Characterization of the mRNA capping apparatus of Candida albicans

The mRNA capping apparatus of the pathogenic fungus Candida albicans consists of three components: a 520- amino acid RNA triphosphatase (CaCet1p), a 449-amino acid RNA guanylyltransferase (Cgt1p), and a 474-amino acid RNA (guanine-N7-)-methyltransferase (Ccm1p). The fungal guanylyltransferase and methyltransferase are structurally similar to their mammalian counterparts, whereas the fungal triphosphatase is mechanistically and structurally unrelated to the triphosphatase of mammals. Hence, the triphosphatase is an attractive antifungal target. Here we identify a biologically active C-terminal domain of CaCet1p from residues 202 to 520. We find that CaCet1p function in vivo requires the segment from residues 202 to 256 immediately flanking the catalytic domain from 257 to 520. Genetic suppression data implicate the essential flanking segment in the binding of CaCet1p to the fungal guanylyltransferase. Deletion analysis of the Candida guanylyltransferase demarcates an N-terminal domain, Cgt1(1-387)p, that suffices for catalytic activity in vitro and for cell growth. An even smaller domain, Cgt1(1-367)p, suffices for binding to the guanylyltransferase docking site on yeast RNA triphosphatase. Deletion analysis of the cap methyltransferase identifies a C-terminal domain, Ccm1(137-474)p, as being sufficient for cap methyltransferase function in vivo and in vitro. Ccm1(137-474)p binds in vitro to synthetic peptides comprising the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II. Binding is enhanced when the C-terminal domain is phosphorylated on both Ser-2 and Ser-5 of the YSPTSPS heptad repeat. We show that the entire three-component Saccharomyces cerevisiae capping apparatus can be replaced by C. albicans enzymes. Isogenic yeast cells expressing "all-Candida" versus "all-mammalian" capping components can be used to screen for cytotoxic agents that specifically target the fungal capping enzymes.     

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.     

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.     

RAM/Fam103a1 is required for mRNA cap methylation

The 7-methylguanosine cap added to the 5' end of mRNA is required for efficient gene expression in eukaryotes. In mammals, methylation of the guanosine cap is catalyzed by RNMT (RNA guanine-7 methyltransferase), an enzyme previously thought to function as a monomer. We have identified an obligate component of the mammalian cap methyltransferase, RAM (RNMT-Activating Mini protein)/Fam103a1, a previously uncharacterized protein. RAM consists of an N-terminal RNMT-activating domain and a C-terminal RNA-binding domain. As monomers RNMT and RAM have a relatively weak affinity for RNA; however, together their RNA affinity is significantly increased. RAM is required for efficient cap methylation in vitro and in vivo, and is indirectly required to maintain mRNA expression levels, for mRNA translation and for cell viability. Our findings demonstrate that RAM is an essential component of the core gene expression machinery.     

mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase from vaccinia virions. Donor and acceptor substrate specificites.

Characterization of the donor and acceptor specificities of mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase isolated from vaccinia virus cores has enabled us to discriminate between alternative reaction sequences leading to the formation of the 5'-terminal m7G(5')pppN-structure. The mRNA guanylyltransferase catalyzes the transfer of a residue of GMP from GTP to acceptors which possess a 5'-terminal diphosphate. A diphosphate-terminated polyribonucleotide is preferred to a mononucleoside diphosphate as an acceptor suggesting that the guanylyltransferase reaction occurs after initiation of RNA synthesis. Although all of the homopolyribonucleotides tested (pp(A)n, pp(G)n, pp(I)n, pp(U)n, and pp(C)n) are acceptors for the mRNA guanylyltransferase indicating lack of strict sequence specificity, those containing purines are preferred. Only GTP and dGTP are donors in the reaction; 7-methylguanosine (m7G) triphosphate specifically is not a donor indicating that guanylylation must precede guanine-7-methylation. The preferred acceptor of the mRNA (guanine-7-)-methyltransferase is the product of the guanylyltransferase reaction, a polyribonucleotide with the 5'-terminal sequence G(5')pppN-. The enzyme can also catalyze, but less efficiently methylation of the following: dinucleoside triphosphates with the structure G(5')pppN, GTP, dGTP, ITP, GDP, GMP, and guanosine. The enzyme will not catalyze the transfer of methyl groups to ATP, XTP, CTP, UTP, or to guanosine-containing compounds with phosphate groups in either positions 2' or 3' or in 3'-5' phosphodiester linkages. The latter specificity provides an explanation for the absence of internal 7-methylguanosine in mRNA. In the presence of PPi, the mRNA guanylyltransferase catalyzes the pyrophosphorolysis of the dinucleoside triphosphate G(5')pppA, but not of m7G(5')pppA. Since PPi is generated in the process of RNA chain elongation, stabilization of the 5'-terminal sequences of mRNA is afforded by guanine-7-methylation.     

Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity.

The replication complexes of all positive strand RNA viruses of eukaryotes are associated with membranes. In the case of Semliki Forest virus (SFV), the main determinant of membrane attachment seems to be the virus-encoded non-structural protein NSP1, the capping enzyme of the viral mRNAs, which has guanine-7-methyltransferase and guanylyltransferase activities. We show here that both enzymatic activities of SFV NSP1 are inactivated by detergents and reactivated by anionic phospholipids, especially phosphatidylserine. The region of NSP1 responsible for binding to membranes as well as to liposomes was mapped to a short segment, which is conserved in the large alphavirus-like superfamily of viruses. A synthetic peptide of 20 amino acids from the putative binding site competed with in vitro synthesized NSP1 for binding to liposomes containing phosphatidylserine. These findings suggest a molecular mechanism by which RNA virus replicases attach to intracellular membranes and why they depend on the membranous environment.     

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.     

Vesicular stomatitis virus mRNA capping machinery requires specific cis-acting signals in the RNA

Many viruses of eukaryotes that use mRNA cap-dependent translation strategies have evolved alternate mechanisms to generate the mRNA cap compared to their hosts. The most divergent of these mechanisms are those used by nonsegmented negative-sense (NNS) RNA viruses, which evolved a capping enzyme that transfers RNA onto GDP, rather than GMP onto the 5' end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we show that mRNA cap formation is further distinct, requiring a specific cis-acting signal in the RNA. Using recombinant VSV, we determined the function of the eight conserved positions of the gene-start sequence in mRNA initiation and cap formation. Alterations to this sequence compromised mRNA initiation and separately formation of the GpppA cap structure. These studies provide genetic and biochemical evidence that the mRNA capping apparatus of VSV evolved an RNA capping machinery that functions in a sequence-specific manner.     

In vitro methylation of undermethylated yeast poly(A)-rich RNA using mRNA(guanine-7-)-methyltransferase purified from wheat germ or yeast

By crossing two strains of Saccharomyces cerevisiae deficient for each of the two methionine adenosyltransferase isoenzymes (ATP: L-methionine S-adenosyltransferase EC 2.5.1.6) respectively, we have constructed a strain strictly auxotrophic for S-adenosylmethionine and used it as a source of undermethylated mRNA suitable for in vitro transmethylation studies. RNA has been phenol-extracted from yeast cells shifted down to S-adenosylmethionine-free medium for 90 min and poly(A)-rich RNA has been prepared by oligo(dT)-cellulose chromatography. Upon incubation in vitro in the presence of methyl-labeled S-adenosylmethionine and mRNA (guanine-7-)-methyltransferase purified from wheat germ or yeast, undermethylated poly(A)-rich RNA became significantly labeled as compared to non-starved cells from the same strain, or from a wild-type control. Cap structures were resolved by paper chromatography afer T2 and P1 RNase digestion, and shown to be a mixture of m7G5'ppp5'G and m7G5'ppp5'A, irrespective of the enzyme source, in agreement with earlier in vivo studies in yeast mRNA capping and methylation.     

The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization

Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.     

Characterization of human, Schizosaccharomyces pombe, and Candida albicans mRNA cap methyltransferases and complete replacement of the yeast capping apparatus by mammalian enzymes.

Human and fission yeast cDNAs encoding mRNA (guanine-N7) methyltransferase were identified based on similarity of the human (Hcm1p; 476 amino acids) and Schizosaccharomyces pombe (Pcm1p; 389 amino acids) polypeptides to the cap methyltransferase of Saccharomyces cerevisiae (Abd1p). Expression of PCM1 or HCM1 in S. cerevisiae complemented the lethal phenotype resulting from deletion of the ABD1 gene, as did expression of the NH2-terminal deletion mutants PCM1(94-389) and HCM1(121-476). The CCM1 gene encoding Candida albicans cap methyltransferase (Ccm1p; 474 amino acids) was isolated from a C. albicans genomic library by selection for complementation of the conditional growth phenotype of S. cerevisiae abd1-ts mutants. Human cap methyltransferase was expressed in bacteria, purified, and characterized. Recombinant Hcm1p catalyzed quantitative S-adenosylmethionine-dependent conversion of GpppA-capped poly(A) to m7GpppA-capped poly(A). We identified by alanine-scanning mutagenesis eight amino acids (Asp-203, Gly-207, Asp-211, Asp-227, Arg-239, Tyr-289, Phe-291, and Phe-354) that are essential for human cap methyltransferase function in vivo. All eight residues are conserved in other cellular cap methyltransferases. Five of the mutant human proteins (D203A, R239A, Y289A, F291A, and F354A) were expressed in bacteria and found to be defective in cap methylation in vitro. Concordance of mutational effects on Hcm1p, Abd1p, and vaccinia capping enzyme underscores a conserved structural basis for cap methylation in DNA viruses, yeast, and metazoans. This is in contrast to the structural and mechanistic divergence of the RNA triphosphatase components of the yeast and metazoan capping systems. Nevertheless, we demonstrate that the entire three-component yeast capping apparatus, consisting of RNA 5'-triphosphatase (Cet1p), RNA guanylyltransferase (Ceg1p), and Abd1p could be replaced in vivo by the two-component mammalian apparatus consisting of a bifunctional triphosphatase-guanylyltransferase Mce1p and the methyltransferase Hcm1(121-476)p. Isogenic yeast strains with fungal versus mammalian capping systems should facilitate rational screens for antifungal drugs that target cap formation in vivo.     

Recognition of RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus

The mRNA-capping process starts with the conversion of a 5′-triphosphate end into a 5′-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5′-5′ phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2′OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2′O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, ^N7MeGpppG, and ^N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2′O-methyltransferase activities.     

Conditional mutants of the yeast mRNA capping enzyme show that the cap enhances, but is not required for, mRNA splicing.

The 5' end of eukaryotic mRNAs are modified by the addition of a 7-methyl guanosine (m7G) cap. The role of the cap in translation has been well established. Additionally, studies conducted in vitro or in microinjected Xenopus oocytes have implicated the cap in RNA processing and transport. To determine the fate of uncapped mRNA in intact yeast cells, conditional alleles of the gene encoding the capping enzyme guanylyltransferase subunit (CEG1) were generated. RNA analysis of temperature-sensitive ceg1 strains revealed an accumulation of unspliced pre-mRNAs and a corresponding decrease in spliced mRNAs at the restrictive temperature. A substantial proportion of spliced mRNA was also uncapped. Therefore, the cap appears to stimulate, but is not absolutely required for, splicing in vivo. In addition, steady-state levels of several mRNAs were decreased, perhaps due to increased degradation of uncapped mRNAs. In contrast to splicing, mRNA polyadenylation and transport to the cytoplasm were unaffected.     

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.     

Conversion of a DNA ligase into an RNA capping enzyme.

In eukaryotes, newly synthesised mRNA is 'capped' by the addition of GMP to the 5" end by RNA capping enzymes. Recent structural studies have shown that RNA capping enzymes and DNA ligases have similar protein folds, suggesting a conserved catalytic mechanism. To explore these similarities we have produced a chimeric enzyme comprising the N-terminal domain 1 of a DNA ligase fused to the C-terminal domain 2 of a mRNA capping enzyme. This report shows that this hybrid enzyme retains adenylation activity, characteristic of DNA ligases but, remarkably, the chimera has ATP-dependent mRNA capping activity. This is the first observation of ATP-dependent RNA capping. These results suggest that nucleotidyltransferases may have evolved from a common ancestral gene.