Covalent Guanylyl Intermediate Formed by HeLa Cell mRNA Capping

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1982, p. 993-1001 0270-7306/82/080993-09$02.00/0

Vol. 2, No. 8

Covalent Guanylyl Intermediate Formed by HeLa Cell mRNA Capping Enzyme DASHOU WANG,t YASUHIRO FURUICHI, AND AARON J. SHATKIN* Roche Institute of Molecular Biology, Nutley, New Jersey 07110 Received 26 February 1982/Accepted 29 April 1982

Guanylyltransferase, an enzyme that catalyzes formation of mRNA 5'-terminal caps, was isolated from HeLa cell nuclei. The partially purified preparation, after incubation with [a-32P]GTP, yielded a single radiolabeled polypeptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The guanylylated product was stable at neutral and alkaline pHs and had a pI of 4 by isoelectric focusing. An apparent molecular weight of -68,000 was estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis under reducing conditions. The formation of a covalently linked, radiolabeled GMP-protein complex and the associated release of PP1 required the presence of [a-32P]GTP and divalent cations and incubation between pH 7 and 9. Reaction with [p-32P]GTP, [a-32P]CTP, [a-32P]UTP, or [a32P]ATP did not label the -68,000-dalton polypeptide. Phosphoamide linkage of the GMP-enzyme complex was indicated by its sensitivity to cleavage by acidic hydroxylamine or HCl and not by NaOH or alkaline phosphatase. Both formation of the GMP-enzyme intermediate and synthesis of cap structures of type GpppApG from GTP and ppApG were remarkably temperature independent; the rates of enzyme activity at 0 to 4°C were 30%o or more of those obtained at 37°C. Radiolabeled GMP-enzyme complex, isolated by heparin-Sepharose chromatography from reaction mixtures, functioned effectively as a GMP donor for cap synthesis with 5'-diphosphorylated oligo- and polynucleotide acceptors. Alternatively, protein-bound GMP could be transferred to PP1 to form GTP. The formation of a guanylylated enzyme intermediate appears to be characteristic of viral and cellular guanylyltransferases that modify eucaryotic mRNA 5' termini. More than 10 years ago, reovirus cores were Eucaryotic mRNAs contain a distinctive 5'terminal cap, m7G5 ppp5'N (14). This structure is shown to catalyze exchange of PP1 with GTP but formed apparently during initiation of transcrip- not with the other individual ribonucleoside trition by modification of nascent 5' ends of all phosphates (21). GTP-specific PPi exchange is in cellular and most viral mRNAs (1). Capping agreement with reaction 1 in the above capping mechanisms were first worked out in detail by series. The same kind of GTP-specific exchange studying the enzyme activities associated with reaction was later reported for the guanylylhuman reovirus (5), vaccinia virus (12), and transferases purified from rat liver (11) and from insect cytoplasmic polyhedrosis virus (3), eu- vaccinia virus (16). Furthermore, the vacciniacaryotic viruses that produce capped mRNAs in derived enzyme formed an enzyme-pG covalent vitro. Several of these activities were purified intermediate that served as a donor of GMP in from vaccinia virions (10), and the correspond- the mRNA capping reaction (15). Enzymeing cellular capping enzymes were subsequently bound GMP could be transferred to either PP1 or isolated from HeLa (17-19) and rat liver (11) the 5' terminus of diphosphorylated polyadenylnuclei. In each case, caps were synthesized by ate [poly(A)], yielding GTP or poly(A) with the stepwise action of guanylyltransferase, fol- GpppA 5' ends. lowed by methyltransferases. For example, the Because 5'-terminal guanylylation is a key enzymatic steps necessary for reovirus mRNA step for generating stable, functional eucaryotic cap synthesis include: mRNAs (4, 14), the properties of cellular and (1) ppGpC + pppG z GpppGpC + PP,

GpppGpC + Adomet -+ m7GGppGpC + Adohcy m7GpppGmpC + Adohcy (3) m7GpppGpC + Adomet (2)

__

where asterisk denotes 32P. t Permanent address: Institute of Microbiology, Academia Sinica, Beijing, China.

viral guanylyltransferases are of some interest and importance. As part of a comparative study of the biochemical effects of mRNA capping on

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WANG, FURUICHI, AND SHATKIN

genetic expression, we purified guanylyltransferase from HeLa cells preliminary to preparing specific antibodies. The molecular weights of the cellular enzyme and the corresponding activities present in vaccinia virus (15), vesicular stomatitis virus (A. K. Banerjee, personal communication), insect cytoplasmic polyhedrosis, virus, and human reoviruses (unpublished data) are different. However, like the virus enzymes, the HeLa cell guanylyltransferase forms a stable, covalent guanylyl-protein complex that is active as an intermediate in the capping reaction. Similar results have been reported recently by Venkatesan and Moss (20), and Mizumoto et al. (lOa) have isolated the guanylylated form of the rat liver enzyme. Formation of a covalent guanylyl-enzyme intermediate appears to be characteristic of eucaryotic mRNA guanylyltransferases that function in the formation of mRNA 5'-terminal caps.

MOL. CELL. BIOL.

5 ,uCi of [a-'2P]GTP (specific activity, 218 to 410 Ci/ mmol), and enzyme protein as indicated. Incubations were stopped after 30 min at 37°C by the addition of 4 RI of 0.1 M EDTA, 50 RI of water, and 0.1 ml of watersaturated phenol. After mixing and centrifugation, the aqueous layer was ether extracted and lyophilized. Samples were redissolved in 30 ,ul of 0.02 M sodium acetate buffer (pH 6) and digested with 5 ,ug of nuclease P1 for 1 h at 37°C. The pH was then raised by the addition of 3 ,ul of 1 M Tris-hydrochloride (pH 8.4), and incubation was continued for 30 min in the presence of 1 U of calf intestine alkaline phosphatase. Digests were applied to Whatman 3MM paper and analyzed together with marker compounds by highvoltage electrophoresis in pyridine acetate buffer (pH 3.5, 3,000 V, 1 h). Nuclease P1 and phosphataseresistant, radiolabeled spots of GpppA located by autoradiography were cut out and quantitated by scintillation counting. One enzyme unit is equivalent to synthesis in 30 min of 1 pmol of GpppApG (18). Demonstration of enzyme-GMP covalent complex. Reaction mixtures consisted of 25 ,u1 of heparinSepharose-purified protein (fraction HS) in buffer C, 25 mM Tris (pH 7.5), 5 mM MgCI2, 0.5 mM MnCI2, 1 MATERIALS AND METHODS mM dithiothreitol, 0.12 ,ug of inorganic pyrophosphatase, and 5 RCi of [a-'2P]GTP (specific activity, 250 to Ceil growth and fractionation. HeLa S3 cells were 410 Ci/mmol) in a total volume of 30 ,ul. Samples were grown in suspension culture in Eagle modified medium incubated at 37°C for 30 min, chilled, and precipitated containing 5% fetal calf serum, and extracts were by the addition of 0.5 ml of 10% trichloroacetic acid fractionated at 0 to 4°C as described previously (18). containing 10%o sodium pyrophosphate. Precipitates Briefly, cells (usually 30 liters at a density of 5 x 10' to collected by centrifugation (12,000 x g, 10 min) were 8 x 10' cells per ml) were collected by centrifugation, washed twice with 0.5 ml of 5% trichloroacetic acid, washed three times with Dulbecco phosphate-buffered followed by 80%o ethanol, and dissolved in 30 RI of 88 saline, resuspended at 108/ml in 0.01 M Tris-hydro- mM Tris-hydrochloride (pH 6.8)-2% sodium dodecyl chloride (pH 7.2) containing 0.01 M NaCl, 1.5 mM sulfate-5% 13-mercaptoethanol-10% glycerol-0.001% MgCl2, and 1 mM dithiothreitol (buffer A), and dis- bromophenol blue dye. After heating for 5 min at rupted after 10 min by 10 strokes in a Dounce homoge- 100°C, samples were analyzed by electrophoresis in a nizer. Nuclei were pelleted, washed, resuspended at 2 1Oo polyacrylamide slab gel with a discontinuous x 108/ml in buffer A with 0.34 M sucrose, and ruptured buffer system containing 0.1% sodium dodecyl sulfate by the addition of 0.3 M (NH4)2SO4 and brief sonica- (8). Coomassie blue-stained gels were dried, and radiotion. After centrifugation (50,000 x g, 45 min), the active proteins located by autoradiography were cut supernatant fraction was treated for 1 h with 0.05 mM out and counted. phenylmethylsulfonyl fluoride to inactivate proteases Preparation of enzymatically active, guanylylated and diluted with 2 volumes of 0.033 M Tris-hydrochlo- capping intermediate. Heparin-Sepharose-purified proride (pH 8) containing 1 mM dithiothreitol, 1 mM tein was incubated as described above for the guanylEDTA, 0.1% Triton X-100, and 10%o glycerol (buffer yltransferase assay, except 200 pCi of [a-32P]GTP was B). The solution was recentrifuged, and the high-speed used with 0.9 ml of protein fraction in a final reaction supernatant fraction (designated S-I) was used for volume of 1 ml. The incubation mixture also included 1 purification of guanylyltransferase. pg of inorganic pyrophosphatase and protease inhibiEnzyme purification. Fraction S-I was applied to a tors phenylmethylsulfonyl fluoride (0.5 mM), Trasylol column (2.6 x 22 cm) of DEAE-cellulose equilibrated (50 U), and soybean trypsin inhibitor (0.1 mg). The with buffer B containing 0.1 M (NH4)2SO4. The flow- reaction was stopped by adding 50 pl of 0.2 M EDTA, through material was collected and dialyzed against and the incubation mixture was applied to a small buffer B to remove the (NH4)2SO4. The dialyzed column (0.6 x 1 cm) of heparin-Sepharose equilibrated sample (S-II) was reapplied to a column (1.5 x 10 cm) with buffer C. After washing with 10 bed volumes of of DEAE-cellulose in buffer B. Rlow-through material buffer C, the bound protein was eluted by application (S-III) was dialyzed against 0.025 M Tris-acetate (pH of a gradient of 0 to 0.5 M NaCl (25 ml each in buffer 7.2)-i mM dithiothreitol-1 mM EDTA-10o glycerol C). Samples of each 0.5-ml fraction were counted, and (buffer C) and applied to a column (1.6 x 2 cm) of the peak fractions of radioactivity eluting at -0.28 M heparin-Sepharose equilibrated with the same buffer. NaCl were pooled. At this stage, traces of [32P]GDP After extensive washing, a gradient of 0 to 0.5 M NaCl and [32P]GTP were detected in the sample by chroma(50 ml each in buffer C) was applied, and eluted tography on polyethyleneimine-cellulose thin-layer proteins were collected in 2-nil fractions. plates in 0.75 M KH2PO4 (pH 4.3). To remove residual Guanylyltrnsferase assay. Reaction mixtures (40 pl) labeled precursor, the sample was diluted threefold consisted of 25 mM Tris buffer (pH 7.5), 5 mM MgCl2, with water and reapplied to a minicolumn of heparin0.5 mM MnCl2, 1 mM dithiothreitol, 4 Fg of bovine Sepharose made in a disposable pipette tip. The colserum albumin, 3.5 ,uM ppApG as the GMP acceptor, umn was washed with buffer C, and protein was eluted

HeLa GMP PROTEIN CAPPING INTERMEDIATE

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TABLE 1. Summary of purification of HeLa cell mRNA guanylyltransferase ~Protein Enzyme activity Fraction' Sac Um)Fold Sp act (U/mg) (total U) puification Fraction" S-I; nuclear soluble 242.7 114 0.5 1 S-II; flow-through from DEAE141.0 165 1.2 2.5 cellulose I S-IlI; flow-through from DEAE67.5 779 11.5 24.5 cellulose II HS; eluates from heparin-Sepharose Pool of fractions 21-36 9.5 256 27.0 57.4 Pool of fractions 29-32 2.5 160 64.0 136.0 a Fractions were prepared from 1.5 x 1010 cells and assayed for guanylyltransferase activity as described in the text.

P(mg)

with 0.3 M NaCl in buffer C. 32P-labeled precursor was usually undetectable in the eluted radiolabeled protein by high-voltage paper electrophoresis and thin-layer chromatography. The isolated GMP-protein complex migrated by isoelectric focusing as a single band with a pl of 4 as compared with amyloglucosidase (pl 3.5) and bovine serum albumin (pl 4.7). Conditions of PP, exchange reaction. Enzyme fractions were incubated at 30°C for 30 min in 50-pJl reaction volumes containing 60 mM Tris-hydrochloride (pH 8.4), 10 mM dithiothreitol, 2.5 mM MgCl2, 1 mM GTP, 200 ,ug of bovine serum albumin, and 3 ,uCi of 32P-labeled sodium pyrophosphate (specific activity, 9.6 Ci/mmol). The reaction was terminated by the addition of 0.5 ml of 10% trichloroacetic acid containing 10% PP,. After centrifugation (12,000 x g, 10 min), a sample (0.4 ml) of the supernatant fraction was added to 0.2 ml of a 10% suspension of activated charcoal in 10% trichloroacetic acid-10% PPi. Samples were shaken for 10 min, collected by filtration onto Whatman GF/A glass filters, and counted by Cerenkov radiation. Materials. Heparin-Sepharose CL-6B was obtained from Pharmacia Fine Chemicals, Inc., and DEAEcellulose (DE-52) was obtained from Whatman, Inc. Thin-layer polyethyleneimine-cellulose plates were Schleicher & Schuell Co. products. Isoelectric focus-

ing was done on Servalyt precote thin-layer plates with a model 1405 electrophoresis cell (Bio-Rad Laboratories). Nuclease Pl, calf intestine alkaline phosphatase, and yeast inorganic pyrophosphatase were purchased from Boehringer Mannheim Corp. [a-32P]GTP and sodium [32P]pyrophosphate were products of Amersham Corp. and New England Nuclear Corp., respectively. Acceptor compounds ppApG and pppApG were kindly provided by J. Tomasz, Institute of Biophysics, Szeged, Hungary. pApG was prepared from ApG by incubation with ATP and polynucleotide kinase and purified by high-voltage paper electrophoresis (5).

RESULTS

Guanylyltransferase was purified more than 100-fold from HeLa cell nuclear lysates by chromatography on DEAE-cellulose and heparinSepharose (Table 1). The total enzyme activity was increased sevenfold by the two DEAEcellulose passes, suggesting removal of inhibitory component(s). A peak of GTP-PPi exchange activity coeluted from heparin-Sepharose with the cap-forming activity (Fig. 1). More than 30% of the capping activity was recovered from the column in samples eluting between 0.2 and 0.3

o

1

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E

-a

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FRACTION NUMBER

FIG. 1. Heparin-Sepharose column chromatography. Flow-through material from the second DEAE-cellulose column was dialyzed against buffer C and applied to a column of heparin-Sepharose. Samples eluted with a linear gradient of 0 to 0.5 M NaCl were collected in 2-mi fractions and assayed for guanylyltransferase and GTPPP, exchange activities as detailed in the text. Protein was estimated by a modification of the Lowry procedure.


996

FRAC-- ON NIJMBER 21 23 25 27 29 31 33 35

MOL. CELL. BIOL.

-68,000, based on its comigration with marker bovine serum albumin. Results of several kinds of experiments demA onstrated that the 32P-labeled guanylyltransfer94ase incubation product corresponds to a poly68peptide linked covalently to GMP. Radiolabeled -- .-4 + !2 -68,000-dalton polypeptide was readily formed 43-a by incubation of the pooled active fractions with [a-32P]GTP (Fig. 3A, lane 3) but was not ob30tained with boiled enzyme (lane 1), [p-32P]GTP (lane 2), or a-32P-labeled CTP, UTP, or ATP (lanes 4 through 6). Weakly labeled, lower21molecular-weight bands obtained with [a32P]CTP (lane 4) and [a-32P]ATP (lane 6) may be due to nonspecific binding or the presence of 21 23 25 27 29 3 33 35 B other potential nucleotide or phosphate acceptors, as also suggested by the more extensive labeling obtained with [y-32P]ATP (Fig. 3A, lane 947). The radioactivity resulting from incubation 68-* ~ with [a-32P]GTP remained protein associated after exposure to fully denaturing conditions, 43including precipitation with trichloroacetic acid, boiling in 1% sodium dodecyl sulfate, and elec30trophoresis in polyacrylamide gels (Fig. 2B). Furthermore, the 3 P-labeled protein was resistant to digestion with calf intestine alkaline 21 phosphatase (25 U/ml, 30 min, 37°C) and remained at the origin during thin-layer chromaFIG. 2. 1Polyacrylamide gel electrophoresis of col- tography (Fig. 3B, lanes 1 and 5) or high-voltage paper electrophoresis (Table 2). Exposure to 0.1 umn fractioins containing guanylyltransferase activity. Protein frac tions shown in Fig. 1 were incubated with M HCl for 10 min at 70°C hydrolyzed the sample [a-32P]GTP under conditions of enzyme-GMP com- and yielded [32P]GMP (Fig. 3B, lane 4; Table 2), plex formaltion. After 30 min, samples were acid but the compound was relatively stable to alkali precipitated and analyzed by sodium dodecyl sulfate- under the same conditions (Fig. 3B, lane 3; polyacrylamfiide gel electrophoresis as described in the Table 2). Incubation in 3.8 M hydroxylamine at text. (A) Coomassie blue-stained proteins; (B) autora- pH 4.7 for 20 min at 37°C released most of the diogram of the same gel. Note that less protein was radioactivity as GMP, indicative of a phospholoaded in fraction 35, as indicated by the staining amide linkage between the nucleotide and propattern. tein (Fig. 3B, lane 2; Table 2; 15, 20). Under the same conditions of acid, alkali, or hydroxylamine treatment, [a-32P]GTP was not extensiveM NaCl, b ut only a small percentage of the total ly hydrolyzed (Fig. 3B, lanes 6 through 8). GTP-PP, exchange activity was present in these Formation of the guanylyl-protein complex fractions. Most of the exchange activity was in was time dependent but remarkably insensitive the flow--through material. Consequently, to incubation temperature (Fig. 4A). After a GpppApG formation from GTP plus ppApG, brief lag, the rates of protein guanylylation in rather thatn GTP-PP1 exchange, was used as the samples kept on ice were similar to those incuassay to Ipurify HeLa cell guanylyltransferase bated at 370C. Divalent cation was required for the reaction, and no guanylylated protein was activity. Analysiss of column fractions by sodium dode- detected by polyacrylamide gel analysis when cyl sulfatU e-polyacrylamide gel electrophoresis the incubation mixture contained 10 mM EDTA indicated that the heparin-Sepharose-purified (Fig. 4B, lane 1). There was also little radiolabelmaterial, i]ncluding the peak fractions of guanyl- ing of polypeptides in mixtures incubated below yltransferaise activity, contained many polypep- pH 7 (Fig. 4B, lanes 2 and 3) or above pH 9 (Fig. tides as dletected by Coomassie blue staining 4B, lane 7). As in the case of protein-pG forma(Fig. 2A). By contrast, a single 32P-labeled band tion (Fig. 4A), transfer of GMP from GTP to was obtaiined by electrophoretic analysis after ppApG to form caps occurred at 0°C at -30% of incubation of the protein fractions with [a- the rate observed at 37°C (Fig. 5). The partially I 32P]GTP (Fig. 2B). The apparent molecular purified guanylyltransferase apparently conweight of the radiolabeled polypeptide was tained nucleotide phosphohydrolase activity, -3

Mr

x

VOL. 2, 1982


A 2

4

3

5

6

7

B

3

4 5 6 7 8

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Mr x '0

9468-

2

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_

FIG. 3. Nucleotide specificity and stability of GMP-protein complex. (A) Heparin-Sepharose-purified pooled protein fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after incubation under conditions of enzyme-GMP complex formation but with the following changes: lane 1, protein heated to 100°C for 5 min before reaction with [a-32P]GTP; lane 2, [_1-32P]GTP in place of [a-32P]GTP; lanes 3 through 6, incubation with a-32P-labeled GTP, CTP, UTP, and ATP, respectively; lane 7, incubation with [-y-32P]ATP. (B) 32P-labeled enzyme-GMP complex was purified by heparin-Sepharose chromatography twice to remove most of the residual radiolabeled precursor. The sample was digested with calf intestine alkaline phosphatase and divided for further treatment as follows: lane 1, 0.2 M sodium acetate buffer (pH 4.8, 37°C, 20 min); lane 2, same as described for lane 1 but with 3.8 M hydroxylamine; lane 3, 0.1 M NaOH (70°C, 10 min); lane 4, 0.1 M HCI (70°C, 10 min); lane 5, 37°C, 20 min in water. As controls, [a-32P]GTP was incubated at 37°C for 20 min in water (lane 6); in 0.1 M HCI at 70°C for 10 min (lane 7); and with 3.8 M hydroxylamine in 0.2 M sodium acetate buffer (pH 4.8) at 37°C for 20 min (lane 8). Samples were spotted on polyethyleneimine-cellulose thin-layer plates, washed with methanol to remove salt, developed in 0.75 M KH2PO4, and analyzed by autoradiography.

since pppApG (and pppGpC [data not shown]) well as ppApG were active for cap synthesis; by contrast, pApG was not a GMP acceptor (Table 3). The guanylyltransferase also formed caps on reovirus mRNA with 5'-terminal ppGpC and on poly(A) consisting of a mixture of 5'diphosphorylated and -triphosphorylated molecules, confirming that the enzyme has mRNAmodifying activity (Table 3). To demonstrate that the guanylylated polypeptide functions as an intermediate in mRNA capping or GTP synthesis or both, column fractions corresponding to the peak of activity shown in Fig. 1 were incubated with [a-32P]GTP in a large-scale reaction. The incubation mixture was applied to heparin-Sepharose to separate the bound radiolabeled protein from unbound residual nucleotide precursors. After two chromatographic passes, all of the 32p in the sample was associated with guanylyl-protein, as determined by thin-layer chromatography and highvoltage paper electrophoresis. Analysis by isoelectric focusing yielded a single radiolabeled band at pH 4 (Fig. 6). The purified guanylyl-protein complex was tested for enzymatic activity by assaying for GTP synthesis in the presence of PPi and for the capacity to form caps by transfer of GMP to ppApG. 32P-labeled samples were incubated for 30 min at 37°C under various conditions and analyzed by high-voltage paper electrophoresis. After incubation in the absence of divalent cation, most of the radioactivity remained covalently associated with the protein and migrated at as

the origin (Fig. 7A, lane 1). Reaction mixtures that were complete except for the inclusion of ppApG or PP, (as a GMP acceptor) yielded a markedly reduced amount of radiolabeled protein (Fig. 7A, lane 2; Table 4). Most of the TABLE 2. Susceptibility of guanylyl-protein complex to chemical cleavage Treatmenta

% complexb Enzyme-pG

% GMP releasedc 0

100d (control) Alkaline 107 0 phosphatase NaOH 90 3 HCI 37 38 Hydroxylamine 19 61 a Samples were digested with 25 U of calf intestine alkaline phosphatase per ml at 37°C for 20 min, with 0.1 M acid or alkali at 70°C for 10 min, or with 3.8 M hydroxylamine in 0.2 M sodium acetate (pH 4.7) at 37°C for 20 min. After spotting on Whatman 3MM paper, residual salt was removed from the origin by washing in methanol, which probably resulted in some loss of hydrolyzed sample and incomplete recoveries. b Percent radioactivity remaining at the origin after high-voltage paper electrophoresis at pH 3.5 (5). The preparation of enzyme-pG complex used in this experiment contained -2,500 cpm of free GTP, which, as shown in Fig. 3B, is not hydrolyzed (less than 10%) by the same chemical treatments. c One hundred percent corresponds to 4,363 cpm. d A background level of 716 cpm in the position of GMP in the untreated control was subtracted from each sample. None


998

MOL. CELL. BIOL.

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FIG. 4. Characteristics of guanylyltransferase. (A) Heparin-Sepharose-purified enzyme was incubated with [a-32P]GTP. After the indicated times at 0 and 37°C, reactions were sampled for sodium dodecyl sulfatepolyacrylamide gel analysis. Radiolabeled GMP-protein complexes located by autoradiography (see inset) were cut and counted. (B) Guanylyltransferase reactions were done at 37°C as described in the text but in the presence of 10 mM EDTA (lane 1) or after adjusting the pH with 0.1 M buffer as follows: lanes 2 and 3, pH 5 and 6 with cacodylate; lanes 4 and 5, pH 7 and 8 with Tris-hydrochloride; lanes 6 and 7, pH 9 and 10 with glycine-NaOH.

radioactivity migrated in the position of GTP and GDP and was shown to be a mixture of about equal amounts of the two nucleotides by thin-layer chromatography. This finding suggests-that the guanylyltransferase or some other protein in the sample contained PPi in a form 3000_

2-000

w

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available as a GMP acceptor (even after heparinSepharose chromatography and dialysis). The GDP presumably resulted from cleavage of the GTP product by some GTPase activity in the preparation. The addition of 0.05 mM sodium pyrophosphate to the incubation mixture stimulated GTP synthesis only slightly, indicating that the endogenous level of PP1 was adequate to support almost maximal transfer of GMP (Fig. 7A, lane 3; Table 4). Consistent with these findings, GTP synthesis was largely prevented by the addition of inorganic pyrophosphatase to the incubation mixture. In the absence of an endogenous or added GMP acceptor, most of the radioactivity remained bound to the protein (Fig. 7A, lane 4; Table 4). TABLE 3. Acceptor activity for cap formation by HeLa cell guanylyltransferasea Sample Cap formed (cpm)

000

pApG ..................... ppApG ....................

TIME (min)

FIG. 5. Time

course

of cap synthesis. Heparin-

Sepharose-purified guanylyltransferase was assayed with [a-32P]GTP as the donor and ppApG as the acceptor at 0°C (0) and 37°C (0). Samples were assayed for GpppA formation by high-voltage paper electrophoresis after digestion with P1 nuclease and phosphatase as described in the text.

81 10,546 5,588

pppApG .......... ......... Reovirus mRNA ...... ....... 5,984 Poly(A) ................... 6,101 a Each dinucleotide was assayed at 3.5 ,uM with 5 ,uCi of [a-32P]GTP having a specific activity of 218 Ci/ mmol; poly(A) was tested at -1 ,uM with 10 ,uCi of [a32P]GTP having the same specific activity. Reovirus mRNA containing 5'-ppGpC (5) was used at -0.7 piM with 50 ,uCi of [a-32P]GTP having a specific activity of 410 Ci/mmol. Analyses were as described in the text.

VOL. 2, 15)82

1.0 F-

_


cpm at the origin (lane 3); poly(A) yielded 1,093 cpm of GpppA and 14 cpm at the origin (lane 4).

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999

on heparin-Sepharose was analyzed by

DISCUSSION Our findings, together with those of Venkatesan and Moss (20) and Mizumoto et al. (10a), indicate that mammalian cell mRNA guanylyltransferase, like the corresponding enzyme isolated from vaccinia virus (15), forms a covalent guanylyl-protein intermediate. Guanylyltransferase activities are associated with human reovirus and insect cytoplasmic polyhedrosis virus, and in each case virion high-molecular-weight polypeptides are labeled by incubation of purified particles with [a-32P]GTP (R. E. Smith, Y. Furuichi, A. LaFiandra, and A. J. Shatkin, unpublished data). In the case of vesicular stomatitis virus, the NS protein has been tentatively identified as guanylyltransferase on the basis of [a-32P]GTP binding (Bishnupada De and Amiya K. Banerjee, personal communication). Covalent protein-GMP complexes are also

eIecLroppforesis on [ervadyL precoLe piaLes wiLn MarKer proteins amyloglucosidase (pI 3.5), bovine serum albumin (pI 4.7), ,B-lactoglobulin (pl 5.3), conalbumin (pl 5.9), and horse myoglobin (pl 7.3). Inset shows autora- A diogram of electrofocused sample.

Results demonstrating that the guanylyl-protein complex is an intermediate in the capping reaction are shown in Fig. 7A, lane 5. Radioactive GMP was efficiently transferred from the protein to ppApG to form GpppApG; a similar level of GTP and GDP mixture was also synthesized. Again, the presence of inorganic pyrophosphatase markedly decreased the formation of GTP and GDP (Fig. 7A, lane 6), and 75% of the radioactivity was transferred to form GpppApG (Table 4). Transfer of GMP from the enzyme intermediate to ppApG in the presence of divalent cations apparently occurred directly and without nucleotide exchange or GTP formation, since the addition of excess nonradioactive GTP had little effect on the yield of radiolabeled GpppApG (Fig. 7A, lane 7; Table 4). Cap formation from ppApG and enzyme-GMP complex also occurred at 0°C (data not shown). Capping of polynucleotides by the heparinSepharose-purified intermediate was obtained in a separate experiment (Fig. 7B). Radiolabeled enzyme-GMP complex was incubated with reovirus mRNA or poly(A) for 30 min at 37°C in the presence of inorganic pyrophosphatase, and the products were analyzed by high-voltage paper electrophoresis after Pl nuclease digestion. GMP transfer did not occur in the absence of an acceptor (lane 1) or when 10 mM EDTA was added to the reaction mixture (lane 2). With reovirus mRNA as the acceptor, the distribution of radioactivity was 962 cpm in GpppG and 104

B

1~- z;_

I

' Q-

-

_

_

_

FIG. 7. Analysis of products formed by isolated GMP-enzyme complex. Radiolabeled guanylylated intermediate was purified by heparin-Sepharose chromatography twice and assayed for guanylyltransferase activity under standard conditions with the indicated changes. (A) Products were analyzed by direct application of incubation mixtures to Whatman 3MM paper, followed by high-voltage electrophoresis and autoradiography. Lane 1, Without Mg2+ or Mn2+; lane 2, standard conditions; lane 3, 0.05 mM PPj; lane 4, 12 ,ug of inorganic pyrophosphatase per ml; lane 5, 3.5 ,uM ppApG; lane 6, 3.5 ,uM ppApG and inorganic pyrophosphatase; lane 7, 3.5 ,uM ppApG, inorganic pyrophosphatase, and 0.5 mM GTP. (B) Products formed in the presence of inorganic pyrophosphatase were analyzed after digestion with Pl nuclease. Lane 1, Without acceptor; lane 2, 3.5 ,uM ppApG plus 10 mM EDTA; lanes 3 and 4, reovirus mRNA and poly(A), respectively, as shown in Table 3.

1000

Lane no. (Fig. 7A)


MOL. CELL. BIOL.

TABLE 4. Formation of GpppApG from guanylated protein and ppApGa 32P-labeled products (%) Total Condltlons cpm Protein-pG GpppApG GTP +

94 2 No Mg2+ Mn2+ 1,220 4 16 6 Complete 1,226 78 Complete + PP, 12 3 1,429 85 Complete + PPaseb 819 88 5 7 Complete + ppApG 15 39 1,244 46 15 75 Complete + ppApG + PPase 1,237 10 Complete + ppApG + PPase + GTP 18 1,140 65 17 a 32P-labeled guanylyl-protein complex was isolated by heparin-Sepharose chromatography from a 1-ml guanylyltransferase reaction mixture. The purified radiolabeled intermediate was incubated under the indicated conditions, and the 32P-labeled products were separated by high-voltage paper electrophoresis. The distribution of radioactivity among protein-pG complex at the origin, GpppApG, and GTP + GDP in Fig. 7A, lanes 1 through 7, was determined by scintillation counting after cutting the paper according to the autoradiogram pattern. b PPase, Inorganic pyrophosphatase.

1 2 3 4 5 6 7

formed by the mRNA guanylyltransferases from Artemia salina, wheat germ, and yeasts (Mizumoto et al., personal communication; J. Keith, personal communication). Synthesis of a characteristic guanylyl-enzyme intermediate by capping enzymes from animal and plant cellular and viral sources is consistent with the following general reaction mechanism for 5' capping of most eucaryotic mRNAs:

observed in cell extracts. The total guanylyltransferase activity was increased sevenfold by passing crude extracts of nuclei through DEAEcellulose (Table 1). It remains to be determined whether guanylyltransferase or other mRNAmodifying enzymes, e.g., methyltransferases, are associated with polypeptides or factors that regulate expression of their activity. It should be possible, by taking advantage of the identity of the 32P-labeled guanylyl-protein intermediate Enz + pppG a Enz-pG + PPi and mRNA capping enzyme, to test whether, under various conditions of cell growth, virus Enz-pG + ppNpN GpppNpN + Enz infection, transformation, etc., guanylyltransfer5' Capping of mRNA may promote transcrip- ase is controlled coordinately with transcription. tion by stabilizing nascent, initiated transcripts ACKNOWLEDGMENTS for chain elongation (4). In a possibly analogous We thank B. Moss for communicating results before publifashion, adenovirus DNA synthesis may be fa- cation, helpful discussions, and providing 5'-diphosphorylated cilitated by the covalent complex formed be- and -triphosphorylated poly(A). Alba LaFiandra and Maureen tween the virus 5'-terminal protein and dCMP, Morgan provided excellent assistance in the preparation of the nucleotide that initiates DNA replication (9). HeLa cell extracts and reovirus mRNA. The same type of nucleotidyl-protein complex may function in genome replication of other LITERATURE CITED DNA viruses (6, 13) and some RNA viruses (7) 1. Banerjee, A. K. 1980. 5'-Terminal cap structure in eucaryand perhaps in uninfected cells as well (2). otic messenger ribonucleic acids. Microbiol. Rev. 44:175As suggested by Venkatesan and Moss (20), 205. the ability to form a stable radiolabeled interme- 2. Coombs, D. H., A. J. Robinson, J. W. Bodnar, C. J. Jones, and G. D. Pearson. 1978. Detection of covalent DNAdiate provides a convenient marker for detecting protein complexes: the adenovirus DNA-terminal protein guanylyltransferases. Because the capping encomplex and HeLa DNA-protein complexes. Cold Spring zyme has not been purified to homogeneity, it Harbor Symp. Quant. Biol. 43:741-753. seemed possible that the catalytic activity re- 3. Furuichl, Y. 1978. "Pretranscriptional capping" in the biosynthesis of cytoplasmic polyhedrosis virus mRNA. sides in another polypeptide that copurifies with Natl. Acad. Sci. U.S.A. 75:1086-1090. the -68,000-molecular-weight GMP donor. This 4. Proc. Furuichl, Y., A. LaFiandra, and A. J. Shatkin. 1977. 5'possibility is made unlikely by the demonstraTerminal structure and mRNA stability. Nature (London) tion that the apparent molecular weight of the 266:235-239. Y., S. Muthukrishnan, J. Tomasz, and A. J. 32P-labeled guanylyl-protein intermediate deter- 5. Furuichl,1976. Mechanism of formation of reovirus mRNA Shatkin. mined by sodium dodecyl sulfate-polyacryl5'-terminal blocked and methylated sequence, amide gel electrophoresis is close to the values m7GpppGmpC. J. Biol. Chem. 251:5043-5053. estimated previously for native guanylyltrans- 6. Gerlich, W. H., and W. S. Robinson. 1980. Hepatitis B virus contains protein attached to the 5' terminus of its ferases from rat liver (-65,000 [11]) and HeLa DNA strand. Cell 21:801-809. cells (-48,500 [18]). However, the intracellular 7. complete Kitamura, N., C. Adler, and E. Wimmer. 1980. Structure configuration of mRNA guanylyltransferase and expression of the picornavirus genome. Ann. N.Y. Acad. Sci. 354:183-201. may be more complex than we and others have

VOL. 2, 1982


8. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 9. Lichy, J. H., M. S. Horwitz, and J. Hurwitz. 1981. Formation of a covalent complex between the 80,000dalton adenovirus terminal protein and 5'-dCMP in vitro. Proc. Natl. Acad. Sci. U.S.A. 78:2678-2682. 10. Martin, S. A., E. Paoletti, and B. Moss. 1975. Purification of mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions. J. Biol. Chem. 250:9322-9329. 10a.Mizumoto, K., Y. Kaziro, and F. Lipmann. 1982. Reaction mechanism of mRNA guanylyltransferase from rat liver: isolation and characterization of a guanylyl-enzyme intermediate. Proc. Natl. Acad. Sci. U.S.A. 79:16931697. 11. Mizumoto, K., and F. Lipmann. 1979. Transmethylation and transguanylylation in 5'-RNA capping system isolated from rat liver nuclei. Proc. Natl. Acad. Sci. U.S.A. 76:4%1-4965. 12. Moss, B., A. Gershowitz, C.-M. Wei, and R. Boone. 1976. Formation of the guanylylated and methylated 5'-terminus of vaccinia virus mRNA. Virology 72:341-351. 13. Revie, D., B. Y. Tseng, R. H. Grafstrom, and M. Goulian. 1979. Covalent association of protein with replicative form DNA of parvovirus H-1. Proc. Natl. Acad. Sci.

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U.S.A. 76:5539-5543. 14. Shatkin, A. J. 1976. Capping of eucaryotic mRNAs. Cell 9:645-653. 15. Shuman, S., and J. Hurwitz. 1981. Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme-guanylate intermediate. Proc. Natl. Acad. Sci. U.S.A. 78:187-191. 16. Shuman, S., M. Surks, H. Furneaux, and J. Hurwitz. 1980. Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. J. Biol. Chem. 255:11588-11598. 17. Venkatesan, S., A. Gershowitz, and B. Moss. 1980. Modification of the 5' end of mRNA. J. Biol. Chem. 255:903908. 18. Venkatesan, S., A. Gershowitz, and B. Moss. 1980. Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei. J. Biol. Chem. 255:2829-2834. 19. Venkatesan, S., and B. Moss. 1980. Donor and acceptor specificities of HeLa cell mRNA guanylyltransferase. J. Biol. Chem. 255:2835-2842. 20. Venkatesan, S., and B. Moss. 1982. Eukaryotic mRNA capping enzyme-guanylate covalent intermediate. Proc. Natl. Acad. Sci. U.S.A. 79:340-344. 21. Wachsman, J. T., D. H. Levin, and G. Acs. 1970. Ribonucleoside triphosphate-dependent pyrophosphate exchange of reovirus cores. J. Virol. 6:563-565.