Proc. Nat. Acad. Sci. USA Vol. 72, No. 3, pp. 1189-1193, March 1975
Methylation-Dependent Translation of Viral Messenger RNAs In Vitro (S-adenosylmethionine/wheat germ/vesicular stomatitis virus/reovirus)
G. W. BOTH, A. K. BANERJEE, AND A. J. SHATKIN Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110
Communicated by Severo Ochoa, January 10, 1975 ABSTRACT Methylated reovirus and vesicular stomatitis virus mRNAs, synthesized in vitro in the presence of S-adenosylmethionine by the virion-associated polymerases (RNA nucleotidyltransferases, EC 2.7.7.6), stimulate protein synthesis by wheat germ extracts to a greater extent than unmethylated mRNAs. Addition of S-adenosylmethionine to a cell-free extract programmed with unmethylated mRNA stimulates protein synthesis and results in methylation of the mRNA. An inhibitor of mRNA methylation, S-adenosylhomocysteine, blocks translation of unmethylated, but not of methylated, mRNAs. Aurintricarboxylic acid, which inhibits polypeptide chain initiation, also prevents mRNA methylation by wheat germ extracts. In contrast, sparsomycin, which inhibits polypeptide chain elongation, does not reduce mRNA methylation. The results indicate that methylation of viral mRNA is required for translation in vitro and suggest that mRNA methylation occurs at the initiation step of protein synthesis.
Many animal viruses contain an RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) that transcribes the viral genome and produces RNA that functions as messenger in infected cells and in cell-free systems (1). Recently, it was reported that purified cytoplasmic polyhedrosis virus (2), reovirus (3), vaccinia virus (4, 5), and vesicular stomatitis virus (VSV) (6) also have an RNA methylase activity. Viral RNA synthesized in vitro in the presence of the methyl group donor S-adenosylmethionine (SAdoMet) is specifically methylated at the 5' end, resulting in molecules with 5'terminal structures of the type m'G(5')ppp(5')Gm... and m7G(5')ppp(5')Am ... (refs. 7-9; G. Abraham, D. P. Rhodes, and A. K. Banerjee, unpublished results). Similar 5' structures have been found in virus-specific mRNAs isolated from cells infected with reovirus (10), simian virus 40 (11), and VSV (12) and in RNA from uninfected cells including Novikoff hepatoma (13), mouse L (14); HeLa (Y. Furuichi, M. M. Morgan, A. J. Shatkin, and J. E. Iarnell, unpublished results), and monkey BSC-1 cells (11). One possible effect of 5'-terminal methylation might be to alter the translational capacity of mRNA. To test this possibility, methylated and unmethylated RNAs synthesized in vitro by purified reovirus and VSV were compared as messengers in a cell-free protein-synthesizing system prepared from wheat germ. The results indicate that methylation of mRNA is required for its translation. MATERIALS AND METHODS
Growth and Purification of Viruses. Reovirus type 3 Dearing strain and VSV (Indiana serotype) were purified from inAbbreviations: VSV, vesicular stomatitis virus; SAdoMet, Sadenosylmethionine; BHK, baby hamster kidney; NaDodSO4, sodium dodecylsulfate; SAdoHcy, S-adenosylhomocysteine; ATA, aurintricarboxylic acid. 1189
fected mouse L cells (15) and baby hamster kidney (BHK) cells (16), respectively.
Synthesis and Purification of Viral mRNAs. The synthesis of reovirus mRNA by purified virus and its isolation by filtration through Sephadex G-50 and sedimentation in glycerol density gradients have been described (17). When methylated mRNA was required, 10 /uM SAdoIet was included in the transcription reaction mixture. VSV mRNA was synthesized as previously described (16) and purified by passage through Sephadex G-100 (6). VSV 12-18S mRNA was isolated by ethanol precipitation after sedimentation in a glycerol gradient (18). Methylated VSV mRNA was synthesized in the presence of 6.8 ,M SAdoMet (6). Translation of Viral mRNAs. Preparation of the wheat germ extract and the conditions for protein synthesis have been described (17, 18). Reovirus and VSV mRNAs were used at concentrations of 100 and 40 Aug/ml, respectively, in a total incubation volume of 12.5,u. Protein synthesis in the presence of L-[5S]methionine (specific activity 200-400 Ci/mmol) was assayed by heating a 1 MuI aliquot of incubation mixture in 2 ml of 10% trichloroacetic acid containing 1% casamino acids (Difco, Detroit, Mich.) at 950 for 15 min. The samples were chilled in ice, collected on Millipore filters, and washed with 5 ml of trichloroacetic acid/casamino acids and 5 ml of 1% acetic acid. The filters were dried and their radioactivity was measured in a toluene-based scintillant.
Methylation of Viral mRNAs by Cell-Free Extracts. Reovirus and VSV mRNAs were translated in the presence of unlabeled methionine and S-adenosyl [methyl-3H ]methionine (specific activity 7.3 Ci/mmol; 500 4s0i/ml; New England Nuclear, Boston, Mass.) at concentrations of 2 Au respectively. The SAdoMet was neutralized with 2 AL unbuffered Trizma (Sigma, St. Louis, Mo.) immediately before use. An aliquot of reaction mixture was diluted with 0.5 ml of buffer [10 mM Tris-HCl, p'i 7.5, 100 mMI NaCl, 5 mM EDTA, 0.5% sodium dodecylsulfate (NaDodSO4)] and extracted with an equal volume of redistilled phenol. The aqueous layer was retained and the phenol layer was re-extracted with 0.5 ml of buffer. The aqueous layers were combined and the RNA was precipitated by the addition of two volumes of ethanol in the presence of 0.3 AlI NaCl before glycerol gradient analysis. Alternatively, the RNA was precipitated with 10% trichloroacetic acid, collected on Aiillipore filters and analyzed for radioactivity. VSV and reovirus mRNAs were sedimented on 5-30% (w/v) glycerol density gradients (17) in the Beckman SW41 rotor at 40 for 17 hr at 33,000 and 28,000 rpm, respectively.
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Proc. Nat. Acad. Sci. USA 72 (1976)
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FIG. 1. Effect of methylation of mRNA on protein synthesis
in vitro. VSV and reovirus mRNAs synthesized in the presence and absence of SAdoMet and poly(A)-containing mRNA isolated from VSV-infected BHK cells (27) were used to stimulate protein synthesis in cell-free extracts of wheat germ. [35S]Methionine incorporated into acid-precipitable material in response to: (a) A- - -A, no RNA; 0----0, poly(A)-containing RNA from VSVinfected BHK cells; VSV mRNA synthesized in vitro in the absence (A-A) and in the presence (@.-L ) of SAdoMet; and (b) A- - -A, no RNA and reovirus mRNA synthesized in vitro in the absence (0-0I) and presence (@ -) of SAdoMet. RESULTS Effect of inethylation on mRNA activity Cell-free extracts of wheat germ are capable of translating
both reovirus and VSV mRNAs with fidelity (17, 18, 27). Therefore the effect of methylation on mRNA activity was studied using this cell-free system. VSV mRNA synthesized in vitro in the presence and absence of SAdoMet was purified and translated in wheat germ extracts as described previously (18). Methylated mRNA stimulated [85S]methionine incorporation into acid-precipitable polypeptide products to a greater extent than an equal amount of unmethylated mRNA (Fig. la); the difference was maximal after incubation for 20 min. In addition, the kinetics of protein synthesis obtained with poly(A)-containing VSV mRNA isolated from infected BHK-21 cells were similar to those observed with mRNA methylated in vitro (Fig. la). Analysis of the polypeptide products by NaDodSO4-polyacrylamide slab gel electrophoresis (18) showed that the same relative amounts of the previously described viral proteins, P63, N, NS, and M, were synthesized in response to both methylated and unmethylated VSV mRNA (ref. 18, and data not shown). Similarly, reovirus mRNA synthesized in the presence of SAdoMet was also a better template for protein synthesis than mRNA made in its absence (Fig. lb). A maximum difference of almost 5fold in the incorporation of [5S ]methionine was observed after 20 min incubation. The relative amounts of the polypeptide products synthesized in response to methylated or unmethylated reovirus mRNA were similar, and the polypeptides synthesized included those described previously (17). Dependence of protein synthesis on methylation Since methylated mRNA was more efficient in directing protein synthesis in vitro, it was of interest to determine whether the activity of unmethylated mRNA was increased in the presence of a methyl group donor. As shown in Fig. 2a, VSV mRNA made by the virion-associated polymerase in the absence of SAdoMet directed the incorporation of [35S]methi-
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FIG. 2. Dependence of in vitro protein synthesis on methylation. (a) VSV RNA synthesized in vitro in the absence of SAdoMet was used as mRNA. Protein synthesis was assayed in the absence (0---O) and in the presence (--) of 4MuM SAdoMet or in the presence of 4 MM SAdoMet plus 320 MAM SAdoHcy (-----A). A- A represents the protein synthesis observed in the absence of added RNA. (b) VSV RNA synthesized in vitro in the presence of SAdoMet was used as mRNA. Protein synthesis directed by this pre-methylated RNA was assayed in the ) of 320 AM absence (0-O) and in the presence (@SAdoHcy. A -A represents protein synthesis without added RNA and A -A protein synthesis directed by unmethylated VSV mRNA.
onine in wheat germ extracts. However, when SAdoMet was added to the translation reaction mixture, both the rate and extent of protein synthesis were stimulated several-fold. Optimum stimulation of translation by SAdoMet occurred at concentrations between 2 and 8 MM and the products, analyzed by gel electrophoresis, were the same as with mRNA that had been methylated during transcription. The SAdoMet analog, S-adenosylhomocysteine (SAdoHcy) has been shown to inhibit methylation of reovirus and VSV mRNA during in vitro transcription (3, 6). Addition of SAdoHcy to wheat germ extracts at an 80-fold higher concentration than SAdoMet also inhibited protein synthesis more than 95%, suggesting that translation of VSV mRNA is completely dependent upon methylation. However, in order to eliminate the possibility that this inhibition was due to some nonspecific effect of SAdoHcy on protein synthesis, the activity of mRNA that was methylated during synthesis in vitro (pre-methylated mRNA) was determined in the cell-free system in the presence and absence of SAdoHcy. In contrast to unmethylated mRNA, translation of pre-methylated VSV mRNA was undiminished by SAdoHcy (Fig. 2b). The results, therefore, indicate that protein synthesis is dependent upon methylation of the viral mRNA and that wheat germ extracts contain an mRNA methylase activity. Furthermore, because translation of unmethylated mRNA occurs in the absence of exogenous SAdoMet, it appears that wheat germ extracts also contain an endogenous methyl group donor. However, it should be noted that even when SAdoMet is present during translation, unmethylated RNA is a less effective messenger than premethylated mRNA (compare Fig. 2a and b). The results obtained with VSV mRNA (Fig. 2) were confirmed with reovirus RNA (data not shown). Methylation of viral mRNA by wheat germ extracts In order to demonstrate directly the methylation of viral
mRNA by wheat germ extracts, 32P-labeled reovirus mRNA
Proc. Nat. Acad. Sci. USA 72
Methylation-Dependent Translation
(1975) (min)
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FIG. 3. Methylation of reovirus mRNA by extracts of wheat germ. Reovirus mRNA synthesized in the presence of 10 MAM SAdoHcy and [a-32P]CTP was used to stimulate protein synthesis in a 200 ;Ml reaction mixture in the presence of unlabeled methionine and 2 gM SAdo [methyl-3H] Met. The mRNA was recovered from (a) 10 MAl and (b-d) 30 MIA aliquots by phenol extraction as described in the Materials and Methods section. (a) Incorporation of 3H radioactivity into acid-precipitable material in the absence of (O---O) of and in the presence (@-@) of reovirus mRNA and in the presence of mRNA plus 160 MM SAdoHcy A--A. Panels b-d show glycerol density gradient profiles of reovirus mRNA after (b) 0 and (c) 20 min of incubation under conditions of translation and (d) after 20 min incubation in the presence of 160 M&M SAdoHcy: O-O, 3H, 0 * 32P. In (c) t-represents [3H]methyl incorporation in the absence of added RNA. In (b-d), radioactivity was measured directly after mixing the fractions with Aquasol. Sedimentation is from right to left.
was used to direct protein synthesis. Reovirus mRNA was synthesized in vitro in the presence of 10 MM SAdoHcy in order to reduce the number of methylated 5' termini in the mRNA (3). Under these conditions, the products consisted of the small (s) and the medium (m) classes of mRNA (see Fig. 3b). When the 32P-labeled reovirus mRNA was translated in wheat germ extracts in the presence of SAdo[methyl-PH]Met, there was a rapid incorporation of [3Hjmethyl into acidprecipitable material (Fig. 3a). The reaction was essentially complete after 10 min, when there was a 7-fold stimulation in the extent of [3H]methyl incorporation as compared to the reaction containing no viral mRNA. By 20 min the rate of ['H]methyl incorporation had decreased and paralleled the endogenous rate, although protein synthesis continued at a maximum rate for at least another hour. Addition of SAdoHcy at a concentration 8-fold greater than SAdoMet reduced
TIME (minutes)
[3Hjmethyl incoporation to a level less than that observed in the absence of mRNA (Fig. 3a). 32P-Labeled reovirus mRNA was recovered from the incubation mixtures by phenol extraction and analyzed by centrifugation in glycerol gradients as described in the Materials and Methods section. The mRNA that had been incubated for 20 min in wheat germ extracts in the presence of SAdo[methyl-3H]Met was 3H-labeled in both the m and s mRNA classes (Fig. 3c). In addition, there was a peak of 3H migrating near the top of the gradient which was also present in wheat germ RNA isolated from the control incubation mixture (Fig. 3c). The viral RNA isolated from the SAdoHcycontaining incubation mixture had little or no 3H under the m and s mRNA peaks (Fig. 3d). Similar results were obtained when VSV 12-18S mRNA synthesized in vitro was used as a template for protein syn-
FRACTION NUMBER
FIG. 4. Methylation of VSV 12-18S mRNA by extracts of wheat germ. VSV mRNA synthesized in vitro without SAdoMet in the presence of [a-32P] GTP was used to stimulate protein synthesis in a 125 M1 reaction mixture in the presence of 4MAM SAdo [methyl-3H] Met. (a) Aliquots of 5 MAl were used to determine the [3H]methyl incorporation into acid-precipitable material as described in Materials and Methods. Incorporation of [3H]methyl without added VSV mRNA, O-O; in the presence of mRNA, *--*; and with mRNA plus 320 MuM SAdoHcy, A A. (b) Glycerol density gradient analysis of VSV mRNA in a 10 Ml aliquot after 20 min of incubation under conditions of translation: [3H]methyl incorporated after 20 min in the presence (0- - -0) and absence (A-A) of VSV [32P]mRNA (0). Radioactivity was measured after acid precipitation.
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Biochemistry: Both et al. 3'
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FIG. 5. Effect of sparsomycin and aurintricarboxylic acid on methylation of reovirus mRNA by wheat germ extracts. Reovirus mRNA synthesized in vitro in the presence of 10 pM SAdoHcy was translated in the presence of SAdo[methyl-'H]Met and .50 jiM ATA or 0.5 uM sparsomycin. [3H]methyl incorporation into acid-precipitable material recovered after phenol extraction of a 15 ,ul aliquot of incubation mixture is shown. [3H]methyl A; in the incorporation in the absence of added RNA, A ; and in the presence presence of reovirus mRNA alone, *of mRNA plus sparsomycin (0- -0) or ATA (A--A). -
thesis. Fig. 4a shows that in the presence of VSV mRNA [3H ]methyl incorporation into acid-precipitable material after 20 min was increased almost 5-fold over that observed in the absence of added mRNA. Again the addition of SAdoHcy reduced [3H]methyl incorporation to less than the endogenous level. Analysis of the VSV mRNA by glycerol gradient sedimentation (Fig. 4b) showed that [3H ]methyl was present in the VSV 12-18S mRNA as well in material near the top of the gradient, which was also present in the products from the endogenous reaction, as noted for reovirus mRNA (Fig. 3). We observed that the VSV mRNA was slightly degraded after 20 min incubation and this probably accounts for the trailing of 3H radioactivity through fractions 18 to 21. Dependence of methylation on initiation of protein synthesis The results indicate that the translation of VSV and reovirus mRNAs in wheat germ extracts requires methylation of the viral mRNA. In order to determine if mRNA methylation is dependent upon translation, two inhibitors of protein synthesis, aurintricarboxylic acid (ATA) and sparsomycin, were tested for their effects on mRNA methylation by wheat germ extracts. At low concentrations, ATA specifically inhibits the initiation of protein synthesis at the level of ribosome binding (19), while sparsomycin inhibits polypeptide chain elongation (20). At concentrations of 5, 20, and 50 uM ATA, reovirus
mRNA-directed incorporation of [3S]methionine into acidprecipitable material in wheat germ extracts was inhibited by 69, 95, and 99.5%, respectively. Similarly, concentrations of 0.5 and 5 ,uM sparsomycin inhibited protein synthesis by 98 and 99.5%, respectively. Examination, by NaDodSO4polyacrylamide gel electrophoresis, of the small amount of [35S]methionine-containing polypeptides synthesized in the presence of 0.5 jiM sparsomycin showed that only low-molecular-weight peptides were present; i.e., polypeptide chain elongation was limited, but initiation of protein synthesis had occurred. Reovirus mRNA was incubated in wheat germ extracts in the presence of SAdo[methyl-3H]Met and 0.5 jIAM
sparsomycin. As shown in Fig. 5, [3H]methyl incorporation into acid-precipitable material was unaffected by the inhibitor of chain elongation. In contrast, in the presence of 50 ,gM ATA, which blocks initiation of protein synthesis completely, [3H]methyl incorporation was reduced to a level less than that seen in the absence of added reovirus mRNA. Furthermore, when the mRNA was recovered by phenol extraction after 20 min of incubation and analyzed by centrifugation in glycerol density gradients, the [3H methyl incorporated in the control and sparsomycin reaction mixtures was associated with the m and s mRNA classes, a result similar to that shown in Fig. 3c. No [3H]methyl was present in these peaks when mRNA was analyzed after incubation in the presence of ATA. The results strongly suggest that methylation of reovirus mRNA in wheat germ extracts is dependent upon the initiation of protein synthesis. DISCUSSION
The results indicate that in extracts of wheat germ cell-free protein synthesis directed by VSV and reovirus mRNA is dependent on methylation of the viral mRNAs. Although the translation of animal virus mRNA in a plant cell extract represents a heterologous translation system, the polypeptides synthesized in vitro include authentic viral proteins (17, 18, 27). In addition, extracts prepared from mouse L cells, which are permissive for the replication of reovirus and VSV, are capable of methylating reovirus mRNA (G. W. Both, S. Muthukrishnan, and A. J. Shatkin, unpublished results). It is, therefore, unlikely that the methylation-dependent translation of viral mRNAs in wheat germ extracts is an artifact due to the disparity of the components of the in vitro system. The enzyme(s) responsible for viral mRNA methylation is apparently present in uninfected L cells and in wheat germ. mRNA methylating activities have been described previously in association with the virion RNA polymerases found in cytoplasmic polyhedrosis virus (2), reovirus (3), vaccinia virus (4, 5), and VSV (6). It is not clear if the virion-associated activities represent unique viral enzymes or cellular constituents packaged during virus maturation. However, the wheat germ and virion methylase(s) both methylate the viral mRNA with great specificity. The 5' end is-the site of mRNA methylation in each case, and 5'-terminal, 3H-labeled 7methylguanosine is obtained from VSV and reovirus mRNA methylated during transcription (7, 12) or during protein synthesis by wheat germ extracts in the presence of SAdo[methyl-3H]Met. However, a 2'--methylated penultimate nucleoside which is found in mRNA methylated during transcription is lacking in mRNA methylated during translation, suggesting that only the 5'-terminal 7-methylguanosine is necessary for efficient translation (S. Muthukrishnan, G. W. Both, Y. Furuichi, and A. J. Shatkin, manuscript in preparation). On the basis of the specific activity of the SAdo [methyl3H]Met, it appears that approximately 18% and 36% of the termini of the reovirus and VSV mRNAs, respectively, are methylated after 20 mm incubation. The concomitant inhibition of viral mRNA methylation and translation by ATA is consistent with an interdependence of the two processes at the initiation step of polypeptide synthesis. The inhibitor blocks protein synthesis at the level of ribosome binding (19), suggesting that in wheat germ extracts the mRNA methylating activity may be ribosome-
Proc. Nat. Acad. Sci. USA 72
(1975)
bound. It will be of interest to test ribosomes and supernatant fractions for mRNA methylase activity. It seems possible that one or more of the known initiation factors (21) may correspond to an mRNA methylase. Reovirus mRNA synthesized in vitro by the virion polymerase in the presence of SAdoMet contains 5'm7GpppGmCp and in the absence of a methyl group donor, ppG-Cp (7). The m7Gp is derived from GTP, apparently by a "capping process" (14). If cell-free extracts can convert viral mRNAs with unblocked, unmethylated 5' termini to chains containing 5'-terminal m7Gp, it may explain, at least in part, the GTP requirement for initiation of polypeptide chains (21). Antibodies have been prepared against many methylated nucleosides including 7-methylguanosine (22). Reovirus and cytoplasmic polyhedrosis virus mRNA methylated during transcription in the presence of SAdoMet can be differentiated from their unmethylated counterparts by employing antibodies for specific immunoprecipitation (A. J. Shatkin, Y. Furuichi, T. Sawicki, and P. R. Srinivasan, unpublished results). Many RNAs in addition to the virion transcription products contain 5'-terminal 7-methylguanosine, including simian virus 40 specific mRNA (11) and RNA from uninfected cells of Novikoff hepatoma (13), mouse L (14), HeLa (Y. Furuichi, M. M. Morgan, A. J. Shatkin, and J. E. Darnell, unpublished results), and BSC-1 monkey (11) cells. Hemoglobin mRNA is resistant to 5' labeling by polynucleotide kinase (R. Williamson, personal communication) and also contains 5'-terminal 7-methylguanosine (S. Muthukrishnan, G. W. Both, Y. Furuichi, and A. J. Shatkin, unpublished results). The possible effects of methylation on the messenger activity of these RNAs should be tested. Rous sarcoma virus genome RNA isolated from purified virions contains unphosphorylated 5' termini (23) and is very inefficient as a messenger in the wheat germ cell-free protein synthesizing system (G. Both, unpublished results). However, RNA similar in sequence to virion RNA apparently functions as mRNA in cells infected with this sarcoma virus (24). It will be of interest to examine the 5' structure of polysome-associated viral RNA and to determine if the presence of SAdoMet will alter the in vitro messenger activity of Rous sarcoma virus RNA. Finally, a well-known phenomenon is the "activation" of pre-formed, dormant mRNA that occurs in oocytes upon fertilization (25, 26). A possibility is that activation of the mRNA requires its methylation.
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We wish to thank Alba LaFiandra, Maureen Morgan, and Dennis Rhodes for excellent technical assistance and S. Muthukrishnan and Y. Furuichi for enthusiastic encouragement and
discussion. 1. Shatkin, A. J. (1974) Annu. Rev. Biochem. 43, 643-665. 2. Furuichi, Y. (1974) Nucl. Acid. Res. 1, 809-822. 3. Shatkin, A. J. (1974) Proc. Nat. Acad. Sci. USA 71, 32043207. 4. Wei, C. W. & Moss, B. (1974) Proc. Nat. Acad. Sci. USA 71, 3014-3018. 5. Urushibara, T., Furuichi, Y., Nishimura, T. & Miura, K.-I. (1975) FEBS Letters 49, 385-389. 6. Rhodes, D. P., Moyer, S. A. & Banerjee, A. K. (1974) Cell 3, 327-333. 7. Furuichi, Y., Morgan, M., Muthukrishnan, S. & Shatkin, A. J. (1975) Proc. Nat. Acad. Sci. USA- 72, 362-366. 8. Wei, C. M. & Moss, B. (1975) Proc. Nat. Acad. Sci. USA 72, 318-322. 9. Furuichi, Y. & Miura, K.-I. (1975) Nature 253, 374-375. 10. Furuichi, Y., Muthukrishnan, S. & Shatkin, A. J. (1975) Proc. Nat. Acad. Sci. USA 72, 742-743. 11. Lavi, S. & Shatkin, A. J. (1975) Fed. Proc. 34, 526-Abstr. 12. Abraham, G., Moyer, S. A., Adler, R. & Banerjee, A. K. (1975) Fed. Proc. 34, 708-Abstr. 13. Reddy, T., Ro-Choi, T. S., Henning, D. & Busch, H. (1974) J. Biol. Chem. 249, 6486-6494. 14. Rottman, F., Shatkin, A. J. & Perry, R. P. (1974) Cell 3, 197-199. 15. Banerjee, A. K. & Shatkin, A. J. (1970) J. Virol. 6, 1-11. 16. Moyer, S. A. & Banerjee, A. K. (1975) Cell 4, 37-43. 17. Both, G. W., Lavi, S. & Shatkin, A. J. (1975) Cell 4, 173180. 18. Both, G. W., Moyer, S. A. & Banerjee, A. K. (1975) Proc. Nat. Acad. Sci. USA 72, 274-278. 19. Marcus, A., Bewley, J. D. & Weeks, D. P. (1970) Science 167, 1735-1736. 20. Smith, A. E. & Wigle, D. T. (1973) Eur. J. Biochem. 35, 566-573. 21. Haselkorn, R. & Rothman-Denes, L. B. (1973) Annu. Rev. Biochem. 42, 397-438. 22. Levin, L. & Gjika, H. (1974) Arch. Biochem. Biophys. 164, 583-589. 23. Silber, R., Malathi, V. G., Schulman, L. H., Hurwitz, J. & Duesberg, P. H. (1973) Biochem. Biophys. Res. Commun. 50, 467-472. 24. Leong, J. -A., Garapin, A.-C., Jackson, N., Fanshier, L., Levinson, W. & Bishop, J. M. (1972) J. Virol. 9, 891-902. 25. Crippa, M., Davidson, E. H. & Mirsky, A. E. (1967) Proc. Nat. Acad. Sci. USA 57, 885-892. 26. Slater, D. W. & Spiegelman, S. (1966) Biochemistry 56, 164-170. 27. Both, G. W., Moyer, S. A. & Banerjee, A. K. (1975) J. Virol. 15, 1012-1019.
Methylation-Dependent Translation of Viral Messenger RNAs In Vitro (S-adenosylmethionine/wheat germ/vesicular stomatitis virus/reovirus)
G. W. BOTH, A. K. BANERJEE, AND A. J. SHATKIN Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110
Communicated by Severo Ochoa, January 10, 1975 ABSTRACT Methylated reovirus and vesicular stomatitis virus mRNAs, synthesized in vitro in the presence of S-adenosylmethionine by the virion-associated polymerases (RNA nucleotidyltransferases, EC 2.7.7.6), stimulate protein synthesis by wheat germ extracts to a greater extent than unmethylated mRNAs. Addition of S-adenosylmethionine to a cell-free extract programmed with unmethylated mRNA stimulates protein synthesis and results in methylation of the mRNA. An inhibitor of mRNA methylation, S-adenosylhomocysteine, blocks translation of unmethylated, but not of methylated, mRNAs. Aurintricarboxylic acid, which inhibits polypeptide chain initiation, also prevents mRNA methylation by wheat germ extracts. In contrast, sparsomycin, which inhibits polypeptide chain elongation, does not reduce mRNA methylation. The results indicate that methylation of viral mRNA is required for translation in vitro and suggest that mRNA methylation occurs at the initiation step of protein synthesis.
Many animal viruses contain an RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) that transcribes the viral genome and produces RNA that functions as messenger in infected cells and in cell-free systems (1). Recently, it was reported that purified cytoplasmic polyhedrosis virus (2), reovirus (3), vaccinia virus (4, 5), and vesicular stomatitis virus (VSV) (6) also have an RNA methylase activity. Viral RNA synthesized in vitro in the presence of the methyl group donor S-adenosylmethionine (SAdoMet) is specifically methylated at the 5' end, resulting in molecules with 5'terminal structures of the type m'G(5')ppp(5')Gm... and m7G(5')ppp(5')Am ... (refs. 7-9; G. Abraham, D. P. Rhodes, and A. K. Banerjee, unpublished results). Similar 5' structures have been found in virus-specific mRNAs isolated from cells infected with reovirus (10), simian virus 40 (11), and VSV (12) and in RNA from uninfected cells including Novikoff hepatoma (13), mouse L (14); HeLa (Y. Furuichi, M. M. Morgan, A. J. Shatkin, and J. E. Iarnell, unpublished results), and monkey BSC-1 cells (11). One possible effect of 5'-terminal methylation might be to alter the translational capacity of mRNA. To test this possibility, methylated and unmethylated RNAs synthesized in vitro by purified reovirus and VSV were compared as messengers in a cell-free protein-synthesizing system prepared from wheat germ. The results indicate that methylation of mRNA is required for its translation. MATERIALS AND METHODS
Growth and Purification of Viruses. Reovirus type 3 Dearing strain and VSV (Indiana serotype) were purified from inAbbreviations: VSV, vesicular stomatitis virus; SAdoMet, Sadenosylmethionine; BHK, baby hamster kidney; NaDodSO4, sodium dodecylsulfate; SAdoHcy, S-adenosylhomocysteine; ATA, aurintricarboxylic acid. 1189
fected mouse L cells (15) and baby hamster kidney (BHK) cells (16), respectively.
Synthesis and Purification of Viral mRNAs. The synthesis of reovirus mRNA by purified virus and its isolation by filtration through Sephadex G-50 and sedimentation in glycerol density gradients have been described (17). When methylated mRNA was required, 10 /uM SAdoIet was included in the transcription reaction mixture. VSV mRNA was synthesized as previously described (16) and purified by passage through Sephadex G-100 (6). VSV 12-18S mRNA was isolated by ethanol precipitation after sedimentation in a glycerol gradient (18). Methylated VSV mRNA was synthesized in the presence of 6.8 ,M SAdoMet (6). Translation of Viral mRNAs. Preparation of the wheat germ extract and the conditions for protein synthesis have been described (17, 18). Reovirus and VSV mRNAs were used at concentrations of 100 and 40 Aug/ml, respectively, in a total incubation volume of 12.5,u. Protein synthesis in the presence of L-[5S]methionine (specific activity 200-400 Ci/mmol) was assayed by heating a 1 MuI aliquot of incubation mixture in 2 ml of 10% trichloroacetic acid containing 1% casamino acids (Difco, Detroit, Mich.) at 950 for 15 min. The samples were chilled in ice, collected on Millipore filters, and washed with 5 ml of trichloroacetic acid/casamino acids and 5 ml of 1% acetic acid. The filters were dried and their radioactivity was measured in a toluene-based scintillant.
Methylation of Viral mRNAs by Cell-Free Extracts. Reovirus and VSV mRNAs were translated in the presence of unlabeled methionine and S-adenosyl [methyl-3H ]methionine (specific activity 7.3 Ci/mmol; 500 4s0i/ml; New England Nuclear, Boston, Mass.) at concentrations of 2 Au respectively. The SAdoMet was neutralized with 2 AL unbuffered Trizma (Sigma, St. Louis, Mo.) immediately before use. An aliquot of reaction mixture was diluted with 0.5 ml of buffer [10 mM Tris-HCl, p'i 7.5, 100 mMI NaCl, 5 mM EDTA, 0.5% sodium dodecylsulfate (NaDodSO4)] and extracted with an equal volume of redistilled phenol. The aqueous layer was retained and the phenol layer was re-extracted with 0.5 ml of buffer. The aqueous layers were combined and the RNA was precipitated by the addition of two volumes of ethanol in the presence of 0.3 AlI NaCl before glycerol gradient analysis. Alternatively, the RNA was precipitated with 10% trichloroacetic acid, collected on Aiillipore filters and analyzed for radioactivity. VSV and reovirus mRNAs were sedimented on 5-30% (w/v) glycerol density gradients (17) in the Beckman SW41 rotor at 40 for 17 hr at 33,000 and 28,000 rpm, respectively.
1190
Biochemistry: Both et al.
Proc. Nat. Acad. Sci. USA 72 (1976)
.-IO
160
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o -80 Li50
00
200 6 K
K
V
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in 25 0
/40
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TIME (minutes)
FIG. 1. Effect of methylation of mRNA on protein synthesis
in vitro. VSV and reovirus mRNAs synthesized in the presence and absence of SAdoMet and poly(A)-containing mRNA isolated from VSV-infected BHK cells (27) were used to stimulate protein synthesis in cell-free extracts of wheat germ. [35S]Methionine incorporated into acid-precipitable material in response to: (a) A- - -A, no RNA; 0----0, poly(A)-containing RNA from VSVinfected BHK cells; VSV mRNA synthesized in vitro in the absence (A-A) and in the presence (@.-L ) of SAdoMet; and (b) A- - -A, no RNA and reovirus mRNA synthesized in vitro in the absence (0-0I) and presence (@ -) of SAdoMet. RESULTS Effect of inethylation on mRNA activity Cell-free extracts of wheat germ are capable of translating
both reovirus and VSV mRNAs with fidelity (17, 18, 27). Therefore the effect of methylation on mRNA activity was studied using this cell-free system. VSV mRNA synthesized in vitro in the presence and absence of SAdoMet was purified and translated in wheat germ extracts as described previously (18). Methylated mRNA stimulated [85S]methionine incorporation into acid-precipitable polypeptide products to a greater extent than an equal amount of unmethylated mRNA (Fig. la); the difference was maximal after incubation for 20 min. In addition, the kinetics of protein synthesis obtained with poly(A)-containing VSV mRNA isolated from infected BHK-21 cells were similar to those observed with mRNA methylated in vitro (Fig. la). Analysis of the polypeptide products by NaDodSO4-polyacrylamide slab gel electrophoresis (18) showed that the same relative amounts of the previously described viral proteins, P63, N, NS, and M, were synthesized in response to both methylated and unmethylated VSV mRNA (ref. 18, and data not shown). Similarly, reovirus mRNA synthesized in the presence of SAdoMet was also a better template for protein synthesis than mRNA made in its absence (Fig. lb). A maximum difference of almost 5fold in the incorporation of [5S ]methionine was observed after 20 min incubation. The relative amounts of the polypeptide products synthesized in response to methylated or unmethylated reovirus mRNA were similar, and the polypeptides synthesized included those described previously (17). Dependence of protein synthesis on methylation Since methylated mRNA was more efficient in directing protein synthesis in vitro, it was of interest to determine whether the activity of unmethylated mRNA was increased in the presence of a methyl group donor. As shown in Fig. 2a, VSV mRNA made by the virion-associated polymerase in the absence of SAdoMet directed the incorporation of [35S]methi-
0
20
40
60-
80
0 20 TIME (minutes)
40
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FIG. 2. Dependence of in vitro protein synthesis on methylation. (a) VSV RNA synthesized in vitro in the absence of SAdoMet was used as mRNA. Protein synthesis was assayed in the absence (0---O) and in the presence (--) of 4MuM SAdoMet or in the presence of 4 MM SAdoMet plus 320 MAM SAdoHcy (-----A). A- A represents the protein synthesis observed in the absence of added RNA. (b) VSV RNA synthesized in vitro in the presence of SAdoMet was used as mRNA. Protein synthesis directed by this pre-methylated RNA was assayed in the ) of 320 AM absence (0-O) and in the presence (@SAdoHcy. A -A represents protein synthesis without added RNA and A -A protein synthesis directed by unmethylated VSV mRNA.
onine in wheat germ extracts. However, when SAdoMet was added to the translation reaction mixture, both the rate and extent of protein synthesis were stimulated several-fold. Optimum stimulation of translation by SAdoMet occurred at concentrations between 2 and 8 MM and the products, analyzed by gel electrophoresis, were the same as with mRNA that had been methylated during transcription. The SAdoMet analog, S-adenosylhomocysteine (SAdoHcy) has been shown to inhibit methylation of reovirus and VSV mRNA during in vitro transcription (3, 6). Addition of SAdoHcy to wheat germ extracts at an 80-fold higher concentration than SAdoMet also inhibited protein synthesis more than 95%, suggesting that translation of VSV mRNA is completely dependent upon methylation. However, in order to eliminate the possibility that this inhibition was due to some nonspecific effect of SAdoHcy on protein synthesis, the activity of mRNA that was methylated during synthesis in vitro (pre-methylated mRNA) was determined in the cell-free system in the presence and absence of SAdoHcy. In contrast to unmethylated mRNA, translation of pre-methylated VSV mRNA was undiminished by SAdoHcy (Fig. 2b). The results, therefore, indicate that protein synthesis is dependent upon methylation of the viral mRNA and that wheat germ extracts contain an mRNA methylase activity. Furthermore, because translation of unmethylated mRNA occurs in the absence of exogenous SAdoMet, it appears that wheat germ extracts also contain an endogenous methyl group donor. However, it should be noted that even when SAdoMet is present during translation, unmethylated RNA is a less effective messenger than premethylated mRNA (compare Fig. 2a and b). The results obtained with VSV mRNA (Fig. 2) were confirmed with reovirus RNA (data not shown). Methylation of viral mRNA by wheat germ extracts In order to demonstrate directly the methylation of viral
mRNA by wheat germ extracts, 32P-labeled reovirus mRNA
Proc. Nat. Acad. Sci. USA 72
Methylation-Dependent Translation
(1975) (min)
TIME
5
C 4
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1191
15
.I
45
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I
I
-
.0A
I
I
II
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-
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2
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I
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I
0O 5
10
IS
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5 FRACTION NUMBER 0
10
15
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25
30
FIG. 3. Methylation of reovirus mRNA by extracts of wheat germ. Reovirus mRNA synthesized in the presence of 10 MAM SAdoHcy and [a-32P]CTP was used to stimulate protein synthesis in a 200 ;Ml reaction mixture in the presence of unlabeled methionine and 2 gM SAdo [methyl-3H] Met. The mRNA was recovered from (a) 10 MAl and (b-d) 30 MIA aliquots by phenol extraction as described in the Materials and Methods section. (a) Incorporation of 3H radioactivity into acid-precipitable material in the absence of (O---O) of and in the presence (@-@) of reovirus mRNA and in the presence of mRNA plus 160 MM SAdoHcy A--A. Panels b-d show glycerol density gradient profiles of reovirus mRNA after (b) 0 and (c) 20 min of incubation under conditions of translation and (d) after 20 min incubation in the presence of 160 M&M SAdoHcy: O-O, 3H, 0 * 32P. In (c) t-represents [3H]methyl incorporation in the absence of added RNA. In (b-d), radioactivity was measured directly after mixing the fractions with Aquasol. Sedimentation is from right to left.
was used to direct protein synthesis. Reovirus mRNA was synthesized in vitro in the presence of 10 MM SAdoHcy in order to reduce the number of methylated 5' termini in the mRNA (3). Under these conditions, the products consisted of the small (s) and the medium (m) classes of mRNA (see Fig. 3b). When the 32P-labeled reovirus mRNA was translated in wheat germ extracts in the presence of SAdo[methyl-PH]Met, there was a rapid incorporation of [3Hjmethyl into acidprecipitable material (Fig. 3a). The reaction was essentially complete after 10 min, when there was a 7-fold stimulation in the extent of [3H]methyl incorporation as compared to the reaction containing no viral mRNA. By 20 min the rate of ['H]methyl incorporation had decreased and paralleled the endogenous rate, although protein synthesis continued at a maximum rate for at least another hour. Addition of SAdoHcy at a concentration 8-fold greater than SAdoMet reduced
TIME (minutes)
[3Hjmethyl incoporation to a level less than that observed in the absence of mRNA (Fig. 3a). 32P-Labeled reovirus mRNA was recovered from the incubation mixtures by phenol extraction and analyzed by centrifugation in glycerol gradients as described in the Materials and Methods section. The mRNA that had been incubated for 20 min in wheat germ extracts in the presence of SAdo[methyl-3H]Met was 3H-labeled in both the m and s mRNA classes (Fig. 3c). In addition, there was a peak of 3H migrating near the top of the gradient which was also present in wheat germ RNA isolated from the control incubation mixture (Fig. 3c). The viral RNA isolated from the SAdoHcycontaining incubation mixture had little or no 3H under the m and s mRNA peaks (Fig. 3d). Similar results were obtained when VSV 12-18S mRNA synthesized in vitro was used as a template for protein syn-
FRACTION NUMBER
FIG. 4. Methylation of VSV 12-18S mRNA by extracts of wheat germ. VSV mRNA synthesized in vitro without SAdoMet in the presence of [a-32P] GTP was used to stimulate protein synthesis in a 125 M1 reaction mixture in the presence of 4MAM SAdo [methyl-3H] Met. (a) Aliquots of 5 MAl were used to determine the [3H]methyl incorporation into acid-precipitable material as described in Materials and Methods. Incorporation of [3H]methyl without added VSV mRNA, O-O; in the presence of mRNA, *--*; and with mRNA plus 320 MuM SAdoHcy, A A. (b) Glycerol density gradient analysis of VSV mRNA in a 10 Ml aliquot after 20 min of incubation under conditions of translation: [3H]methyl incorporated after 20 min in the presence (0- - -0) and absence (A-A) of VSV [32P]mRNA (0). Radioactivity was measured after acid precipitation.
1192
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Both et al. 3'
)
)
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ICD)
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FIG. 5. Effect of sparsomycin and aurintricarboxylic acid on methylation of reovirus mRNA by wheat germ extracts. Reovirus mRNA synthesized in vitro in the presence of 10 pM SAdoHcy was translated in the presence of SAdo[methyl-'H]Met and .50 jiM ATA or 0.5 uM sparsomycin. [3H]methyl incorporation into acid-precipitable material recovered after phenol extraction of a 15 ,ul aliquot of incubation mixture is shown. [3H]methyl A; in the incorporation in the absence of added RNA, A ; and in the presence presence of reovirus mRNA alone, *of mRNA plus sparsomycin (0- -0) or ATA (A--A). -
thesis. Fig. 4a shows that in the presence of VSV mRNA [3H ]methyl incorporation into acid-precipitable material after 20 min was increased almost 5-fold over that observed in the absence of added mRNA. Again the addition of SAdoHcy reduced [3H]methyl incorporation to less than the endogenous level. Analysis of the VSV mRNA by glycerol gradient sedimentation (Fig. 4b) showed that [3H ]methyl was present in the VSV 12-18S mRNA as well in material near the top of the gradient, which was also present in the products from the endogenous reaction, as noted for reovirus mRNA (Fig. 3). We observed that the VSV mRNA was slightly degraded after 20 min incubation and this probably accounts for the trailing of 3H radioactivity through fractions 18 to 21. Dependence of methylation on initiation of protein synthesis The results indicate that the translation of VSV and reovirus mRNAs in wheat germ extracts requires methylation of the viral mRNA. In order to determine if mRNA methylation is dependent upon translation, two inhibitors of protein synthesis, aurintricarboxylic acid (ATA) and sparsomycin, were tested for their effects on mRNA methylation by wheat germ extracts. At low concentrations, ATA specifically inhibits the initiation of protein synthesis at the level of ribosome binding (19), while sparsomycin inhibits polypeptide chain elongation (20). At concentrations of 5, 20, and 50 uM ATA, reovirus
mRNA-directed incorporation of [3S]methionine into acidprecipitable material in wheat germ extracts was inhibited by 69, 95, and 99.5%, respectively. Similarly, concentrations of 0.5 and 5 ,uM sparsomycin inhibited protein synthesis by 98 and 99.5%, respectively. Examination, by NaDodSO4polyacrylamide gel electrophoresis, of the small amount of [35S]methionine-containing polypeptides synthesized in the presence of 0.5 jiM sparsomycin showed that only low-molecular-weight peptides were present; i.e., polypeptide chain elongation was limited, but initiation of protein synthesis had occurred. Reovirus mRNA was incubated in wheat germ extracts in the presence of SAdo[methyl-3H]Met and 0.5 jIAM
sparsomycin. As shown in Fig. 5, [3H]methyl incorporation into acid-precipitable material was unaffected by the inhibitor of chain elongation. In contrast, in the presence of 50 ,gM ATA, which blocks initiation of protein synthesis completely, [3H]methyl incorporation was reduced to a level less than that seen in the absence of added reovirus mRNA. Furthermore, when the mRNA was recovered by phenol extraction after 20 min of incubation and analyzed by centrifugation in glycerol density gradients, the [3H methyl incorporated in the control and sparsomycin reaction mixtures was associated with the m and s mRNA classes, a result similar to that shown in Fig. 3c. No [3H]methyl was present in these peaks when mRNA was analyzed after incubation in the presence of ATA. The results strongly suggest that methylation of reovirus mRNA in wheat germ extracts is dependent upon the initiation of protein synthesis. DISCUSSION
The results indicate that in extracts of wheat germ cell-free protein synthesis directed by VSV and reovirus mRNA is dependent on methylation of the viral mRNAs. Although the translation of animal virus mRNA in a plant cell extract represents a heterologous translation system, the polypeptides synthesized in vitro include authentic viral proteins (17, 18, 27). In addition, extracts prepared from mouse L cells, which are permissive for the replication of reovirus and VSV, are capable of methylating reovirus mRNA (G. W. Both, S. Muthukrishnan, and A. J. Shatkin, unpublished results). It is, therefore, unlikely that the methylation-dependent translation of viral mRNAs in wheat germ extracts is an artifact due to the disparity of the components of the in vitro system. The enzyme(s) responsible for viral mRNA methylation is apparently present in uninfected L cells and in wheat germ. mRNA methylating activities have been described previously in association with the virion RNA polymerases found in cytoplasmic polyhedrosis virus (2), reovirus (3), vaccinia virus (4, 5), and VSV (6). It is not clear if the virion-associated activities represent unique viral enzymes or cellular constituents packaged during virus maturation. However, the wheat germ and virion methylase(s) both methylate the viral mRNA with great specificity. The 5' end is-the site of mRNA methylation in each case, and 5'-terminal, 3H-labeled 7methylguanosine is obtained from VSV and reovirus mRNA methylated during transcription (7, 12) or during protein synthesis by wheat germ extracts in the presence of SAdo[methyl-3H]Met. However, a 2'--methylated penultimate nucleoside which is found in mRNA methylated during transcription is lacking in mRNA methylated during translation, suggesting that only the 5'-terminal 7-methylguanosine is necessary for efficient translation (S. Muthukrishnan, G. W. Both, Y. Furuichi, and A. J. Shatkin, manuscript in preparation). On the basis of the specific activity of the SAdo [methyl3H]Met, it appears that approximately 18% and 36% of the termini of the reovirus and VSV mRNAs, respectively, are methylated after 20 mm incubation. The concomitant inhibition of viral mRNA methylation and translation by ATA is consistent with an interdependence of the two processes at the initiation step of polypeptide synthesis. The inhibitor blocks protein synthesis at the level of ribosome binding (19), suggesting that in wheat germ extracts the mRNA methylating activity may be ribosome-
Proc. Nat. Acad. Sci. USA 72
(1975)
bound. It will be of interest to test ribosomes and supernatant fractions for mRNA methylase activity. It seems possible that one or more of the known initiation factors (21) may correspond to an mRNA methylase. Reovirus mRNA synthesized in vitro by the virion polymerase in the presence of SAdoMet contains 5'm7GpppGmCp and in the absence of a methyl group donor, ppG-Cp (7). The m7Gp is derived from GTP, apparently by a "capping process" (14). If cell-free extracts can convert viral mRNAs with unblocked, unmethylated 5' termini to chains containing 5'-terminal m7Gp, it may explain, at least in part, the GTP requirement for initiation of polypeptide chains (21). Antibodies have been prepared against many methylated nucleosides including 7-methylguanosine (22). Reovirus and cytoplasmic polyhedrosis virus mRNA methylated during transcription in the presence of SAdoMet can be differentiated from their unmethylated counterparts by employing antibodies for specific immunoprecipitation (A. J. Shatkin, Y. Furuichi, T. Sawicki, and P. R. Srinivasan, unpublished results). Many RNAs in addition to the virion transcription products contain 5'-terminal 7-methylguanosine, including simian virus 40 specific mRNA (11) and RNA from uninfected cells of Novikoff hepatoma (13), mouse L (14), HeLa (Y. Furuichi, M. M. Morgan, A. J. Shatkin, and J. E. Darnell, unpublished results), and BSC-1 monkey (11) cells. Hemoglobin mRNA is resistant to 5' labeling by polynucleotide kinase (R. Williamson, personal communication) and also contains 5'-terminal 7-methylguanosine (S. Muthukrishnan, G. W. Both, Y. Furuichi, and A. J. Shatkin, unpublished results). The possible effects of methylation on the messenger activity of these RNAs should be tested. Rous sarcoma virus genome RNA isolated from purified virions contains unphosphorylated 5' termini (23) and is very inefficient as a messenger in the wheat germ cell-free protein synthesizing system (G. Both, unpublished results). However, RNA similar in sequence to virion RNA apparently functions as mRNA in cells infected with this sarcoma virus (24). It will be of interest to examine the 5' structure of polysome-associated viral RNA and to determine if the presence of SAdoMet will alter the in vitro messenger activity of Rous sarcoma virus RNA. Finally, a well-known phenomenon is the "activation" of pre-formed, dormant mRNA that occurs in oocytes upon fertilization (25, 26). A possibility is that activation of the mRNA requires its methylation.
Methylation-Dependent Translation
1193
We wish to thank Alba LaFiandra, Maureen Morgan, and Dennis Rhodes for excellent technical assistance and S. Muthukrishnan and Y. Furuichi for enthusiastic encouragement and
discussion. 1. Shatkin, A. J. (1974) Annu. Rev. Biochem. 43, 643-665. 2. Furuichi, Y. (1974) Nucl. Acid. Res. 1, 809-822. 3. Shatkin, A. J. (1974) Proc. Nat. Acad. Sci. USA 71, 32043207. 4. Wei, C. W. & Moss, B. (1974) Proc. Nat. Acad. Sci. USA 71, 3014-3018. 5. Urushibara, T., Furuichi, Y., Nishimura, T. & Miura, K.-I. (1975) FEBS Letters 49, 385-389. 6. Rhodes, D. P., Moyer, S. A. & Banerjee, A. K. (1974) Cell 3, 327-333. 7. Furuichi, Y., Morgan, M., Muthukrishnan, S. & Shatkin, A. J. (1975) Proc. Nat. Acad. Sci. USA- 72, 362-366. 8. Wei, C. M. & Moss, B. (1975) Proc. Nat. Acad. Sci. USA 72, 318-322. 9. Furuichi, Y. & Miura, K.-I. (1975) Nature 253, 374-375. 10. Furuichi, Y., Muthukrishnan, S. & Shatkin, A. J. (1975) Proc. Nat. Acad. Sci. USA 72, 742-743. 11. Lavi, S. & Shatkin, A. J. (1975) Fed. Proc. 34, 526-Abstr. 12. Abraham, G., Moyer, S. A., Adler, R. & Banerjee, A. K. (1975) Fed. Proc. 34, 708-Abstr. 13. Reddy, T., Ro-Choi, T. S., Henning, D. & Busch, H. (1974) J. Biol. Chem. 249, 6486-6494. 14. Rottman, F., Shatkin, A. J. & Perry, R. P. (1974) Cell 3, 197-199. 15. Banerjee, A. K. & Shatkin, A. J. (1970) J. Virol. 6, 1-11. 16. Moyer, S. A. & Banerjee, A. K. (1975) Cell 4, 37-43. 17. Both, G. W., Lavi, S. & Shatkin, A. J. (1975) Cell 4, 173180. 18. Both, G. W., Moyer, S. A. & Banerjee, A. K. (1975) Proc. Nat. Acad. Sci. USA 72, 274-278. 19. Marcus, A., Bewley, J. D. & Weeks, D. P. (1970) Science 167, 1735-1736. 20. Smith, A. E. & Wigle, D. T. (1973) Eur. J. Biochem. 35, 566-573. 21. Haselkorn, R. & Rothman-Denes, L. B. (1973) Annu. Rev. Biochem. 42, 397-438. 22. Levin, L. & Gjika, H. (1974) Arch. Biochem. Biophys. 164, 583-589. 23. Silber, R., Malathi, V. G., Schulman, L. H., Hurwitz, J. & Duesberg, P. H. (1973) Biochem. Biophys. Res. Commun. 50, 467-472. 24. Leong, J. -A., Garapin, A.-C., Jackson, N., Fanshier, L., Levinson, W. & Bishop, J. M. (1972) J. Virol. 9, 891-902. 25. Crippa, M., Davidson, E. H. & Mirsky, A. E. (1967) Proc. Nat. Acad. Sci. USA 57, 885-892. 26. Slater, D. W. & Spiegelman, S. (1966) Biochemistry 56, 164-170. 27. Both, G. W., Moyer, S. A. & Banerjee, A. K. (1975) J. Virol. 15, 1012-1019.
