Characterization of Human, Schizosaccharomyces pombe,
and Candida albicans mRNA Cap Methyltransferases
and Complete Replacement of the Yeast Capping Apparatus by
Mammalian Enzymes*
Nayanendu
Saha
,
Beate
Schwer§, and
Stewart
Shuman
¶
From the
Molecular Biology Program, Sloan-Kettering
Institute and the § Department of Microbiology and
Immunology, Weill Medical College of Cornell University, New York,
New York 10021
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ABSTRACT |
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.
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INTRODUCTION |
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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EXPERIMENTAL PROCEDURES |
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-
-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 (MAT
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 |
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.
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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
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.
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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
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
29,
55,
and
80 and
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 Pcm1p
93
deletion is denoted by the arrow in Fig. 1.) The lethal
NH2-terminal
106 and
117 deletions of Pcm1p
correspond to
135 and
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.
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HCM1 and HCM1(121-476) were also cloned into
CEN TRP1 vectors. Expression of the
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
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-
-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-
-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.
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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
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
120. Panel A, polypeptide
composition. Aliquots (10 µg) of the peak glycerol gradient fractions
of wild type (WT) Hcm1p and deletion mutant 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 120 enzyme as
specified.
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|
A His-tagged version of the NH2-terminal truncated protein
Hcm1(121-476)p (referred to as
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
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
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
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
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
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. cerevisiae 2µ URA3 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-
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.
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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
ceg1 or
cet1 strains (11,
14, 32, 33). This prompted us to test the ability of the mammalian
capping enzyme to complement a
cet1
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
cet1
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
( cet1 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.
HCM1 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.
HCM1 120 CET1 CEG1 is YBS40 transformed with
HCM1(121-476).
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|
We constructed a
cet1
ceg1
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 |
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
cet1
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
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.
 |
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