From the Molecular Biology Program, Sloan-Kettering
Institute, New York, New York 10021, § Amgen Institute and
Ontario Cancer Institute, Toronto, Ontario M5G 2C1, Canada, and the
¶ Microbiology Department, Cornell University Medical College, New
York, New York 10021
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ABSTRACT |
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We have conducted a biochemical and genetic analysis of mouse mRNA capping enzyme (Mce1), a bifunctional 597-amino acid protein with RNA triphosphatase and RNA guanylyltransferase activities. The principal conclusions are as follows: (i) the mammalian capping enzyme consists of autonomous and nonoverlapping functional domains; (ii) the guanylyltransferase domain Mce1(211-597) is catalytically active in vitro and functional in vivo in yeast in lieu of the endogenous guanylyltransferase Ceg1; (iii) the guanylyltransferase domain per se binds to the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD), whereas the triphosphatase domain, Mce1(1-210), does not bind to the CTD; and (iv) a mutation of the active site cysteine of the mouse triphosphatase elicits a strong growth-suppressive phenotype in yeast, conceivably by sequestering pre-mRNA ends in a nonproductive complex or by blocking access of the endogenous yeast triphosphatase to RNA polymerase II. These findings contribute to an emerging model of mRNA biogenesis wherein RNA processing enzymes are targeted to nascent polymerase II transcripts through contacts with the CTD. The phosphorylation-dependent interaction between guanylyltransferase and the CTD is conserved from yeast to mammals.
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INTRODUCTION |
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mRNA capping occurs by a series of three enzymatic reactions in which the 5' triphosphate terminus of the primary transcript is cleaved to a diphosphate by RNA triphosphatase, capped with GMP by RNA guanylyltransferase, and methylated at the N-7 position of guanine by RNA (guanine 7) methyltransferase (1). In vivo, the capping reactions occur cotranscriptionally, i.e. the substrates for the capping enzymes are nascent RNA chains engaged within RNA polymerase II (pol II)1 elongation complexes. There must exist a mechanism to target cap formation in vivo to transcripts made by pol II, because the capping enzymes have no inherent specificity for modifying pre-mRNAs in vitro. We and others (2-4) have suggested that targeting is achieved through direct physical interaction of one or more components of the capping apparatus with the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of pol II. This model is supported by the finding that recombinant yeast guanylyltransferase and methyltransferase proteins bind specifically and independently to the phosphorylated CTD in vitro (2). Are such direct protein-protein interactions conserved in higher eukaryotes? Does the triphosphatase component of the capping apparatus also interact with the CTD?
We know that the physical and functional organizations of the
triphosphatase and guanylyltransferase components of the capping apparatus have diverged in fungi versus metazoans. The
guanylyltransferases of Saccharomyces cerevisiae (Ceg1; 459 amino acids), Schizosaccharomyces pombe (Pce1; 402 amino
acids), and Candida albicans (Cgt1; 449 amino acids) are
monofunctional polypeptides that cap diphosphate-terminated RNAs
(5-7). Transfer of GMP from GTP to the 5' diphosphate terminus of RNA
occurs in a two-stage reaction involving a covalent enzyme-GMP intermediate (8). The GMP is linked to the enzyme through a phosphoamide (P-N) bond to the -amino group of a lysine residue within a conserved motif, KXDG, found in all known cellular
and DNA virus-encoded capping enzymes (9). The fungal
guanylyltransferases display ~38% amino acid sequence identity
overall. They are also functionally homologous, insofar as
PCE1 and CGT1 can complement lethal
ceg1 mutations in S. cerevisiae (6, 7). The
S. cerevisiae RNA triphosphatase is a 549-amino acid
polypeptide encoded by the CET1 gene (10). The Ceg1 and Cet1
polypeptides interact in vivo and in vitro.
Metazoan capping enzymes are bifunctional polypeptides with triphosphatase and guanylyltransferase activities (11, 12). Yagi et al. (12) isolated triphosphatase and guanylyltransferase domain fragments of the Artemia salina capping enzyme by partial proteolysis with trypsin. However, it was not clear from this work whether the functional domains overlapped structurally. The first metazoan-capping enzyme gene was isolated recently from Caenorhabditis elegans (13, 14). The 573-amino acid nematode protein consists of a carboxyl-terminal domain homologous to yeast Ceg1 and an amino-terminal domain that has strong similarity to the superfamily of protein phosphatases that act via a covalent phosphocysteine intermediate.
We recently isolated a mouse cDNA encoding the mammalian homologue of the C. elegans capping enzyme (2). The 597-amino acid mouse capping enzyme (Mce1) also consists of an amino-terminal phosphatase domain and a carboxyl-terminal guanylyltransferase domain. Here, we report that Mce1 is functional in vitro and in vivo. An autonomous RNA triphosphatase domain (amino acids 1-210) and an autonomous guanylyltransferase domain (amino acids 211-597) have been purified and characterized. We found that the guanylyltransferase domain per se binds to the pol II CTD and that this interaction requires CTD phosphorylation. The triphosphatase domain of mouse capping enzyme does not bind to the CTD. The phosphorylation-dependent interaction between guanylyltransferase and the CTD is conserved from yeast to mammals and provides a general mechanism for targeting caps to nascent pol II transcripts in vivo.
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MATERIALS AND METHODS |
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Yeast Expression Plasmids-- The MCE1 cDNA was cloned 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 (pYX-132 was purchased from Novagen). The DNA insert of the resulting plasmid pYX1-MCE1 extended from the Mce1 translation start codon to an XhoI site located in the 3' UTR. pYX1-MCE1 encodes the full-length 597-amino acid Mce1 polypeptide fused in-frame with an amino-terminal 12-amino acid leader peptide (MGSHHHHHHSGH). MCE1 expression in this plasmid was under the control of a constitutive yeast TPI promoter. Amino-terminal deletion mutants of MCE1 were constructed by PCR amplification with mutagenic primers that introduced an NdeI restriction site and a methionine codon in lieu of the codons for Ser-210 or Gln-259 or an NdeI site at Met-276. The PCR products were digested with NdeI and BglII and then inserted into pYX1-MCE1 so as to replace a 1.0-kilobase pair NdeI-BglII fragment of the wild type MCE1 gene with restriction fragments encoding deleted Mce1 polypeptides. The mutated genes were named according to the amino acid coordinates of their polypeptide products, i.e. MCE1(211-597), MCE1(260-597), and MCE1(276-597). Alanine-substitution mutations (C126A and K294A) in MCE1 were programmed by synthetic oligonucleotides using the two-stage overlap extension method (15). pYX1-MCE1 served as the template for the first round of amplification. The second-stage PCR products of the C126A and K294A reactions were digested with NdeI and BglII or NdeI and HindIII, respectively, and then ligated into the corresponding sites in pYX1-MCE1 in place of the wild type fragments. The presence of the desired mutations was confirmed in every case by dideoxy sequencing. We sequenced the entire restriction fragment insert in each pXY1 plasmid to exclude the introduction of unwanted mutations. A CEN TRP1 vector, pYX1-CEG1, encoding yeast Ceg1 under the control of a TPI promoter, was constructed by inserting an NcoI-HindIII fragment of plasmid pGYCE-360 (16) into pYX132.
Test of MCE1 Function by Plasmid Shuffle--
Strain YBS2
(MATa ura3 trp1 lys2 leu2 ceg1::hisG
pGYCE-360), which is deleted at the chromosomal CEG1 locus,
is viable when it maintains an extrachromosomal copy of CEG1
on a CEN URA3 plasmid (pGYCE-360) (16). YBS2 was transformed
with pXY1-based plasmids bearing wild type and mutant alleles of
MCE1. Trp+ transformants were selected on medium lacking
tryptophan. Individual colonies were patched on medium lacking
tryptophan. Cells from single patches were then streaked on medium
containing 0.75 mg/ml of 5-fluoroorotic acid (5-FOA). The plates were
incubated at 30 °C. Mutations scored as lethal were those that did
not support colony formation after 7 days. Individual colonies of the
viable MCE1 alleles were picked from the 5-FOA plate and
patched to plates lacking tryptophan. All 5-FOA survivors were
confirmed to be Ura.
Bacterial Expression Plasmids-- NdeI-XhoI fragments of pYX1-MCE1, pYX1-MCE1(211-597), pYX1-MCE1(260-597), and pYX1-MCE1(276-597) were inserted into to the T7-based expression plasmid pET16b (Novagen). The carboxyl-terminal deletion mutant MCE1(1-210) was constructed by PCR amplification from an MCE1 template with an antisense primer that introduced a translation stop at codon 211 and a flanking XhoI restriction site. The PCR product was digested with NdeI and XhoI and inserted into pET16b.
Expression and Purification of Recombinant Mce1 Protein--
A
25-ml culture of E. coli BL21(DE3)/pET-MCE1 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 17 °C for 17 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 5 ml of Buffer A (50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 10% sucrose). Lysozyme was added to a final
concentration of 50 µg/ml; the suspension was incubated on ice for 10 min and then sonicated for 30 s. Triton X-100 was added to a final
concentration of 0.1%, and sonication was repeated to reduce the
viscosity of the lysate. Insoluble material was removed by
centrifugation for 45 min at 18,000 rpm in a Sorvall SS34 rotor. The
soluble extract was applied to a 0.5-ml column of Ni-NTA-agarose
(Qiagen) that had been equilibrated with Buffer A containing 0.1%
Triton X-100. The column was washed with the same buffer and then
eluted step-wise with Buffer B (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 10% glycerol) containing 50, 100, 200, 500, and
1000 mM imidazole. The polypeptide compositions of the
column fractions were monitored by SDS-polyacrylamide gel electrophoresis (PAGE). The recombinant Mce1 polypeptide was retained on the column and recovered in the 200 and 500 mM imidazole
eluates. These two fractions were pooled and dialyzed against Buffer C (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM DTT, 10% glycerol, 0.05% Triton X-100).
Expression and Purification of Mce1(1-210) and
Mce1(211-597)--
500-ml cultures of E. coli BL21(DE3)
harboring pET-MCE1(1-210) or pET-MCE1(211-597) were grown at 37 °C
until the A600 reached 0.5. The cultures were
adjusted to 0.4 mM
isopropyl--D-thiogalactopyranoside and incubated for
3 h at 37 °C. Cells were harvested by centrifugation and stored
at
80 °C. Thawed bacteria were resuspended in 25 ml of Buffer A. Soluble lysates were prepared as described in the preceding section and
then applied to 4-ml columns of Ni-NTA-agarose, which were eluted
step-wise with 50, 100, 200, 500, and 1000 mM imidazole.
The Mce1(1-210) and Mce1(211-597) polypeptides were retained on the
Ni-NTA-agarose columns and recovered in the 200 and 500 mM
imidazole eluates. The preparations were dialyzed against Buffer C. Enzyme fractions were stored at
80 °C and thawed on ice just prior
to use. Protein concentrations were determined using the Bio-Rad dye
binding assay with bovine serum albumin as a standard.
Glycerol Gradient Sedimentation-- Aliquots (200 µl) of the dialyzed Ni-agarose preparations of Mce1, Mce1(1-210), and Mce1(211-597) were applied to a 4.8-ml 15-30% glycerol gradients containing 50 mM Tris-HCl (pH 8.0), 0.3 M NaCl, 2 mM DTT, 1 mM EDTA, and 0.1% Triton X-100. The gradients were centrifuged at 50,000 rpm for 24 h at 4 °C in a Beckman SW50 rotor. Fractions (0.2 ml) were collected from the bottom of the tube. Marker proteins catalase, bovine serum albumin, soybean trypsin inhibitor, and cytochrome C were sedimented in a parallel gradient.
Enzyme-GMP Complex Formation--
Standard reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 8.0), 5 mM
DTT, 5 mM MgCl2, [32P]GTP as
specified, and enzyme were incubated for 5 min at 37 °C. The
reactions were halted by adding SDS to a final concentration of 1%.
The samples were electrophoresed through a 12% polyacrylamide gel
containing 0.1% SDS. Enzyme-[32P]GMP complexes were
visualized by autoradiographic exposure of the dried gel and was
quantitated by scanning the gel with a FUJIX BAS1000 Bio-Imaging
Analyzer.
RNA Triphosphatase Assay--
RNA triphosphatase activity was
assayed by liberation of 32Pi from
-32P-labeled triphosphate-terminated poly(A) (17).
Standard reaction mixtures (10 µl) containing 50 mM
Tris-HCl (pH 7.5), 5 mM DTT, 10 pmol (of triphosphate
termini) of [
-32P]poly(A), and enzyme as specified
were incubated for 15 min at 37 °C. Reactions was halted by addition
of 1 µl of 12 N formic acid. Aliquots of the mixtures
were applied to a polyethyleneimine-cellulose TLC plate that was
developed with 0.75 M potassium phosphate (pH 4.3). The
release of 32Pi from
[
-32P]poly(A) was quantitated by scanning the TLC
plate with a FUJIX BAS1000 Bio-Imaging Analyzer.
CTD Affinity Chromatography--
Recombinant glutathione
S-transferase (GST)-CTD, consisting of
glutathione-S-transferase fused to the first 15 tandem heptad repeats
from the mouse pol II CTD (consensus sequence YSPTSPS), was purified
and coupled to glutathione agarose as described (2). GST-CTD was
phosphorylated in vitro by incubation with HeLa cell extract
and ATP (2). The phosphorylation reaction resulted in the addition of
approximately 3 phosphates per molecule of GST-CTD. The GST-CTD-PO4
polypeptide was adsorbed to glutathione agarose and the resin was
washed with buffer containing 1 M NaCl prior to use in
affinity chromatography. The GST-CTD and GST-CTD-PO4 resins contained
3.0 mg of fusion protein per ml of agarose. Affinity chromatography was
performed by mixing 25 µl of GST-CTD or GST-CTD-PO4 resins with
180-200-µl samples of Mce1 proteins in binding buffer containing 20 mM HEPES (pH 7.9), 0.1 mM EDTA, 1 mM DTT, 20% glycerol, 0.1 M NaCl, 0.5 µM microcystine, 1 mM -glycerophosphate,
0.1% Nonidet P-40. After mixing for 1 h at 4 °C, the agarose
beads were collected by centrifugation, and the supernatant was
removed. The resins were washed three or four times with 400-500 µl
of binding buffer, and then the bound material was eluted with 100 µl
of binding buffer containing 0.9 M NaCl. Aliquots of the
input sample and the 0.9 M NaCl eluates were analyzed by
SDS-PAGE.
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RESULTS |
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Mammalian Capping Enzyme-- We have isolated a mouse cDNA that encodes Mce1, a putative mRNA capping enzyme (GenBankTM accession no. AF034568). The size of the Mce1 polypeptide (597-amino acids; 68 kDa) agrees with the size of the 68-kDa enzyme-GMP complex formed by the capping enzyme isolated from mouse cell extracts (18). The amino acid sequence of Mce1 suggests that it is a bifunctional enzyme consisting of an amino-terminal phosphatase domain and a C-terminal guanylyltransferase domain. In this respect, it resembles the C. elegans capping enzyme (13, 14), to which it displays 43% sequence identity. The carboxyl-terminal portion of Mce1 contains the defining sequence motifs of the covalent nucleotidyl transferase superfamily (9), including the KXDGXR sequence that constitutes the active site. The lysine side chain within this motif (Lys-294 in Mce1) reacts with GTP to form a covalent enzyme-GMP intermediate. The amino-terminal portion of Mce1 contains the (I/V)HCXAGXGR(S/T)G signature motif of the dual-specificity protein phosphatase/protein tyrosine phosphatase enzyme family. These proteins catalyze phosphoryl transfer from a protein phosphomonoester substrate to the thiol of a cysteine on the enzyme to form a covalent phosphocysteine intermediate, which is then attacked by water to liberate phosphate (19). The cysteine within the signature motif is the active site of phosphoryl transfer and is thus essential for reaction chemistry. Cys-126 is predicted to be the active site of phosphoryl transfer by Mce1.
Catalytic Activity of Mouse Capping Enzyme--
The biochemical
properties of the mammalian capping enzyme were examined after
expressing Mce1 and its component domains in bacteria. First, the
full-length MCE1 coding sequence was inserted into an
inducible T7 RNA polymerase-based pET vector such that a histidine-rich
amino-terminal leader (His-tag) was fused to the 597-amino acid Mce1
polypeptide. The pET-Mce1 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 68-kDa polypeptide
corresponding to His-tagged Mce1 was detected by SDS-PAGE in whole-cell
and soluble lysates of
isopropyl--D-thiogalactopyranoside-induced bacteria
(Fig. 1A and data not shown).
To assay guanylyltransferase activity of the expressed Mce1 protein, we
incubated soluble protein from induced bacteria in the presence of
[
-32P]GTP and a divalent cation. This resulted in the
formation of a 32P-labeled nucleotidyl-protein adduct that
migrated as a 68-kDa species during SDS-PAGE (Fig. 1A).
Three other polypeptides in the range of 45-55 kDa were labeled with
GMP to a lesser extent. We detected no label transfer from GTP to
polypeptides of these sizes in extracts prepared from bacteria that
lacked the MCE1 gene (not shown). Purification of the
His-tagged 68-kDa Mce1 guanylyltransferase was achieved by adsorption
to Ni-agarose and elution with 200-500 mM imidazole (Fig.
1A). The imidazole eluate fractions were highly enriched
with respect to Mce1, as judged by SDS-PAGE (Fig. 1A, left
panel). Note that the lower molecular weight polypeptides with
guanylyltransferase activity were recovered in the Ni-agarose flow-through and wash fractions (Fig. 1A, right panel),
which suggested that these were proteolytic fragments of Mce1 that
lacked the amino-terminal His-tag.
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Autonomous Guanylyltransferase and Triphosphatase
Domains--
To evaluate whether the carboxyl-terminal portion of
Mce1 constituted an autonomous functional guanylyltransferase domain, we expressed the protein segment from residues 211-597 as a His-tagged fusion protein. The choice of residue 211 as a domain breakpoint was
based on its location 84 amino acids from the putative Mce1 active site
Lys-294; the active site lysine of the monofunctional Chlorella virus guanylyltransferase is located 82 amino
acids from its amino terminus (20, 21). We found that
isopropyl--D-thiogalactopyranoside-induced bacteria
accumulated substantial amounts of a soluble 46-kDa polypeptide corresponding to Mce1(211-597), which reacted with
[
-32P]GTP to form a 47-kDa radiolabeled enzyme-GMP
adduct (Fig. 1B). Mce1(211-597) adsorbed to Ni-agarose and
was eluted at 200-500 mM imidazole (Fig. 1B).
Mce1(211-597) sedimented as a discrete 2.8 S peak during
glycerol gradient centrifugation (Fig. 2B). We surmise that
Mce1 the guanylyltransferase component is a monomer in solution.
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Characterization of the RNA Triphosphatase Domain--
The
standard RNA triphosphatase reaction contained 50 mM
Tris-HCl (pH 7.5) and 1 µM 32P-labeled
poly(A). The extent of
-phosphate hydrolysis during a 15-min
incubation at 37 °C was proportional to input Mce1(1-210) (Fig.
4A). In the linear range of
enzyme dependence, 1 pmol of 32Pi was released
per fmol of protein. The specific activity of the Ni-agarose
preparation was 37 units/µg. (One unit of enzyme releases 1 nmol of
32Pi from
32P poly(A) in 15 min.) A kinetic analysis showed that the initial rate of Pi
release was proportional to enzyme concentration (Fig. 4B).
Mce1(1-210) hydrolyzed 1.2-2 molecules of Pi/enzyme/s at steady state. RNA triphosphatase activity was optimal in 50 mM Tris buffer at pH 7.0-7.5; the extents of
-phosphate
release at pH 6.0 and pH 9.5 were 70 and 35%, respectively, of the
activity seen at pH 7.5 (not shown). Activity was optimal in the
absence of a divalent cation and was unaffected by EDTA. Inclusion of divalent cations elicited a concentration-dependent
inhibition of RNA triphosphatase activity (Fig. 4C). 75%
inhibition was observed at 0.5 mM MgCl2 or
MnCl2. Mce1(1-210) did not catalyze release of
32Pi from [
-32P]ATP (not
shown).
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Characterization of the Guanylyltransferase Domain-- Formation of the covalent enzyme-GMP complex by Mce1(211-597) was dependent on the concentration of GTP from 0.01 to 2 µM (Fig. 5A). The yield of guanylated enzyme plateaued at 5-10 µM GTP; we estimated that at least 30% of the enzyme molecules were labeled with GMP in vitro. (It has been our experience that a significant fraction of any recombinant guanylyltransferase purified from bacteria is already guanylylated and thus cannot be labeled during the in vitro reaction.) Enzyme-GMP complex formation was strictly dependent on a divalent cation cofactor. Either magnesium or manganese sufficed; activity was optimal at 1-2 mM MnCl2 or 5-10 mM MgCl2 (Fig. 5B). The rate of enzyme-guanylate formation was slightly greater in the presence of manganese versus magnesium (Fig. 5C). The Ni-agarose preparation of Mce1(211-597) displayed trace levels of RNA triphosphatase activity. The specific activity (0.02 units/µg of protein) was 0.05% that of Mce1(1-210). The residual triphosphatase was resolved from the guanylyltransferase activity during glycerol gradient sedimentation of Mce1(211-597), suggesting that this phosphatase was an E. coli contaminant. We surmise that the guanylyltransferase domain of Mce1 has no triphosphatase activity per se.
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Mce1 Activity in Vivo-- To test whether the mouse capping enzyme was functional in vivo, we examined the ability of the wild type full-length MCE1 cDNA to complement a ceg1 null mutation by using the plasmid shuffle technique (6, 16). MCE1 was cloned into a yeast CEN TRP1 expression plasmid under the control of a constitutive yeast TPI promoter. The MCE1 plasmid was introduced into YBS2, a yeast strain in which the chromosomal CEG1 locus is deleted and that is dependent for growth on maintenance of an extrachromosomal copy of CEG1 on a CEN URA3 plasmid. Trp+ transformants were plated on medium containing 5-FOA to select against retention of the wild type CEG1 gene. Cells bearing the TRP1 MCE1 plasmid grew readily on 5-FOA, whereas cells containing the TRP1 vector plasmid were incapable of growth on 5-FOA. A mutated allele, MCE1-K294A, in which the active site nucleophile of the guanylyltransferase Lys-294 was substituted by alanine, was completely incapable of supporting yeast growth in the plasmid shuffle assay (Fig. 6A). We conclude that the mammalian capping enzyme is functional as a guanylyltransferase in vivo.
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Interaction of Mce1 with the Pol II CTD-- We used affinity chromatography to analyze the binding of mouse capping enzyme and its constituent domains to the pol II CTD. The test samples were incubated in parallel with a glutathione-Sepharose resin containing an immobilized ligand composed of either (i) glutathione S-transferase fused to the first 15 tandem heptad repeats of the mouse pol II CTD (consensus sequence YSPTSPS) or (ii) GST-CTD fusion protein that had been phosphorylated in vitro using HeLa nuclear extract as a source of kinase activity. The phosphorylation reaction resulted in the addition of approximately 3 phosphates per molecule of GST-CTD. After phosphorylation in vitro, the GST-CTD resin was washed extensively with 1 M NaCl to remove residual non-CTD proteins before performing affinity chromatography with mouse capping enzyme. The 68-kDa full-length Mce1 protein (residues 1-597) was prepared by in vitro translation in the presence of [35S]methionine, and the translation product was subjected to affinity chromatography. We found that 35S-labeled Mce1 bound specifically to the phosphorylated CTD (Fig. 7A, lane P) but not to the nonphosphorylated CTD control (lane C). The 46-kDa Mce1(211-597) guanylyltransferase domain by itself also bound specifically to phosphorylated CTD (Fig. 7A). In contrast, the RNA triphosphatase domain Mce1(1-210) did not interact with either phosphorylated or nonphosphorylated CTD (Fig. 7A). We conclude that the triphosphatase domain has no intrinsic affinity for the CTD and that its recruitment to the pol II transcription complex is mediated via its connection in cis to the guanylyltransferase. Guanylyltransferase deletion mutants Mce1(260-597) and Mce1(276-597) were unable to bind the phosphorylated CTD (Fig. 7A). Apparently, deletion of the segment of Mce1 from residues 211-259 disrupts CTD-PO4 recognition as well as guanylyltransferase activity.
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Cis Negative Effect of an Active Site Mutation of the RNA Triphosphatase on Mce1 Function Yeast-- Yeast cells bearing MCE1-C126A, an allele with an alanine substitution at the active site cysteine of the triphosphatase domain, formed very tiny colonies on 5-FOA plates, even after 6 days of incubation. This contrasted starkly with the behavior of MCE1 cells, which formed colonies of normal size after 2 days on 5-FOA. MCE1-C126A cells displayed a severe growth defect when plated on rich medium at 30 °C (Fig. 6B) and were unable to grow on YPD plates at 37 °C (not shown). The growth suppressive effects of an inactivating point mutation in the triphosphatase were far more severe than a complete deletion of the triphosphatase domain (Fig. 6B). We hypothesize that the C126A mutation interferes with the endogenous yeast triphosphatase, either by sequestering the nascent pre-RNA ends in a nonproductive complex or by blocking access of the yeast triphosphatase to RNA polymerase II.
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DISCUSSION |
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Functional Domains of Mammalian Capping Enzyme--
Fungi and
Chlorella virus encode monofunctional guanylyltransferases,
whereas mammals encode a bifunctional
triphosphatase-guanylyltransferase enzyme. Here, we demonstrate that
the triphosphatase and guanylyltransferase domains of mammalian capping
enzyme are distinct and nonoverlapping. The guanylyltransferase domain
is a 46-kDa monomer that extends from Mce1 residue 211 to residue 597. The recombinant domain is catalytically active in vitro and
is capable of genetically complementing the function of the yeast
guanylyltransferase Ceg1 in vivo. A recent study of the
C. elegans enzyme Cel1 documented triphosphatase activity of
an amino-terminal domain (13), but the guanylyltransferase activity
could not be demonstrated. We found that the proximal margin of the
active guanylyltransferase domain is located 84 amino acids upstream of
the presumptive active site nucleophile Lys-294. This is in keeping
with the positions of the N termini of the yeast and
Chlorella virus guanylyltransferases 67-82 residues upstream of their respective lysine nucleophiles (6, 7, 16, 20, 22).
The fact that the in vivo activity of Mce1 is abrogated by
the K294A mutation supports our designation of this residue as the
active site. Substitutions of the equivalent residues of Ceg1 and Pce1
are also lethal in vivo (6, 16, 22). The function of the
Mce1 guanylyltransferase domain is abolished when residues 211-259 are
deleted. Deletion to a similar point of the yeast guanylyltransferase
Ceg1 is also lethal (16). In the crystal structure of
Chlorella virus guanylyltransferase, the analogous
amino-terminal segment is located on the protein surface and consists
of two antiparallel -sheets followed by an
-helix (21). This
region contains no residues that make direct contact with GTP in the
enzyme-GTP cocrystal (21). The fact that recombinant Mce1(260-597)
expressed in bacteria was predominantly insoluble complicated efforts
to distinguish whether the loss of activity upon amino-terminal
truncation was caused by defective protein folding or a primary defect
in catalysis.
Evolution of the Capping Apparatus--
The linear order of
amino-terminal RNA triphosphatase and carboxyl-terminal
guanylyltransferase domains in Mce1 is superficially similar to the
arrangement of the triphosphatase and guanylyltransferase active sites
in the vaccinia capping enzyme (24-27). However, the amino acid
sequences of the Mce1 and vaccinia amino-terminal segments are
unrelated, and the properties of the vaccinia triphosphatase differ
from those of the metazoan triphosphatases in two key respects: (i)
lack of domain autonomy, and (ii) divalent cation dependence. The
vaccinia triphosphatase and guanylyltransferase active sites are
distinct, but the two activities are not partitioned into discrete and
separable structural modules as they are in Mce1. Instead, moieties on
the vaccinia enzyme that are essential for triphosphatase and
guanylyltransferase activity overlap within a 545-amino acid
polypeptide (23, 26, 27). The triphosphatase activity of the vaccinia
capping enzyme depends absolutely on a divalent cation cofactor,
whereas the rat liver and brine shrimp triphosphatases, as well as the
recombinant C. elegans and mouse triphosphatases, require no
divalent cation for activity (in fact, they are inhibited by divalent
cations). The sequences of the C. elegans and mouse enzymes
indicate that -phosphate cleavage occurs through a phosphoenzyme
intermediate. Mutation of the active site cysteine of the C. elegans triphosphatase domain abolished enzyme activity (13). We
have been unable to detect a phosphoenzyme intermediate for the
vaccinia triphosphatase, which suggests that covalent catalysis does
not apply. Mutational analysis of the vaccinia triphosphatase provides
additional evidence for a distinct mechanism. We have identified four
acidic side chains that are essential for catalysis by vaccinia
triphosphatase and are conserved among the poxvirus and African swine
fever virus capping enzymes (26, 27). One or more of these acidic
residues is likely to bind the essential metal ion(s). The RNA
triphosphatase isolated from S. cerevisiae, also depends
completely on a divalent cation cofactor (28). The amino acid sequence
of yeast Cet1 bears no similarity to the mammalian triphosphatase
domains, but it does display local similarities to the vaccinia
triphosphatase. We surmise that higher eukaryotes have diverged from
vaccinia and yeast with respect to both mechanism and structure of the
triphosphatase component of the capping machinery. In contrast, the
reaction mechanism and structure of the guanylyltransferase components are conserved in yeasts, metazoans, and DNA viruses (14, 21).
Interaction of the Capping Apparatus with the CTD of RNA Polymerase II-- Cap formation in vivo is targeted to the nascent chains synthesized by pol II. Placing a mammalian pol II transcription unit under the control of a pol III promoter results in a failure to cap the transcript (29). We have elaborated a solution to the problem of how pol II transcripts are specifically singled out for capping whereby the capping enzymes are targeted to pre-mRNA by binding to the phosphorylated CTD of the largest subunit of RNA polymerase II (2). The CTD, which is unique to pol II, consists of a tandem array of a heptapeptide repeat with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The mammalian pol II large subunit has 52 tandem repeats, whereas the S. cerevisiae subunit has 27 copies. The pol II largest subunit exists in two forms, a nonphosphorylated IIA form and a phosphorylated IIO form, which are interconvertible and functionally distinct. In vivo, the pol IIO enzyme contains as many as 50 phosphorylated amino acids (primarily phosphoserine) within the CTD (30). During transcription initiation, pol IIA is recruited to the DNA template by the general transcription factors. The pol IIA CTD undergoes extensive phosphorylation and conversion to IIO during the transition from preinitiation complex to stable elongation complex. Several CTD kinase activities have been implicated in CTD hyperphosphorylation, each of which contains a cyclin and cyclin-dependent kinase subunit pair. The cdk7 and cyclin H subunits of the general transcription factor TFIIH catalyze phosphorylation of Ser-5 of the CTD heptapeptide (31). Other CTD kinases include the cdk8/cylin C pair found in the pol II holoenzyme (32, 33), CTDK-I, a heterotrimeric kinase with cdk-like and cyclin-like subunits (34), and P-TEFb, a regulator of polymerase elongation with a cdc2-like subunit (35, 36).
We recently showed that the recombinant S. cerevisiae and S. pombe guanylyltransferases Ceg1 and Pce1 bind specifically to the phosphorylated form of the CTD (2). Moreover, recombinant yeast cap methyltransferase Abd1 also binds specifically to CTD-PO4 (2). Phosphorylation at Ser-5 of the heptad repeat was necessary and sufficient to confer guanylyltransferase and methyltransferase binding capacity to the CTD (2). Here, we have extended this analysis to mammalian capping enzyme. The key finding is that the guanylyltransferase domain by itself binds to CTD-PO4, whereas the triphosphatase domain has no evident capacity to bind to the CTD. The CTD binding studies reported herein employed a CTD-PO4 ligand composed of recombinant GST-CTD-PO4 that was phosphorylated in vitro. Preliminary experiments show that the purified mouse guanylyltransferase domain Mce1(211-597) also binds to a chemically synthesized 42-amino acid phosphopeptide consisting of 6 tandem repeats of the CTD heptad sequence (YSPTSPS) in which all six Ser-5 residues are Ser-PO4.2 The purified triphosphatase domain Mce1(1-210) does not bind to the 42-amino acid CTD phosphopeptide. These findings suggest that the mammalian RNA triphosphatase is targeted to the nascent pre-mRNA by virtue of its connection in cis to the guanylyltransferase. Lower eukaryotes may have adopted an alternative strategy to achieve the same end. Although the guanylyltransferase and triphosphatase components of budding yeast are encoded separately, the Ceg1 and Cet1 proteins interact in trans to form a heteromeric enzyme complex that can be isolated from yeast extracts (28). Because Ceg1 by itself can bind CTD-PO4, it may well chaperone Cet1 to the pol II elongation complex. If this is the case, then our genetic complementation data raise the prospect that the mouse guanylyltransferase domain can engage to some degree in cross-species interaction with yeast triphosphatase. Alternatively, Cet1 may have its own capacity to bind the pol II transcription complex, be it through the CTD or some other constituent of the complex. In either case, it appears that poisoning the mouse triphosphatase active site in the full-length Mce1 enzyme antagonizes the yeast capping system in vivo in a way that deletion of the triphosphatase domain does not. Recruitment of guanylyltransferase to the phosphorylated CTD neatly accounts for pol II specificity of capping and also provides a means of traffic control whereby CTD-interacting factors bind and dissociate from polymerase at the appropriate times in the transcription cycle without getting in each other's way. During preinitiation complex formation, the unphosphorylated CTD interacts with several general transcription factors and the SRB/mediator component of the pol II holoenzyme (37). Phosphorylation of the CTD presumably destabilizes these contacts and makes the CTD available for a novel set of interactions with the capping enzymes. Our findings contribute to an emerging picture of the CTD as a landing pad for macromolecular assemblies that regulate mRNA synthesis and processing (38, 39). Other recent studies indicate that protein components of the pre-mRNA splicing and 3' cleavage-polyadenylation assemblies also bind to the CTD (40, 41). The role of CTD phosphorylation in those interactions remains to be clarified. Thus far, only the capping enzymes display a strict requirement for CTD phosphorylation. ![]() |
FOOTNOTES |
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* 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.
1 The abbreviations used are: pol, polymerase; CTD, carboxyl-terminal domain; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; GST, glutathione S-transferase; 5-FOA, 5-fluoroorotic acid.
2 C. K. Ho and S. Shuman, unpublished data.
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