to bind the methyl donor S-adenosylmethionine did not significantly perturb viral growth and gene expression. Analysis of the mRNA cap structure revealed that alterations to the predicted active site residues decreased [guanine-N-7]- and [ribose-2'-O]-MTase activity below the limit of detection of our assay. In contrast, the alanine substitution at G1674 had no apparent consequence. These data show that the predicted MTase active-site residues K1651, D1762, K1795, and E1833 within domain VI of the VSV L protein are essential for mRNA cap methylation. A model of mRNA processing consistent with these data is presented.     

A system for receptor functional analysis based on c-fos mRNA expression: analysis of GnRH receptors as a test system

This report describes the establishment of a system for assessing receptor activation by RT-PCR-based detection of c-fos mRNA induction. In this system, COS-7 cells were transiently transfected with GnRH receptor expression plasmid, and ligand-induced c-fos expression was quantified by the RT-competitive PCR method. The results were compared with those of a conventional inositol phosphate (IP) assay. Changes in c-fos expression levels were observed in a dose- and ligand-dependent manner. Similar tendencies were observed in ligand selectivity between c-fos expression and IP production. The novel system developed and established in the present study is sensitive by using RT-PCR and convenient because it requires only basic methods of cell culture and molecular biology. It also has the merit that it does not need any specific measuring devices or radioactive substances. Given the ability of c-fos to respond to diverse stimuli, the present system may be applicable for various receptors for bioactive substances in addition to GnRH receptor, and useful for various purposes including screening ligands for orphan receptors.     

Mutational analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase reveals essential contributions of the N-terminal peptide that closes over the active site

RNA guanine-N7 methyltransferase catalyzes the third step of eukaryal mRNA capping, the transfer of a methyl group from AdoMet to GpppRNA to form m⁷GpppRNA. Mutational and crystallographic analyses of cellular and poxvirus cap methyltransferases have yielded a coherent picture of a conserved active site and determinants of substrate specificity. Models of the Michaelis complex suggest a direct in-line mechanism of methyl transfer. Because no protein contacts to the guanine-N7 nucleophile, the AdoMet methyl carbon (Cε) or the AdoHcy sulfur (Sδ) leaving group were observed in ligand-bound structures of cellular cap methyltransferase, it was initially thought that the enzyme facilitates catalysis by optimizing proximity and geometry of the donor and acceptor. However, the structure of AdoHcy-bound vaccinia virus cap methyltransferase revealed the presence of an N-terminal “lid peptide” that closes over the active site and makes multiple contacts with the substrates, including the AdoMet sulfonium. This segment is disordered in the vaccinia apoenzyme and is not visible in the available structures of cellular cap methyltransferase. Here, we conducted a mutational analysis of the vaccinia virus lid peptide (⁵⁴⁵DKFRLNPEVSYFTNKRTRG⁵⁶³) entailing in vivo and in vitro readouts of the effects of alanine and conservative substitutions. We thereby identified essential functional groups that interact with the AdoMet sulfonium (Tyr555, Phe556), the AdoMet adenine (Asn550), and the cap triphosphate bridge (Arg560, Arg562). The results suggest that van der Waals contacts of Tyr555 and Phe556 to the AdoMet Sδ and Cε atoms, and the electron-rich environment around the sulfonium, serve to stabilize the transition state of the transmethylation reaction.     

Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis

The Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase Ecm1 has been characterized structurally but not biochemically. Here we show that purified Ecm1 is a monomeric protein that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to GTP. The reaction is cofactor-independent and optimal at pH 7.5. Ecm1 also methylates GpppA, GDP, and dGTP but not ATP, CTP, UTP, ITP, or m(7)GTP. The affinity of Ecm1 for the cap dinucleotide GpppA (K 0.1 mm) is higher than that for GTP (K(m) 1 mm) or GDP (K(m) 2.4 mm). Methylation of GTP by Ecm1 in the presence of 5 microm AdoMet is inhibited by the reaction product AdoHcy (IC(50) 4 microm) and by substrate analogs sinefungin (IC(50) 1.5 microm), aza-AdoMet (IC(50) 100 microm), and carbocyclic aza-AdoMet (IC(50) 35 microm). The crystal structure of an Ecm1.aza-AdoMet binary complex reveals that the inhibitor occupies the same site as AdoMet. Structure-function analysis of Ecm1 by alanine scanning and conservative substitutions identified functional groups necessary for methyltransferase activity in vivo. Amino acids Lys-54, Asp-70, Asp-78, and Asp-94, which comprise the AdoMet-binding site, and Phe-141, which contacts the cap guanosine, are essential for cap methyltransferase activity in vitro.     

Structural and functional analysis of EcoRI DNA methyltransferase by proteolysis

Native EcoRI DNA methyltransferase (Mtase, Mr 38,050) is proteolyzed by trypsin to generate an intermediate 36-kDa fragment (p36) followed by the formation of two polypeptides of Mr 23,000 and 13,000 (p23 and p13, respectively). Protein sequence analysis of the tryptic fragments indicates that p36 results from removal of the first 14 or 16 amino acids, p23 spans residues 15-216, and p13 spans residues 217-325. The relative resistance to further degradation of p23 and p13 suggests stable domain structures. This is further supported by the generation of similar fragments with SV8 endoprotease which has entirely different peptide specificities. Our results suggest the Mtase is a two-domain protein connected by a highly flexible interdomain hinge. The putative hinge region encompasses previously identified peptides implicated in AdoMet binding [Reich, N.O., & Everett, E. (1990) J. Biol. Chem. 265, 8929-8934] and catalysis [Everett et al. (1990) J. Biol. Chem. 265, 17713-17719]. Protection studies with DNA, S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy), and sinefungin (AdoMet analogue) show that the Mtase undergoes significant conformational changes upon ligand binding. Trypsinolysis of the AdoMet-bound form of the Mtase generates different fragments, and the AdoMet-bound form is over 800 times more stable than unbound Mtase. The sequence-specific ternary complex (Mtase-DNA-sinefungin) is 2000 times more resistant to degradation by trypsin; cleavage eventually generates 26- and 12-kDa fragments which span residues 104-325 and 1-103, respectively (p26 and p12). The first 14 or 16 amino acids of the Mtase are not essential since p36 retains activity. Activity analysis of the p26 and p12 mixture also indicates retention of activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Poxvirus mRNA cap methyltransferase. Bypass of the requirement for the stimulatory subunit by mutations in the catalytic subunit and evidence for intersubunit allostery

The guanine-N7 methyltransferase domain of vaccinia virus mRNA capping enzyme is a heterodimer composed of a catalytic subunit vD1-(540-844) and a stimulatory subunit vD12. The poxvirus enzyme can function in vivo in Saccharomyces cerevisiae in lieu of the essential cellular cap methyltransferase Abd1. Coexpression of both poxvirus subunits is required to complement the growth of abd1delta cells. We performed a genetic screen for mutations in the catalytic subunit that bypassed the requirement for the stimulatory subunit in vivo. We thereby identified missense changes in vicinal residues Tyr-752 (to Ser, Cys, or His) and Asn-753 (to Ile), which are located in the cap guanine-binding pocket. Biochemical experiments illuminated a mechanism of intersubunit allostery, whereby the vD12 subunit enhances the affinity of the catalytic subunit for AdoMet and the cap guanine methyl acceptor by 6- and 14-fold, respectively, and increases kcat by a factor of 4. The bypass mutations elicited gains of function in both vD12-independent and vD12-dependent catalysis of cap methylation in vitro when compared with wild-type vD1-(540-844). These results highlight the power of yeast as a surrogate model for the genetic analysis of interacting poxvirus proteins and demonstrate that the activity of an RNA processing enzyme can be augmented through selection and protein engineering.     

Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth.

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.     

An integrated system for genetic analysis

Background  

Large-scale genetic mapping projects require data management systems that can handle complex phenotypes and detect and correct high-throughput genotyping errors, yet are easy to use.     

Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins

Cap-binding proteins have been routinely isolated using m⁷GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m⁷GTP. Here, we report the synthesis of new affinity resins, m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m⁷GpppA-Sepharose, bind recombinant mouse eIF4E^(28-217) specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m⁷GpCH₂ppA-Sepharose. Our data prove the applicability of these novel resins, especially m⁷GpCH₂ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.     

A system for receptor functional analysis based on c-fos mRNA expression: Analysis of GnRH receptors as a test system

This report describes the establishment of a system for assessing receptor activation by RT-PCR-based detection of c-fos mRNA induction. In this system, COS-7 cells were transiently transfected with GnRH receptor expression plasmid, and ligand-induced c-fos expression was quantified by the RT-competitive PCR method. The results were compared with those of a conventional inositol phosphate (IP) assay. Changes in c-fos expression levels were observed in a dose- and ligand-dependent manner. Similar tendencies were observed in ligand selectivity between c-fos expression and IP production. The novel system developed and established in the present study is sensitive by using RT-PCR and convenient because it requires only basic methods of cell culture and molecular biology. It also has the merit that it does not need any specific measuring devices or radioactive substances. Given the ability of c-fos to respond to diverse stimuli, the present system may be applicable for various receptors for bioactive substances in addition to GnRH receptor, and useful for various purposes including screening ligands for orphan receptors.