Characterization of Human, Schizosaccharomyces pombe, and Candida albicans mRNA Cap Methyltransferases and Complete Replacement of the Yeast Capping Apparatus by Mammalian Enzymes*

Nayanendu SahaDagger , Beate Schwer§, and Stewart ShumanDagger

From the Dagger  Molecular Biology Program, Sloan-Kettering Institute and the § Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The m7GpppN cap of eukaryotic mRNA is formed by a series of three enzymatic reactions in which the 5'-triphosphate end of nascent pre-mRNA is hydrolyzed to a 5'-diphosphate by RNA triphosphatase, then capped with GMP by GTP:RNA guanylyltransferase, and methylated by RNA (guanine-N7) methyltransferase (1). RNA capping is essential for cell growth. Mutations of the triphosphatase, guanylyltransferase, or methyltransferase components of the yeast capping apparatus which abrogate catalytic activity are lethal in vivo (2-12).

The physical and functional organizations of the capping apparatus differ in significant respects in fungi, metazoans, protozoa, and viruses. For example, fungi and mammals use distinct strategies to assemble a bifunctional enzyme with triphosphatase and guanylyltransferase activities. In yeast, separate triphosphatase (Cet1p) and guanylyltransferase (Ceg1p) polypeptides interact to form a heteromeric complex (10, 11, 13), whereas in mammals, autonomous triphosphatase and guanylyltransferase domains are linked in cis within a single polypeptide (Mce1p) (14, 15). The triphosphatase and guanylyltransferase components of the vaccinia virus and baculovirus capping enzymes also reside within single polypeptides (16-19). The active sites of the guanylyltransferases are conserved among fungi, mammals, protozoa, and DNA viruses (6, 18, 20, 21), but the triphosphatase components diverge with respect to structure and mechanism. There are at least two distinct classes of RNA triphosphatases: (i) the divalent cation-dependent RNA triphosphatase/NTPase family (exemplified by yeast Cet1p, baculovirus LEF-4, and vaccinia D1) (11, 12, 16-19, 21) and (ii) the divalent cation-independent RNA triphosphatases, e.g. the metazoan cellular enzymes and the baculovirus enzyme BVP (14, 15, 22-26).

The enzyme RNA (guanine-N7) methyltransferase (referred to hereafter as cap methyltransferase) catalyzes the transfer of a methyl group from AdoMet1 to the GpppN terminus of RNA to produce m7GpppN-terminated RNA and AdoHcy (1). The Saccharomyces cerevisiae cap methyltransferase is the product of the ABD1 gene (7). ABD1 encodes a 436-amino acid polypeptide. A catalytic domain of Abd1p from residues 110-426 suffices for yeast cell growth (8, 9); this segment of Abd1p is homologous to the methyltransferase catalytic domain of the vaccinia virus capping enzyme (7). A key distinction between the yeast and poxvirus cap methyltransferases is their physical linkage, or lack thereof, to the other cap-forming enzymes. The poxvirus methyltransferase active site is encoded within the same polypeptide as the triphosphatase and guanylyltransferase (27, 28), whereas the yeast methyltransferase is a monomeric protein that is not associated with the other capping activities during fractionation of yeast extracts (7). Mutational analyses of the yeast and vaccinia cap methyltransferases have identified conserved residues that are critical for cap methylation (8, 9, 29). In the case of Abd1p, mutations that abolished methyltransferase activity in vitro were lethal in vivo. At present, Abd1p is the only cap methyltransferase that has been identified from a fungal source.

There is scant information available on the cap-methylating enzymes from metazoan organisms. An RNA (guanine-N7) methyltransferase with a native size of 130 kDa (estimated by gel filtration) was isolated from rat liver by Mizumoto and Lipmann (30). A cap methyltransferase with a native size of 56 kDa (gauged by sedimentation and gel filtration) was partially purified from HeLa cells by Ensinger and Moss (31). No metazoan cap methyltransferase has yet been purified to homogeneity. Identification of the gene encoding a metazoan cap methyltransferase and characterization of the recombinant protein offer the quickest route to an understanding of cap methylation in higher organisms. A putative cap methyltransferase from Caenorhabditis elegans was identified on phylogenetic grounds (9). Alignment of the sequence of the predicted 402-amino acid C. elegans protein (GenBank accession Z81038) with the yeast cap methyltransferase Abd1p revealed side chain identity or similarity at 149/402 positions. Although it remains to be shown that the nematode protein has cap methyltransferase activity, the extensive sequence conservation suggested that other metazoans might also encode homologs of Abd1p.

Here, we report the identification of Schizosaccharomyces pombe, Candida albicans, and human cDNAs or genes that encode cap methyltransferases (Pcm1p, Ccm1p, and Hcm1p, respectively) that can substitute for yeast Abd1p in vivo. (cDNAs encoding the human protein were reported recently by other investigators (34, 38) but without evidence of activity in vivo.) We present a physical characterization of the recombinant human cap methyltransferase produced in bacteria, and we define by deletion analysis a catalytic domain of Hcm1p which is active in vivo and in vitro. We also identify by alanine scanning eight amino acid residues that are essential for Hcm1p function and are conserved in other cellular cap-methylating enzymes. Finally, we establish the potential for using yeast to identify therapeutic drugs that differentially target fungal and metazoan cap-forming enzymes.

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INTRODUCTION
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HCM1 cDNA Expression Vectors-- Plasmid pHG0376 containing the 6,203-bp human brain cDNA KIAA0398 (GenBank accession AB007858) inserted into pBluescript II-SK+ was a generous gift of Dr. Takahiro Nagase (Kasuza DNA Research Institute, Japan). A DNA fragment containing the 476-amino acid open reading frame was amplified by polymerase chain reaction (PCR) from the pHG0376 template using Pfu DNA polymerase and oligonucleotide primers designed to introduce an NdeI restriction site at the translation start codon and a BamHI site 3' of the stop codon. The PCR product was digested with NdeI and BamHI and inserted into the T7 RNA polymerase-based expression plasmid pET16b to generate plasmid pET-His-Hcm1. The DNA sequence of the insert of pET-His-Hcm1 was identical to that of the KIAA0398 clone. Amino-terminal deletion mutants of HCM1 were constructed by PCR amplification with mutagenic sense strand primers that introduced an NdeI restriction site and a methionine codon in lieu of the codon for Gly-120 or an NdeI restriction site at the Met-179 codon. The PCR products were digested with NdeI and BamHI and then inserted into pET16b to yield plasmids pET-His-Hcm1(121-476) and pET-His-Hcm1(179-476). The inserts of each plasmid were sequenced to verify that no unwanted coding changes were introduced during amplification and cloning.

Hcm1p Expression and Purification-- pET-His-Hcm1 was transformed into Escherichia coli BL21(DE3). A 1-liter culture of E. coli BL21(DE3)/pET-His-Hcm1 was grown at 37 °C in Luria-Bertani medium containing 0.1 mg/ml ampicillin until the A600 reached 0.5. The culture was adjusted to 0.4 mM isopropyl-beta -D-thiogalactopyranoside, and incubation was continued at 30 °C for 4 h. Cells were harvested by centrifugation, and the pellet was stored at -80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria were resuspended in 50 ml of lysis buffer (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10% sucrose). Cell lysis was achieved by the addition of lysozyme and Triton X-100 to final concentrations of 50 µg/ml and 0.1%, respectively. The lysate was sonicated to reduce viscosity, and insoluble material was removed by centrifugation for 30 min at 18,000 rpm in a Sorvall SS34 rotor. The soluble extract was mixed for 1 h with 2 ml of Ni+-nitrilotriacetic acid-agarose resin (Qiagen) that had been equilibrated with lysis buffer. The suspension was poured into a column and washed with lysis buffer. The column was eluted stepwise with IMAC buffer (20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol) containing 25, 50, 200, and 500 mM imidazole. The polypeptide composition of the column fractions was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant Hcm1p protein was retained on the column and recovered in the 200 mM imidazole eluate. An aliquot of this fraction (100 µg of protein) was applied to a 4.8-ml 15-30% glycerol gradient containing 0.5 M NaCl in buffer A (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM dithiothreitol, and 0.1% Triton X-100). The gradient was centrifuged at 50,000 rpm for 15 h at 4 °C in a Beckman SW50 rotor. Fractions (~0.2 ml) were collected from the bottom of the tube. Protein concentration was determined using the Bio-Rad dye reagent with bovine serum albumin as the standard.

A modified strategy was employed to optimize the expression of the Hcm1(121-476)p protein in soluble form in bacteria. A 1-liter culture of E. coli BL21(DE3) bearing the pET-based plasmid was grown at 37 °C until the A600 reached 0.5. The culture was adjusted to 2% ethanol, and incubation was continued at 17 °C for 24 h. The recombinant Hcm1(121-476)p protein was purified from the soluble lysate by Ni+-nitrilotriacetic acid-agarose chromatography and glycerol gradient sedimentation as described above for wild type Hcm1p. Hcm1(179-476)p was expressed in bacteria but was recovered exclusively in the insoluble pellet fraction of the cell lysate.

Yeast HCM1 Expression Plasmids-- NdeI-BamHI restriction fragments containing the wild type HCM1 gene and the NH2-terminal deletion mutants were excised from the respective pET16b-based plasmids and inserted into a customized yeast expression vector pYX1-His, a derivative of pYX132 (CEN TRP1) in which six consecutive histidine codons and a unique NdeI site are inserted between the NcoI and BamHI sites of pYX132. (pYX132 was purchased from Novagen.) The single copy expression plasmids were named pYX-Hcm1, pYX-Hcm1(121-476), and pYX-Hcm1(179-476). NcoI-XhoI fragments containing the wild type HCM1 gene and the NH2-terminal deletion mutants were excised from the respective pYX1-based CEN plasmids and inserted into the yeast expression vector pYX232 (2µ TRP1). In these vectors, expression of the human methyltransferase is under the control of the yeast TPI1 promoter.

Alanine Mutants of Hcm1(121-476)p-- Alanine substitution mutations in the HCM1(121-476) gene were programmed by synthetic oligonucleotides using the two-stage PCR-based overlap extension strategy as described (8). An NdeI-BamHI restriction fragment of each PCR-amplified gene was inserted into pET16b. The presence of the desired mutation was confirmed in every case by sequencing the entire insert. The Hcm1p (121-476)-Ala mutant proteins were expressed in bacteria and purified as described above for the "wild type" Hcm1(121-476) protein. The mutated genes were transferred from the pET vectors to the yeast expression plasmid pYX1-His. Then, the mutant genes were excised from their respective pYX1-based CEN plasmids and inserted into the 2µ plasmid pYX232.

Yeast PCM1 Expression Plasmids-- The complete S. pombe open reading frame encoding Pcm1p (a putative homolog of Abd1p; GenBank accession AL031603) was amplified by PCR from a S. pombe cDNA library (39) using an antisense primer that introduced a BamHI site immediately 3' of the translation stop codon. The PCR product was digested with NdeI (which cleaved at a unique in-frame site overlapping codons His-5 and Met-6) and BamHI, then inserted into a customized yeast expression vector pYN132, a derivative of pYX132 in which a unique NdeI site replaced the NcoI site of pYX132. The resulting plasmid was named pYN-Pcm1(6-389). Amino-terminal deletion mutants of PCM1 were constructed by PCR amplification with mutagenic sense strand primers that introduced an NdeI restriction site at the codon for Met-30 or Met-56 or an NdeI restriction site and a methionine codon in lieu of the codon for Gly-80, Val-93, Tyr-106, or Leu-117. The PCR products were digested with NdeI and BamHI and then inserted into pYN132 to yield plasmids pYN-Pcm1(30-389), pYN-Pcm1(56-389), pYN-Pcm1(81-389), pYN-Pcm1(94-389), pYN-Pcm1(107-389), and pYN-Pcm1(118-389). In these vectors, expression of the S. pombe polypeptide is under the control of the TPI1 promoter.

Expression Plasmids for S. cerevisiae Capping Enzymes-- Plasmids p360-ABD1 (CEN URA3 ABD1) and p358-ABD1 (CEN TRP1 ABD1) contain the yeast cap methyltransferase gene under the control of its natural promoter (7). Plasmids p360-CET1/CEG1 (CEN URA3 CET1 CEG1) and p358-CET1/CEG1 (CEN TRP1 CET1 CEG1) contain the yeast RNA triphosphatase and guanylyltransferase genes under the control of their natural promoters; the CET1 and CEG1 genes are arrayed head to head and transcribed divergently. Plasmids p360-CET1/CEG1/ABD1 (CEN URA3 CET1 CEG1 ABD1) and p358-CET1/CEG1/ABD1 (CEN TRP1 CET1 CEG1 ABD1) were constructed by insertion of ABD1 (under the control of its natural promoter) into p360-CET1/CEG1 and p358-CET1/CEG1, respectively; the ABD1 gene was placed next to CEG1 in a tail-to-tail orientation.

Yeast Strains-- Yeast strain YBS50 (MATa leu2 ade2 trp1 his3 ura3 can1 ceg1::hisG cet1::LEU2 p360-CET1/CEG1) is deleted at the chromosomal loci encoding RNA triphosphatase and guanylyltransferase. YBS40 (MATa leu2 ade2 trp1 his3 ura3 can1 abd1::hisG p360-ABD1) and YBS10 (MATalpha ura3 trp1 lys2 his3 leu2 abd1::LEU2 p360-ABD1) are deleted at the chromosomal locus encoding cap methyltransferase. YBS52 (MATa leu2 ade2 trp1 his3 ura3 can1 ceg1::hisG cet1::LEU2 abd1::KAN p360-CET1/CEG1/ABD1) is deleted at the chromosomal loci encoding all three components of the yeast capping apparatus. YBS50, YBS40, and YBS52 were derived by targeted gene disruptions in the diploid strain W303 followed by tetrad dissection and genotyping of haploid progeny. YBS10 was derived by targeted gene disruption in strain YPH399. Gene disruptions were confirmed by Southern blotting.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cDNAs Encoding Human and Fission Yeast Cap Methyltransferases-- The 476-amino acid polypeptide encoded by human cDNA KIAA0398 was uncovered during a screen of the NCBI data base for potential homologs of the yeast cap-methylating enzyme Abd1p. The putative human cap methyltransferase (which we named Hcm1p) displays extensive sequence conservation with the 426-amino acid Abd1p protein (176 positions of identity or similarity) (Fig. 1). Abd1p contains an 11-amino acid COOH-terminal extension that has no counterpart in Hcm1p; note that the COOH-terminal decapeptide of Abd1p is dispensable for cap methyltransferase activity in vitro and in vivo (9). Hcm1p also displays extensive sequence similarity to the candidate cap methyltransferase from C. elegans: there are 179 identical or conserved side chains with a 347-amino acid overlap that extends from Hcm1p residue 130 to its COOH terminus and from positions 8 to 356 of the C. elegans polypeptide (Fig. 1).


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Fig. 1.   Amino acid sequence conservation in human and yeast cap methyltransferases. The complete amino acid sequences of the 476-amino acid Hcm1p protein (hcm) and the 389-amino acid Pcm1p protein (pcm) are aligned with the complete sequence of yeast Abd1p (abd) and with the predicted 402-amino acid C. elegans C25A1.f gene product (cel) from residue 1 to 373. Gaps in the sequences are indicated by dashes. The carboxyl termini of Hcm1p, Pcm1p, and Abd1p are indicated by asterisks. Residues that are identical or similar in at least three of the four proteins are shown in shaded boxes. Amino acids essential for Abd1p function are denoted by dots.

We also identified on phylogenetic grounds a putative S. pombe cap methyltransferase (which we named Pcm1p) encoded by a gene on fission yeast chromosome III. We determined that this gene is expressed in vivo, insofar as we were able to PCR amplify the complete PCM1 coding sequence from a S. pombe cDNA library. The 389-amino acid S. pombe polypeptide displays extensive sequence similarity to S. cerevisiae Abd1p (201 positions of identity or side chain similarity) (Fig. 1). It is noteworthy that all six amino acids that have been shown to be essential for cap methylation by Abd1p (denoted by dots above the aligned sequences in Fig. 1) are conserved in Hcm1p and Pcm1p (8, 9).

S. pombe Pcm1p Is Functional in Vivo in Lieu of S. cerevisiae Abd1p-- We took advantage of an in-frame NdeI restriction site at codons His-5 and Met-6 in the PCM1 cDNA to clone a restriction fragment encoding Pcm1(6-389)p into a yeast CEN TRP1 plasmid such that its expression was under the control of the constitutive S. cerevisiae TPI1 promoter. The PCM1(6-389) plasmid was introduced into a yeast strain in which the chromosomal ABD1 locus was deleted. Growth of the Delta abd1 strain is contingent on maintenance of an extrachromosomal ABD1 gene on a CEN URA3 plasmid. Trp+ transformants were plated on medium containing 5-FOA to select against the URA3 ABD1 plasmid. Control cells transformed with a TRP1 ABD1 plasmid grew on 5-FOA, whereas cells transformed with the TRP1 vector were incapable of growth on 5-FOA (Fig. 2A). The instructive finding was that cells bearing the PCM1(6-389) plasmid grew on 5-FOA (Fig. 2A). Thus, the S. pombe cap methyltransferase was functional in vivo in lieu of the endogenous S. cerevisiae enzyme.


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Fig. 2.   S. pombe Pcm1p replaces S. cerevisiae Abd1p in vivo. Yeast strain YBS40 was transformed with CEN TRP1 plasmids containing the indicated alleles of PCM1. Control transformations were performed using the TRP1 vector alone and p358-ABD1. Trp+ isolates were streaked on agar plates containing 0.75 mg/ml 5-FOA. The plates were photographed after incubation for 3 days at 30 °C.

Several NH2-terminal deletion mutants of PCM1 were cloned into the CEN TRP1 vector and tested for function in vivo by plasmid shuffle. PCM1(30-389) and PCM1(56-389) complemented growth of the Delta abd1 strain on 5-FOA (Fig. 2A), as did alleles PCM1(81-389) and PCM1(94-389) (Fig. 2B). However, the more extensively truncated alleles PCM1(107-389) and PCM1(118-389) were lethal in vivo (Fig. 2B). Based on the sequence alignment in Fig. 1, the viable NH2-terminal Delta 29, Delta 55, and Delta 80 and Delta 93 deletions of Pcm1p would be analogous to deletions of 53, 69, 102, and 121 amino acids from the NH2 terminus of Abd1p. (The NH2 terminus of the viable Pcm1pDelta 93 deletion is denoted by the arrow in Fig. 1.) The lethal NH2-terminal Delta 106 and Delta 117 deletions of Pcm1p correspond to Delta 135 and Delta 147 deletions of Abd1p. We showed previously that deleting 52 or 109 amino acids from the NH2 terminus of Abd1p had no effect on yeast cell growth, whereas deletion of 142 or 155 residues was lethal (8, 9). We recently found that an NH2-terminal deletion of 120 amino acids from Abd1p is viable when the truncated protein is expressed under the control of the TPI1 promoter.2 Thus, the NH2-terminal margins of the functional domains of the budding and fission yeast cap methyltransferases are fairly similar.

Human Cap Methyltransferase Is Functional in Vivo in Yeast-- Full-length HCM1 and an NH2-terminal deletion mutant HCM1(121-476) were cloned into a yeast 2µ TRP1 plasmid such that their expression was under the control of the yeast TPI1 promoter. The HCM1 plasmids were introduced into an abd1 deletion strain and tested for function by plasmid shuffle. Cells bearing the HCM1 or HCM1(121-476) plasmids grew on 5-FOA, as did cells transformed with a TRP1 ABD1 control plasmid, whereas cells transformed with the TRP1 vector did not grow on 5-FOA (Fig. 3A). We conclude that human cap methyltransferase was functional in vivo in lieu of the endogenous yeast enzyme and that deletion of 120 amino acids from the NH2 terminus (comparable to a deletion of 92 amino acids from the NH2 terminus of yeast Abd1p) was without effect. Thus, the dispensability of the NH2-terminal domains is a shared feature of the human and fungal cap methyltransferases. A more extensively truncated allele HCM1(179-476) did not support growth on 5-FOA (not shown).


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Fig. 3.   Human cap methyltransferase replaces yeast Abd1p in vivo. Yeast strain YBS10 was transformed with 2µ TRP1 plasmids (panel A) or CEN TRP1 plasmids (panel B) containing the wild type HCM1 gene or NH2-terminal deletion mutants as specified. Control transformations were performed using the TRP1 vectors alone and p358-ABD1. Trp+ isolates were streaked on agar plates containing 0.75 mg/ml 5-FOA. The plates were photographed after incubation for 3 days at 30 °C.

HCM1 and HCM1(121-476) were also cloned into CEN TRP1 vectors. Expression of the Delta 120 protein in single copy complemented the abd1 deletion, whereas expression of the full-length Hcm1p protein did not (Fig. 3B). The copy number dependence of complementation by full-length HCM1 may be indicative of lower levels of Hcm1p expression in yeast compared with the Delta 120 derivative.

Cap Methyltransferase Activity of Recombinant Hcm1p-- We expressed the Hcm1p protein in bacteria. An inducible T7 RNA polymerase-based vector was constructed in which a short histidine-rich amino-terminal leader segment was fused to the Hcm1p polypeptide. The expression plasmid was introduced into E. coli BL21(DE3), a strain that contains the T7 RNA polymerase gene under the control of a lacUV5 promoter. A 57-kDa polypeptide corresponding to His-Hcm1p was detectable by SDS-PAGE in soluble extracts of isopropyl-beta -D-thiogalactopyranoside-induced bacteria (not shown). The His-tag allowed for rapid enrichment of Hcm1p based on the affinity of the tag for immobilized nickel. The bacterial lysate was applied to Ni+-nitrilotriacetic acid-agarose, and adsorbed material was step-eluted with increasing concentrations of imidazole. SDS-PAGE analysis revealed a prominent 57-kDa Coomassie Blue-stained species in the 200 mM imidazole eluate (see Fig. 6A). This polypeptide was not recovered when a lysate of isopropyl-beta -D-thiogalactopyranoside-induced BL21(DE3) carrying the pET vector alone was subjected to the same Ni+-nitrilotriacetic acid-agarose chromatography procedure (data not shown).

RNA (guanine-N7) methyltransferase activity of the Ni+-nitrilotriacetic acid-agarose Hcm1p preparation was detected by the conversion of 32P cap-labeled poly(A) to methylated cap-labeled poly(A) in the presence of AdoMet (7). The reaction products were digested to cap dinucleotides with nuclease P1 and then analyzed by polyethyleneimine-cellulose thin layer chromatography, which resolves the GpppA cap from the methylated cap m7GpppA. The radiolabeled product synthesized by Hcm1p (Fig. 4, lane 1) comigrated with m7GpppA generated in a parallel reaction mixture containing purified recombinant vaccinia virus cap methyltransferase (Fig. 4, lane 6). Cap methylation by Hcm1p depended on inclusion of S-adenosylmethionine in the reaction mixture (Fig. 4, lane 2). S-Adenosylhomocysteine did not support cap methylation (Fig. 4, lane 3) and was partially inhibitory in the presence of AdoMet (Fig. 4, lane 4).


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Fig. 4.   Cap methyltransferase activity of recombinant Hcm1p. The complete reaction mixture (lane 1) contained (in 10 µl) 50 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 16 fmol of cap-labeled poly(A), 50 µM AdoMet, and ~50 fmol of Hcm1p (Ni+-nitrilotriacetic acid-agarose 0.2 M imidazole eluate fraction). Reaction components were varied as follows: omit AdoMet (lane 2); omit AdoMet, include 50 µM AdoHcy (lane 3); include 50 µM AdoMet plus 500 µM AdoHcy (lane 4); omit Hcm1p (lane 5); omit Hcm1p, include purified recombinant vaccinia capping enzyme (lane 6). After incubation at 37 °C for 10 min, the reaction mixtures were heated at 95 °C for 5 min and then adjusted to 50 mM sodium acetate (pH 5.5). The samples were incubated with 5 µg of nuclease P1 for 60 min at 37 °C. The digests were then spotted on polyethyleneimine-cellulose TLC plates that were developed with 0.2 M (NH4)2SO4. An autoradiograph of the chromatogram is shown. The chromatographic origin and the positions of cap dinucleotides m7GpppA and GpppA are indicated on the right.

Hcm1p was purified further by centrifugation of the Ni+-nitrilotriacetic acid-agarose fraction through a 15-30% glycerol gradient. Cap methyltransferase activity sedimented as a single peak coincident with the peak of the Hcm1p polypeptide (Fig. 5A). The apparent sedimentation coefficient of 4 S (relative to markers analyzed in parallel) suggested that the recombinant human cap methyltransferase is a monomer. The S value of the recombinant human enzyme determined by glycerol gradient sedimentation agrees with the value of 3.8 determined by sucrose gradient sedimentation of the cap methyltransferase isolated from HeLa cell extracts (31).


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Fig. 5.   Glycerol gradient sedimentation. The Ni+-nitrilotriacetic acid-agarose fractions of Hcm1p (panel A) and the NH2-terminal deletion mutant Delta 120 (panel B) were sedimented in glycerol gradients as described under "Experimental Procedures." Fractions were collected from the bottom of the tubes (fraction 1). Aliquots (20 µl) of alternate fractions were analyzed by SDS-PAGE along with an aliquot of the material that had been applied to the gradient (lane Ni). The gels were fixed and stained with Coomassie Blue. The positions and sizes (kDa) of coelectrophoresed marker polypeptides are indicated at the left of each gel. Methyltransferase reaction mixtures contained 16 fmol of cap-labeled poly(A), 50 µM AdoMet, and 1 µl of a 1/500 dilution of the indicated fractions from the glycerol gradients. The reaction products were digested with nuclease P1 and analyzed by TLC. The extent of cap methylation (m7GpppA/(m7GpppA + GpppA)) was determined by scanning the chromatogram with a FUJIX BAS1000 PhosphorImager. The peaks of marker proteins catalase, bovine serum albumin (BSA), and cytochrome c, which were centrifuged in a parallel gradient, are indicated by arrowheads.

Characterization of the enzyme was performed using the peak glycerol gradient fraction of Hcm1p. Methylation of capped poly(A) varied linearly with input enzyme and was quantitative at saturation (Fig. 6B). MgCl2 strongly inhibited activity in a concentration-dependent fashion; methylation was reduced by an order of magnitude by 1 mM magnesium (not shown). Similar inhibition by magnesium was noted for the yeast and vaccinia cap methyltransferases (7, 27, 29). The extent of methylation varied with AdoMet concentration. Half-maximal activity was observed at ~25 µM AdoMet (not shown). The reaction product AdoHcy inhibited cap methylation in a concentration-dependent manner; cap methylation in the presence of 10 µM AdoMet was reduced by 80% in the presence of 100 µM AdoHcy (not shown).


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Fig. 6.   Cap methylation by Hcm1p and Delta 120. Panel A, polypeptide composition. Aliquots (10 µg) of the peak glycerol gradient fractions of wild type (WT) Hcm1p and deletion mutant Delta 120 were analyzed by SDS-PAGE. Polypeptides were visualized by staining with Coomassie Blue. The positions and sizes (kDa) of marker polypeptides are indicated at the left. Panel B, methyltransferase activity. Reaction mixtures contained 20 fmol of cap-labeled poly(A), 50 µM AdoMet, and wild type or Delta 120 enzyme as specified.

A His-tagged version of the NH2-terminal truncated protein Hcm1(121-476)p (referred to as Delta 120) was expressed in E. coli. and purified from a soluble bacterial lysate by Ni+-nitrilotriacetic acid-agarose chromatography and glycerol gradient sedimentation (Fig. 5B). (The more extensively truncated protein Hcm1(179-476)p was insoluble when expressed in bacteria and therefore not amenable to purification.) The Delta 120 protein sedimented as a discrete peak of 4 S coincident with cap methyltransferase activity.

SDS-PAGE analysis of the peak glycerol gradient fractions of full-length Hcm1p and Delta 120 revealed that the proteins were of comparable purity and that the truncated version migrated more rapidly than the full-sized Hcm1p, as expected (Fig. 6A). The specific activity of Delta 120 in cap methylation, calculated from the slope of the titration curve in the linear range of enzyme dependence (Fig. 6B), was 50% of the activity of full-length Hcm1p.

Identification of Essential Residues of Human Cap Methyltransferase by Alanine Scanning-- Mutational analysis of S. cerevisiae Abd1p by alanine scanning has led to the identification of six individual amino acid residues that are essential for Abd1p function in vivo (8, 9); these are denoted by dots in the sequence alignment in Fig. 1. To gauge whether these residues are also critical for the human cap methyltransferase, we introduced alanine substitutions at the six corresponding positions of Hcm1(121-476)p: Asp-203, Gly-207, Asp-211, Asp-227, Arg-239, and Tyr-289. In addition, we mutated two other aromatic residues of Hcm1p, Phe-291 and Phe-354 (denoted by arrowheads in Fig. 1), which are conserved in Abd1p, Pcm1p, and the C. elegans homolog. The eight HCM1-Ala alleles were cloned into CEN TRP1 vectors under the control of the TPI1 promoter, transformed into a Delta abd1 strain, and then tested for function in vivo by plasmid shuffle. All eight mutations were lethal, i.e. the Trp+ transformants could not form colonies on medium containing 5-FOA (not shown). The eight HCM1-Ala alleles were also cloned into 2µ TRP1 vectors under the control of the TPI1 promoter; all eight were lethal even in high copy (not shown).

Five of the Hcm1(121-476)p-Ala mutants, D203A, R239A, Y289A, F291A, and F354A were expressed in bacteria and purified from soluble extracts by Ni+-nitrilotriacetic acid-agarose chromatography and glycerol gradient sedimentation in parallel with wild type Hcm1(121-476)p. (The other three Ala mutants were insoluble when expressed in bacteria.) SDS-PAGE analysis indicated that the glycerol gradient preparations were all highly enriched with respect to the Hcm1(121-476)p polypeptide (Fig. 7A). The methyltransferase activity of the recombinant proteins was assayed as a function of protein concentration (Fig. 7B). All five mutations, which were lethal in vivo, elicited severe defects in cap methylation activity in vitro. The specific activity of the F291A protein was 5% that of wild type Delta 120, whereas the activities of the D203A, R239A, Y289A, and F354A mutants were less than 1% of the wild type value (Fig. 7B).


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Fig. 7.   Effects of alanine mutations on cap methyltransferase activity. Panel A, polypeptide composition. Aliquots (2.5 µg) of the peak glycerol gradient fractions of wild type (WT) Hcm1(121-476)p and the indicated Hcm1(121-476)-Ala mutants were analyzed by SDS-PAGE. Polypeptides were visualized by staining with Coomassie Blue. The positions and sizes (kDa) of marker polypeptides are indicated at the left. Panel B, methyltransferase activity. Reaction mixtures contained 20 fmol of cap-labeled poly(A), 50 µM AdoMet, and wild type or mutant enzymes as specified. Activity is plotted as a function of input enzyme. Each datum is the average of two (wild type) or three (Ala mutants) separate titration experiments.

Isolation of the Gene Encoding C. albicans Cap Methyltransferase by Selection for Complementation of abd1-ts Mutants-- We sought to identify the cap methyltransferase gene from the pathogenic yeast C. albicans by screening for complementation of the conditional growth defect of S. cerevisiae abd1-ts mutants3 after transforming three different ts mutant strains (abd1-5, abd1-8, and abd1-15) with a C. albicans genomic DNA library that had been cloned into an S. cerevisiaeURA3 plasmid vector (40). Ura+ transformants were selected for growth at the nonpermissive temperature of 34 °C; positive isolates were then rescreened for growth at the even higher restrictive temperature of 37 °C. 2µ plasmid DNA recovered from nine individual isolates that grew at 37 °C was amplified in E. coli, and then the plasmid inserts were analyzed by digestion with a battery of restriction endonucleases that cut in the polylinker flanking the library insertion site. An apparently identical 6.6-kilobase pair insert was present in the 2µ plasmids from all nine isolates that had been rescued to grow at 37 °C. This 2µ plasmid (named pCCM1-6.6; Fig. 8A) retested faithfully in complementation of the ts growth phenotype when retransfromed into the abd1-5 strain (Fig. 8B). Hence, we presumed that the plasmid contains the Candida cap methyltransferase gene (which we named CCM1). Excision of a 2.7-kilobase pair SphI fragment from the right end of pCCM1-6.6 yielded the 2µ plasmid pCCM1-3.9 (Fig. 8A), which was as effective as the original isolate in rescuing growth of abd1-5 at 37 °C (Fig. 8B). Removal of a 1-kilobase pair EcoRI fragment from the left end of the insert (to yield the 2µ plasmid pCCM1-Delta Eco) resulted in loss of complementing activity (not shown), which implies that the CCM1 gene spans the EcoRI site of the insert. Removal of a 0.3-kilobase pair HindIII fragment from the right end of the insert had no effect on complementation (not shown). Restriction fragments derived from the insert of pCCM1-3.9 were subcloned into pBluescript and then sequenced.


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Fig. 8.   Cloning of the C. albicans cap methyltransferase gene by genetic complementation in S. cerevisiae. Panel A, the C. albicans genomic DNA inserts of the 2µ library plasmid pCCM1-6.6 and the subclone pCCM1-3.9 are illustrated. Restriction sites used in subcloning and gene mapping are indicated. The CCM1 open reading frame is depicted as a black bar, with the orientation of the coding strand indicated by an arrow. Panel B, complementation of abd1-5 by CCM1. Yeast strain ad1-5 was transformed with 2µ URA3 plasmids pCCM1-6.6, pCCM1-3.9, YEp24 (vector), and YEp24-ABD1. Transformants were streaked on agar medium lacking uracil and incubated at either 25 °C or 37 °C. Panel C, the sequence of the 474-amino acid Ccm1p protein is shown. The Ccm1p residues corresponding to the 8 amino acids found to be essential for Hcm1p function are denoted by dots.

The CCM1 gene comprises a continuous open reading frame of 1,422 nucleotides extending from left to right across the EcoRI site (Fig. 8A) (GenBank accession AF133529). CCM1 encodes a 474-amino acid polypeptide (Fig. 8C). The genomic insert in pCCM1-6.6 extends 254 base pairs upstream of the translation start site; this element apparently includes a C. albicans promoter element that can drive CCM1 expression in S. cerevisiae. The predicted amino acid sequence reveals Ccm1p to be an obvious homolog of Abd1p (209 positions of identity or similarity), Pcm1p (184 positions of identity or similarity), and Hcm1p (140 positions of identity or similarity). All 8 residues defined above as essential for the human cap methyltransferase are conserved in the C. albicans protein (Fig. 8C). Ccm1p is the first example of a cap methyltransferase from a fungal genus which is pathogenic for humans.

Complete Replacement of the Yeast Capping Apparatus by Mammalian Enzymes-- The physical and functional organizations of the capping apparatus differ in significant respects in fungi, metazoans, protozoa, and viruses. Hence, the capping enzymes are potential targets for antifungal, antiviral, and antiprotozoal drugs that would interfere with capping of pathogen mRNAs but spare the mammalian host capping enzymes. A plausible strategy for drug discovery would be to identify compounds that block cell growth contingent on pathogen-encoded capping activities without affecting the growth of otherwise identical cells bearing the capping enzymes of the host organism. For this approach to be feasible, the capping systems of interest must be interchangeable in vivo. Thus, we sought to construct yeast strains in which the entire fungal capping apparatus was replaced by mammalian enzymes.

We and others have shown that expression of the mammalian triphosphatase-guanylyltransferase in yeast can complement the growth of singly deleted Delta ceg1 or Delta cet1 strains (11, 14, 32, 33). This prompted us to test the ability of the mammalian capping enzyme to complement a Delta cet1 Delta ceg1 double deletion strain (YBS50), growth of which depends on maintenance of a CEN URA3 CET1 CEG1 plasmid. Control experiments showed that transformation of YBS50 with a CEN TRP1 CET1 CEG1 plasmid permitted the cells to grow on 5-FOA, whereas a CEN TRP1 plasmid containing only CET1 or only CEG1 was unable to rescue growth on 5-FOA (Fig. 9A). Expression of MCE1 (the gene encoding the bifunctional mouse capping enzyme) on a CEN TRP1 plasmid under control of the yeast TPI1 promoter fully complemented the Delta cet1 Delta ceg1 double-deletion (Fig. 9A).


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Fig. 9.   Replacement of the yeast capping apparatus by mammalian capping enzymes. Panel A, yeast strain YBS50 (Delta cet1 Delta ceg1) was transformed with CEN TRP1 plasmids containing the indicated genes. Trp+ isolates were streaked on agar containing 0.75 mg/ml 5-FOA. The plate was photographed after incubation for 3 days at 30 °C. Panel B, isogenic strains deleted at one or more yeast capping enzyme loci were transformed with CEN TRP1 plasmids as specified below and then selected on 5-FOA. FOA-resistant isolates were streaked on a YPD agar plate. The plate was photographed after incubation for 3 days at 30 °C. HCM1Delta 120 MCE1 is YBS52 transformed with HCM1(121-476) and MCE1. ABD1 CET1 CEG1 is YBS52 transformed with ABD1 CET1 and CEG1 on a single plasmid. ABD1 MCE1 is YBS50 transformed with MCE1. HCM1Delta 120 CET1 CEG1 is YBS40 transformed with HCM1(121-476).

We constructed a Delta cet1 Delta ceg1 Delta abd1 triple deletion strain (YBS52), growth of which is sustained by a CEN URA3 CET1 CEG1 ABD1 plasmid. Control plasmid shuffle experiments showed that YBS52 cells transformed with a CEN TRP1 CET1 CEG1 ABD1 plasmid grew on 5-FOA, whereas plasmids containing only CET1, only CEG1, or only ABD1 did not complement growth on 5-FOA (not shown). Cotransformation with MCE1 plus HCM1(121-476) complemented the triple deletion; neither MCE1 alone nor HCM1(121-476) alone permitted growth of YBS52 on 5-FOA. 5-FOA-resistant isolates were then streaked on YPD plates at 30 °C. Using colony size as a rough estimate of growth, we surmised that cells containing either MCE1 in place of CET1 plus CEG1, or HCM1(121-476) in place of ABD1 grew as well as the strain containing an all-yeast capping apparatus (Fig. 9B). However, colony size was smaller when all three yeast genes were replaced by MCE1 plus HCM1(121-476) on CEN plasmids (Fig. 9B). Colony size was larger when the yeast genes were replaced with CEN MCE1 plus 2µ HCM1(121-476) (not shown), implying that the human methyltransferase was limiting for growth in single copy in this background. To gauge better the growth of isogenic yeast cells containing yeast versus mammalian capping enzyme components, we measured their doubling times in YPD medium in suspension cultures at 30 °C. The growth rates of cells expressing either Mce1p or Hcm1(121-476)p in lieu of the yeast enzymes (generation times 1.6-1.8 h) were similar to those of cells with an all-yeast capping system (1.4 h). Cells with an all-mammalian capping system expressed from single copy plasmids grew more slowly (doubling time 3.3 h), but the defect was suppressed by high copy expression of the human methyltransferase (doubling time 2.2 h). Thus, we have demonstrated that the entire three-component yeast capping system can be replaced by the two-component mammalian system.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we have identified S. pombe and human cDNAs encoding mRNA cap methyltransferase and have isolated by in vivo selection the cap methyltransferase gene from C. albicans. While this work was in progress, Pillutla et al. (34) demonstrated cap methyltransferase activity of a polypeptide encoded by a human cDNA of identical size and sequence which they assembled from ESTs and completed by primer-directed amplification of the 5'- and 3'-ends. Tsukamoto et al. (38) identified two other human cDNAs encoding the same polypeptide, which differed from the 6,203-nucleotide cDNA by virtue of presumptive alternative splicing events within the 3'-untranslated region. Neither of these other studies provided a physical characterization of the human cap methyltransferase, and in neither case was activity demonstrated in vivo via genetic complementation.

We find that the properties of recombinant human cap methyltransferase are similar to those of the monomeric yeast enzyme Abd1p. Indeed, we show that Hcm1p or a catalytically active COOH-terminal domain Hcm1(121-476)p can support yeast cell growth in lieu of the endogenous cap methyltransferase. The dispensability of the NH2 terminus for Hcm1p function in vivo and in vitro echoes previous findings for Abd1p (9). The nonessential NH2-terminal segments of Hcm1p, Pcm1p, Ccm1p, and Abd1p are much less well conserved than the COOH-terminal catalytic domains. The NH2 terminus of Hcm1p, which is quite hydrophilic, may play an ancillary role in metazoan cells (e.g. intracellular localization, protein turnover, protein-protein interaction) which is not called for when the human enzyme is expressed in yeast.

Structure-function relationships for the human cap-methylating enzyme were illuminated by testing the effects of point mutations on cell growth and on methyltransferase activity in vitro. From the results of this analysis, we conclude that: (i) the catalytic activity of Hcm1p is required for in vivo complementation (as expected); (ii) all six residues identified previously as being essential for Abd1p function in vivo are also essential for the human cap methyltransferase; and (iii) two new residues are identified (Phe-291 and Tyr-289) which are important for Hcm1p function.

The fact that all eight positions implicated in Hcm1p function have identical or structurally conserved side chains at the corresponding loci in Abd1p, Hcm1p, Ccm1p, and the C. elegans protein (Fig. 1) suggests that the cellular cap-methylating enzymes have a common structure and mechanism. Five of the eight amino acids important in Hcm1p (Gly-207, Asp-211, Asp-227, Arg-239, Tyr-289, and Phe-291) are also conserved in the vaccinia virus cap methyltransferase, whereas a slightly different set of five of eight of the important Hcm1p residues (Asp-203, Gly-207, Asp-211, Asp-227, Arg-239, and Tyr-289) is conserved in the capping enzyme of African swine fever virus (9). Available mutational data for vaccinia cap methyltransferase are not as extensive as for the cellular enzymes and are confined to analysis of the effects of amino acid substitutions on vaccinia methyltransferase activity in vitro (there being no facile way to test the function of the viral methyltransferase in vivo). Nonetheless, there are concordant findings for alanine replacements at vaccinia capping enzyme Gly-600 and Tyr-683 (defective in vitro), Abd1p Gly-174 and Tyr-254 (defective in vivo and in vitro), and Hcm1p Gly-207 (defective in vivo), and Tyr-289 (defective in vivo and in vitro) (9, 29). A discordant note is that the vaccinia F685A mutation had little effect on methyltransferase activity in vitro, whereas the homologous Hcm1p F291A mutation was deleterious in vitro and in vivo. It is possible that the introduction of an alanine at the Phe position exerts its lethality on the human protein via an indirect effect on the universally essential tyrosine side chain located two residues upstream (Fig. 1). Note that the vaccinia cap methyltransferase is a heterodimeric protein that consists of a catalytic subunit (homologous to Abd1p, Pcm1p, Ccm1p, and Hcm1p) and a stimulatory subunit that has no known cellular counterpart. Hence, the subunit interaction may obscure or ameliorate the reliance of the vaccinia methyltransferase on certain residues that appear to be more critical for the activity of the monomeric cellular methyltransferases. Further clarification of the roles of the eight important residues identified in this study awaits successful crystallization of one of the cap-methylating enzymes.

With all of the genes encoding the components of the yeast and mammalian capping systems in hand, we have shown that the yeast capping apparatus can be replaced in toto by mammalian counterparts. This result bears on the recently elaborated model for targeting caps to pre-mRNAs, which posits that the capping and methylating enzymes are recruited to the RNA polymerase II elongation complex via their binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of the RNA polymerase (14, 32, 35, 36). In both fungi and mammals, the guanylyltransferase subunit or domain is capable per se of binding to CTD-PO4, whereas the RNA triphosphatase subunit or domain does not (14, 35).4 Genetic evidence supports the idea that the triphosphatase is chaperoned to the pre-mRNA by virtue of its physical association with guanylyltransferase. This association occurs via subunit heteromerization in the case of yeast and covalent linkage of autonomous domains in the case of mammals (11, 13, 14). Interference with Ceg1p-Cet1p heteromerization is a potential mechanism for blocking gene expression in fungi without impacting on mammalian cells. Complementation of a Delta cet1 Delta ceg1 double deletion by MCE1 suggests that the mammalian guanylyltransferase domain can recognize the yeast transcription complex. The yeast methyltransferase Abd1p is itself capable of binding to CTD-PO4 (35). Hence, complementation of Delta abd1 by human cap methyltransferase may imply that the human methyltransferase also interacts with CTD-PO4. The affinity of Hcm1p for CTD-PO4 may be lower than that of Abd1p, insofar as Hcm1p must be expressed in high copy in yeast to sustain cell growth. Pillutla et al. (34) reported that the human cap methyltransferase does not bind by itself to polymerase II but does bind to polymerase II in the presence of the mammalian triphosphatase-guanylyltransferase. They suggest that the human cap methyltransferase forms a complex with the triphosphatase-guanylyltransferase in vitro in the absence of polymerase II, as assayed by immune adsorption (34). To our knowledge, an interaction of the methyltransferase and guanylyltransferase in solution has not been reported. Two groups have found that cap methyltransferase is readily separated from the 68-kDa guanylyltransferase during purification of the latter enzyme from mammalian cells (30, 37). Our preliminary experiments using defined CTD phosphopeptide ligands indicate that purified recombinant Hcm1(121-476)p can bind weakly to CTD-PO4 by itself and that the affinity of the human methyltransferase for the phosphopeptide is not enhanced significantly by recombinant mammalian guanylyltransferase.5

The availability of isogenic yeast strains containing fungal versus mammalian capping systems provides an attractive means of drug discovery aimed at blocking cap formation in fungi. Any compound that is selectively cytotoxic to the CET1 CEG1 ABD1 strain but not to the MCE1 HCM1 strain is a strong candidate for a specific inhibitor of fungal capping. Secondary screens for cytotoxicity comparing strains in which only a subset of the yeast capping activities are replaced by a mammalian enzyme could pinpoint which of the gene products is targeted by such a compound. A similar strategy could be applied to the identification of antiviral or antiprotozoal drugs that target virus-encoded or protozoa-encoding capping enzymes, provided that viral and protozoal capping enzymes can function or be engineered to function in yeast.

    ACKNOWLEDGEMENTS

We thank Dr. Eric Phizicky of the University of Rochester for providing the C. albicans DNA library.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM52470.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Molecular Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, NY 10021. Tel.: 212-639-7145; Fax: 212-717-3623; E-mail: s-shuman{at}ski.mskcc.org.

2 N. Saha and S. Shuman, unpublished data.

3 B. Schwer, X. Mao, and S. Shuman; manuscript in preparation.

4 K. Ho and S. Shuman, unpublished data.

5 K. Ho, N. Saha, and S. Shuman, unpublished data.

    ABBREVIATIONS

The abbreviations used are: AdoMet, adenosylmethionine; AdoHcy, adenosylhomocysteine; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CTD, carboxyl-terminal domain; 5-FOA, 5-fluoroorotic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Shuman, S. (1995) Prog. Nucleic Acids Res. Mol. Biol. 50, 101-129[Medline] [Order article via Infotrieve]
  2. Schwer, B., and Shuman, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4328-4332[Abstract]
  3. Fresco, L. D., and Buratowski, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6624-6628[Abstract]
  4. Shibagaki, Y., Gotoh, H., Kato, M., and Mizumoto, K. (1995) J. Biochem. 118, 1303-1309[Abstract]
  5. Shuman, S., Liu, Y., and Schwer, B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12046-12050[Abstract/Free Full Text]
  6. Wang, S. P., Deng, L., Ho, C. K., and Shuman, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9573-9578[Abstract/Free Full Text]
  7. Mao, X., Schwer, B., and Shuman, S. (1995) Mol. Cell. Biol. 15, 4167-4174[Abstract]
  8. Mao, X., Schwer, B., and Shuman, S. (1996) Mol. Cell. Biol. 16, 475-480[Abstract]
  9. Wang, S. P., and Shuman, S. (1997) J. Biol. Chem. 272, 14683-14689[Abstract/Free Full Text]
  10. Tsukamoto, T., Shibagaki, Y., Imajoh-Ohmi, S., Murakoshi, T., Suzuki, M., Nakamura, A., Gotoh, H., and Mizumoto, K. (1997) Biochem. Biophys. Res. Commun. 239, 116-122[CrossRef][Medline] [Order article via Infotrieve]
  11. Ho, C. K., Schwer, B., and Shuman, S. (1998) Mol. Cell. Biol. 18, 5189-5198[Abstract/Free Full Text]
  12. Ho, C. K., Pei, Y., and Shuman, S. (1998) J. Biol. Chem. 273, 34151-34156[Abstract/Free Full Text]
  13. Yamada-Okabe, T., Mio, T., Matsui, M., Kashima, Y., Arisawa, M., and Yamada-Okabe, H. (1998) FEBS Lett. 435, 49-54[CrossRef][Medline] [Order article via Infotrieve]
  14. Ho, C. K., Sriskanda, V., McCracken, S., Bentley, D., Schwer, B., and Shuman, S. (1998) J. Biol. Chem. 273, 9577-9585[Abstract/Free Full Text]
  15. Wen, Y., Yue, Z., and Shatkin, A. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12226-12231[Abstract/Free Full Text]
  16. Yu, L., and Shuman, S. (1996) J. Virol. 70, 6162-6168[Abstract]
  17. Myette, J. R., and Niles, E. G. (1996) J. Biol. Chem. 271, 11936-11944[Abstract/Free Full Text]
  18. Gross, C. H., and Shuman, S. (1998) J. Virol. 72, 10020-10028[Abstract/Free Full Text]
  19. Jin, J., Dong, W., and Guarino, L. A. (1998) J. Virol. 72, 10011-10019[Abstract/Free Full Text]
  20. Ho, C. K., Van Etten, J. L., and Shuman, S. (1996) J. Virol. 70, 6658-6664[Abstract]
  21. Silva, E., Ullu, E., Kobayashi, R., and Tschudi, C. (1998) Mol. Cell. Biol. 18, 4612-4619[Abstract/Free Full Text]
  22. Yu, L., Martins, A., Deng, L., and Shuman, S. (1997) J. Virol. 71, 9837-9843[Abstract]
  23. Myette, J. R., and Niles, E. G. (1996) J. Biol. Chem. 271, 11945-11952[Abstract/Free Full Text]
  24. Takagi, T., Moore, C. R., Diehn, F., and Buratowski, S. (1997) Cell 89, 867-873[Medline] [Order article via Infotrieve]
  25. Gross, C. H., and Shuman, S. (1998) J. Virol. 72, 7057-7063[Abstract/Free Full Text]
  26. Takagi, T., Taylor, G. S., Kusakabe, T., Charbonneau, H., and Buratowski, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9808-9812[Abstract/Free Full Text]
  27. Higman, M. A., Christen, L. A., and Niles, E. G. (1994) J. Biol. Chem. 269, 14974-14981[Abstract/Free Full Text]
  28. Mao, X., and Shuman, S. (1994) J. Biol. Chem. 269, 24472-24479[Abstract/Free Full Text]
  29. Mao, X., and Shuman, S. (1996) Biochemistry 35, 6900-6910[CrossRef][Medline] [Order article via Infotrieve]
  30. Mizumoto, K., and Lipmann, F. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4961-4965[Abstract]
  31. Ensinger, M. J., and Moss, B. (1976) J. Biol. Chem. 251, 5283-5291[Abstract]
  32. Yue, Z., Maldonado, E., Pillutla, R., Cho, H., Reinberg, D., and Shatkin, A. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12898-12903[Abstract/Free Full Text]
  33. Yamada-Okabe, T., Doi, R., Shimmi, O., Arisawa, M., and Yamada-Okabe, H. (1998) Nucleic Acids Res. 26, 1700-1706[Abstract/Free Full Text]
  34. Pillutla, R. C., Yue, Z., Maldonado, E., and Shatkin, A. J. (1998) J. Biol. Chem. 273, 21443-21446[Abstract/Free Full Text]
  35. McCracken, S., Fong, N., Rosonina, E., Yankulov, K., Brothers, G., Siderovski, D., Hessel, A., Foster, S., Shuman, S., and Bentley, D. L. (1997) Genes Dev. 11, 3306-3318[Abstract/Free Full Text]
  36. Cho, E., Takagi, T., Moore, C. R., and Buratowski, S. (1997) Genes Dev. 11, 3319-3326[Abstract/Free Full Text]
  37. Venkatesan, S., Gershowitz, A., and Moss, B. (1980) J. Biol. Chem. 255, 2829-2834[Abstract/Free Full Text]
  38. Tsukamoto, T., Shibagaki, Y., Niikura, Y., and Mizumoto, K. (1998) Biochem. Biophys. Res. Commun. 251, 27-34[CrossRef][Medline] [Order article via Infotrieve]
  39. Fikes, J. D., Becker, D. M., Winston, F., and Guarente, L. (1990) Nature 346, 291-294[CrossRef][Medline] [Order article via Infotrieve]
  40. Janbon, G., Rustchenko, E. P., Klug, S., Scherer, S., and Sherman, F. (1997) Yeast 10, 985-990[CrossRef]


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