Relative Importance of 7-Methylguanosine in Ribosome Binding and Translation of Vesicular Stomatitis Virus mRNA in Wheat Germ and Reticulocyte Cell-free Systems *

Vesicular stomatitis virus mRNAs with these four types of 5’-termini, (a) m7G5’ppp5’(m)Am, (b) pp$‘(m)Am, (c) m7G5’pppS’Am, and Cd) G5’ppp5’A, were prepared and their translation and ribosome binding analyzed in wheat germ and reticulocyte cell-free protein synthesis systems. The relative efficiencies of translation of individual vesicular stomatitis virus (VSV) mRNAs having type 2 termini ranged from 23 to 29% of the control (type 1) RNA in the reticulocyte system and 6 to 7% of control RNA in the wheat germ system. A similar difference between the two systems was seen in ribosome-binding experiments in which type 2 RNA formed an 80 S initiation complex with high efficiency (70% of control type 1 RNA) in the reticulocyte system, but with low efficiency (17% of control RNA) in the wheat germ system. Similar differences in the importance of m7G in translation in the two systems were seen when VSV mRNAs synthesized in vitro with type 3 and type 4 termini were analyzed. However, the analysis of type 4 RNA (which was synthesized in uitro in the presence of S-adenosylhomocysteine) was complicated by the presence of abnormally large poly(A) at its 3’-end. Another series of experiments showed that compounds such as 5’pm7G and m7G”‘ppp5’Np are potent and specific inhibitors of translation of all types of VSV mRNAs in the wheat germ system (greater than 98% inhibition) but cause less than 20% inhibition of translation in the reticulocyte system. Taken together, all of the results indicate that a 5’-terminal m7G is far more important in translation of VSV mRNAs in the heterologous plant cell-free system than in the reticulocyte lysate system.

Vesicular stomatitis virus mRNAs with these four types of 5'-termini, (a) m7G5'ppp5'(m)Am, (b) pp$'(m)Am, (c) m7G5'-pppS'Am, and Cd) G5'ppp5'A, were prepared and their translation and ribosome binding analyzed in wheat germ and reticulocyte cell-free protein synthesis systems. The relative efficiencies of translation of individual vesicular stomatitis virus (VSV) mRNAs having type 2 termini ranged from 23 to 29% of the control (type 1) RNA in the reticulocyte system and 6 to 7% of control RNA in the wheat germ system. A similar difference between the two systems was seen in ribosome-binding experiments in which type 2 RNA formed an 80 S initiation complex with high efficiency (70% of control type 1 RNA) in the reticulocyte system, but with low efficiency (17% of control RNA) in the wheat germ system. Similar differences in the importance of m7G in translation in the two systems were seen when VSV mRNAs synthesized in vitro with type 3 and type 4 termini were analyzed. However, the analysis of type 4 RNA (which was synthesized in uitro in the presence of S-adenosylhomocysteine) was complicated by the presence of abnormally large poly(A) at its 3'-end. Another series of experiments showed that compounds such as 5'pm7G and m7G"'ppp5'Np are potent and specific inhibitors of translation of all types of VSV mRNAs in the wheat germ system (greater than 98% inhibition) but cause less than 20% inhibition of translation in the reticulocyte system. Taken together, all of the results indicate that a 5'-terminal m7G is far more important in translation of VSV mRNAs in the heterologous plant cell-free system than in the reticulocyte lysate system. 32P-labeled m'GpppAm was isolated from an RNase Pl digest of 32P-labeled VSV mRNA by electrophoresis at pH 3.5 on DEAE-paper, as described previously (22). Gel Electrophoresis and Radioautography -Pancreatic RNase (50 /Ig) was added to all reactions, followed by incubation at 37" for 5 min. Generally, 3 ~1 of the reaction were analyzed on a 13% polyacrylamide slab gel as detailed by Laemmli (27). The gels were fixed (281, dried, and subjected to radioautography with Kodak Royal Blue x-ray film. The films were scanned with a Joyce-Loebl microdensitometer; the areas under the peaks were determined with a K & E planimeter. In these cases, the time of exposure of the film was such that the absorbance of the most intense VSV band (generally the N protein) was less than 1.0 absorbance unit. Under these conditions, the absorbance of a band is proportional to the amount of radioactivity in the gel, as was shown by appropriate reconstruction experiments with [35Slmethionine-labe1ed VSV virions. (1 x lo+ M), other components described previously (26), and 1.5 pg of VSV mRNA or periodate-oxidized and p-eliminated VSV mRNA, as indicated. Incubation was at 30" for 60 min; 3 pl of the reaction was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the fixed, dried gel was exposed to Kodak Royal Blue x-ray film for 47 h. Globin has migrated off the bottom of the gel. 1, no additions; 2,97 pM pm'@ 3,250 ELM pm'@ 4,500 FM pm'G, 5, control VSV mRNA; 6, as 5, plus 97 PM pm'G, 7, as5, plus 250 PM pm'G, 8, as 5, plus 500 ELM pm'G, 9, periodate-oxidized, p-eliminated VSV mRNA; 10, as 9, plus 97 pM pm'G; 11, as 9, plus 250 FM pm7G, 12, as 9, plus 500 PM pm7G.

Translation
of Modified VSV mRNAs -Oxidation of VSV mRNA with periodate, followed by p elimination with aniline, yields the 5' terminus pppmAmpmAmpCp on all mRNA species (22). This structure has only partial base methylation of the terminal adenosine and partial base and ribose methylation of the second adenosine. For simplicity, we will indicate this structure with only the ribose methylation of the penultimate nucleotide.
Previously we showed that this RNA is translated by unfractionated reticulocyte lysate 25 to 30% as efficiently as is normal VW mRNA (5'-end m7G5'ppp5'AmpAp. . .) and that these extracts did not add an m7G residue to the RNA (22). Translation of control and peliminated RNA is shown in Fig. 1, lanes 5 and 9. Table I shows the relative amounts of the three major VSV proteins produced in a similar experiment. In wheat germ extracts, by contrast, periodate-oxidized and p-eliminated RNA is translated only 6% as efficiently as is control RNA (Table I; Fig. 2).
Somewhat similar results were obtained with VSV mRNA synthesized by virion transcriptase in the presence of AdoHcy (Table II). The 5'-end of this unmethylated RNA is GS'ppp5'ApAp, whereas that of RNA made in the presence of AdoMet is m7GS'ppp5'AmpAp.
. . (4, 12,241. While methylated RNA has a normal-sized sequence of about 200 adenylic acid residues (poly(A)) at the 3'-end, that made in the presence of AdoHcy has a heterogeneous poly(A) sequence with an average size of about 700 nucleotides, but otherwise the two prepa-rations of RNA are identical (24). In reticulocyte extracts, unmethylated RNA, which we will refer to as AdoHcy RNA, directs synthesis of 13% as much of the three predominant VSV proteins as does control methylated RNA (AdoMet RNA) (Table II). We do not know to what extents the lack of methylation and the very long poly(A1 contribute to the reduction in translation. Wheat germ extracts, by contrast, translate this RNA only 1 to 2% as efficiently as they do normal VSV RNA (Table II; Fig. 5).

Attachment
of Modified VSV mRNAs to Ribosomes -The above studies suggested that wheat germ ribosomes are much less efficient than reticulocyte ribosomes in their ability to translate VSV mRNAs lacking a Y-terminal m7G residue. These conclusions were corroborated by two types of experiments in which the binding of labeled mRNAs to ribosomes was followed. As was shown previously, a large fraction of control VSV mRNA is incorporated into polysomes following incubation in a reticulocyte lysate under conditions of protein synthesis (22). These polysomes contained, on the average, 3.7 ribosomes per mRNA ( Fig. 3; Table III). About half as much periodate-oxidized, P-eliminated RNA is incorporated into polysomes, and the attached mRNAs contain about 60% the number of ribosomes, on the average, as does control RNA ( Fig. 3; Table I). The variable decrease in the amount of peliminated RNA bound to ribosomes is due presumably to partial degradation of the RNA caused by the chemical treatment (22). In wheat germ extracts, by contrast, only 8% as much P-eliminated RNA as control RNA is incorporated into polysomes. Whereas the average size of polysomes formed by normal VSV mRNA in the two extracts is about the same, the size of polysomes formed by p-eliminated RNA is significantly 7-Methylguanosine and Translation of VSV mRNA Incubation was at 25" for 120 min. Two microliters of the reaction was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the fixed, dried gel was exposed to Kodak Royal Blue x-ray film for 15 days. 1, no additions; 2, plus 132 PM pm'G, 3, plus 672 PM pm7G, 4, plus Sindbis 26 S mRNA; 5, as 4, plus 132 FM pm'@ 6, as4, plus 672 PM pm'G; 7, control VSV mRNA; 8, as 7, plus 132 PM pm'@ 9, as 7, plus 672 PM pm'G; 10, periodateoxidized, P-eliminated VSV mRNA; 11, as 10, plus 132 PM pm'@ 12, as 10, plus 672 PM pm'G.
smaller in wheat germ extracts than in reticulocyte extracts ( Fig. 3; Table III). The ability of labeled mRNAs to participate in cell-free protein synthesis can also be determined if the reactions con- tain 1 mglml of anisomycin, a specific inhibitor of polypeptide chain elongation. Under these conditions, a large fraction of control VSV mRNA is incorporated into 80 S initiation complexes (Fig. 4;Ref. 22). These complexes remain stable in a solution of 0.5 M NaCl and 0.03 M magnesium acetate, conditions which will dissociate 40 S to 60 S ribosome couples which do not contain mRNA and initiator tRNA. Fig. 4, using different mRNA preparations from the experiment in Fig. 3, shows that the p-eliminated is bound to reticulocyte ribosomes to almost the same extent as is control VSV. Wheat germ ribosomes, by contrast, incorporate only 8% of the P-eliminated' RNA into monoribosomes. Taken together with results on cellfree protein synthesis (Figs. 1 and 2; Tables I and II), these results suggest that wheat germ ribosomes are far less efficient than reticulocyte ribosomes at binding to or initiating protein synthesis on VSV mRNA molecules lacking a terminal 5'-m7G residue. We showed previously that the rate of attachment of reticulocyte ribosomes to P-eliminated RNA molecules to form an 80 S initiation complex is about one-third that of control RNA (22). So little P-eliminated VSV mRNA binds to wheat germ ribosomes that we have not attempted to measure the rate of formation of the 80 S complex.
Inhibitors Containing pm7G-Hickey et al. showed that pm7G, when used at high concentrations, was an inhibitor of translation of reovirus mRNA, tobacco mosaic virus RNA, and globin mRNA by wheat germ extracts (18). All of these RNAs contain the Y-sequence m7G5'ppp5'NpmNp.
This compound also blocked binding of these mRNAs to wheat germ ribosomes (18). In contrast, translation of satellite tobacco necrosis virus RNA, which has the 5'-terminal sequence pppApGp or pp-ApGp, is not affected by pm7G. It was concluded that pm'G specifically blocks binding of the 5'-m7G5'ppp5'Nmp sequence on mRNA to a ribosome subunit or essential initiation factor. Figs. 5 and 6 and Table IV show that a similar result is obtained for translation of VSV mRNA by wheat germ extracts; pm7G, but not pG or Gp, is an inhibitor of translation of normal VSV mRNA. It is of interest that translation of AdoHcy RNA (5' end G5'ppp5'AmpAp) is also affected by pm7G, although less than is control RNA, 650 pM pm7G blocks translation of AdoMet RNA over 98%, but that of AdoHcy RNA only 62% (Fig. 5; Table IV). Fig. 2 shows that translation of periodate-oxidized, p-eliminated RNA in wheat germ ex- In control reactions (which were not incubated) only 2 to 2.5% of the radioactivity was found in the monosome or polysome regions of the gradient (data not shown). Analysis of data from this experiment are in Table III. tracts is inhibited by pm'G to the same extent as is that of normal RNA; densitometer scans of these radioautograms showed that a concentration of pm7G (0.1 mM) which inhibits translation of control RNA 96% inhibited translation of treated RNA over 90%.
Figs. 5 and 6 also illustrate that several other analogues of 5'-ends of mRNAs, m7G5'ppp"'G, m7G5'ppp5'Gm, m7G5'-ppp5'Am, are also potent inhibitors of translation of VSV mRNA by wheat germ extracts. Equivalent inhibition of VSV protein synthesis by these compounds is achieved at about one-fifth the concentration required of pm'G (Fig. 6). Similar compounds lacking an m7G residue, G5'ppp5'G and G5'ppp5'Gm, are essentially inactive as inhibitors (Fig. 5). m7GpppAm, we determined that half of the compound remains intact after 22 min of incubation in a reticulocyte lysate (Fig.  8), whereas no significant inhibition of translation of VSV mRNA is apparent after either 10 min (data not shown) or 20 min (Fig. 7) of incubation. The smaller peak of material seen in the unincubated sample (13 cm) is possibly m7G5'ppp5'(m)Am containing an m7G in the open ring form which is generated during elution of the compound from DEAE-paper with triethylamine bicarbonate.
By contrast, none of the aforementioned compounds have any discernible effect on translation of endogenous globin mRNA by reticulocyte lysates (data not shown). Neither do they have any significant effect on translation of exogenous VW mRNA by these lysates (Figs. 6 and 7). In particular, a concentration of pm'G or m7G5'ppp5'Am which inhibits translation of VSV mRNA by a wheat germ lysate over 90% inhibits translation in a reticulocyte extract less than 10% (Fig. 7). The absence of significant inhibition by m7GpppAm in the reticulocyte lysate is not due to its degradation. By adding 32P-labeled The most significant aspect of this work is that the effect on translation of a 5'-terminal m7G residue in VSV mRNA is much less pronounced in the reticulocyte lysate system than in the wheat germ system. It is important to emphasize that in both extracts a large fraction of VSV mRNA will bind to ribosomes and participate in protein synthesis (Figs. 3 and 4). However, because the translation of animal virus mRNAs is being investigated, the results obtained from the reticulocyte lysate system would presumably be most relevant to an understanding of the importance of m7G in translation of VSV mRNA in infected cells. Indeed, the rates of chain initiation and elongation on endogenous globin mRNA by reticulocyte  Reactions contained 1 mg/ml of anisomycin, a specific inhibitor of polypeptide chain elongation (25) but were otherwise identical with those of Fig. 3. However, different preparations of RNA were used. Incubation gradient centrifugation and counting of the samples was as in Fig. 3. In reactions not incubated, 2.1 to 2.6% of the 32P-labeled RNA was found to sediment faster than 40 S (data not shown). The percentage of added "2P-labeled mRNA bound to ribosomes after the incubation was a, control RNA, reticulocyte extract, 71%; b, periodate-oxidized, P-eliminated RNA, reticulocyte extract, 48%; c, control RNA, wheat germ extract, 48%; d, periodate-oxidized, p-eliminated wheat germ extract, 8%.  efficiency varying from 23 to 29% of control RNA (depending on the protein examined) in the reticulocyte system but only 5 to 7% of control RNA in the wheat germ system.
A second type of experiment which demonstrated the differences between the reticulocyte and wheat germ systems employed the RNA synthesized in vitro by the VSV virion transcriptase in its methylated (5' end m7G5'ppp5'Amp) and unmethylated form (5' end GS'ppp5'Ap). Tbe studies on these Inhibition of wheat germ protein synthesis by analogs of 5' ends of messenger RNA Reactions are described in the legend to Fig. 5, and a radioautogram of a gel analysis of the protein products is shown in Fig. 5. Tabulated here is the radioactivity incorporated into acid-precipitable polypeptides from a 5-~1 sample of the reaction after 120 min of incubation.
RNAs are complicated by the finding that the unmethylated RNA does not contain the correct RNA species because of the presence of large heterogeneous poly(A1 on all mRNA species (24). Translation of these RNAs again revealed a discrepancy between the two systems because the AdoHcy RNA was translated with an eficiency of 12% of AdoMet RNA in the reticulocyte system but with only 1 to 2% efficiency in the wheat germ system. Standard reactions for reticulocyte cell-free protein synthesis (50 ~1) were used (see Fig. 1). They contained 1 x 10m4 M each of 20 nonradioactive amino acids, no mRNA or radioactive amino acids, 130 /ILM unlabeled m7GpppAm, and 1800 cpm of :12P-labeled m'GpppAm.
After the indicated periods of incubation at 30", aliquots ___ of 10 ~1 were added to 40 ~1 of cold water and spotted at the origin of a sheet of Whatman No. 3MM paper. Electrophoresis was for 60 min at 20 V/cm. The strip was cut into l-cm wide pieces, and each was counted with a toluene/2,5-diphenyloxazole scintillant in a liquid scintillation spectrometer. Authentic pm7G and m?GpppAm were analyzed in parallel and detected by their fluorescence with ultraviolet light. a, product after 30 min of incubation; b, product after 10 min of incubation; c, product after no incubation; d, percentage of m7GpppAm remaining after different periods of incubation.
5' end. A study of this question will require p elimination and the removal of poly(A) from the RNAs synthesized in vitro followed by in uitro translation studies. A third type of experiment illustrating a discrepancy between the wheat germ and reticulocyte systems in the response to m7G comes from studies of 5' end analogues pm'G, m7G5'ppp5'Am, and m7GS'pppS'G(m) which inhibit translation of normal VSV mRNA greater than 95% in the wheat germ system but do not have significant effects on translation in the reticulocyte system even when used at very high concentrations. One aspect of these studies which is somewhat surprising is that these compounds also inhibit translation of periodate-oxidized and P-eliminated VSV mRNA in the wheat germ system (Fig. 2), suggesting that their effect may not be entirely specific for m7G-containing RNA. Our studies on VSV mRNA translation in the wheat germ system essentially confirm and extend the studies of Both et al., who concluded that a 5'-terminal m7G is obligatory for ribosome binding and translation of VSV and reovirus mRNAs (14-17). However, the results from the reticulocyte system are considerably different (22) and show that other aspects of the VSV mRNA structure are completely sufficient for correct ribosome recognition of the appropriate translation start sites. Presumably, this discrepancy between the two systems reflects the absence or relatively low concentrations of appropriate initiation factors in the heterologous plant cell system and a more complete reliance on a factor(s) which recognizes the pm7G of at least heterologous mRNAs. The virtually complete inhibition of VSV mRNA translation by 5' end analogues (an effect first described by Hickey et al. (18)) in the wheat germ system compared to less than 20% inhibition by these compounds in the reticulocyte system also suggests that at least some animal cells may not rely heavily on an mRNA recognition system involving m7G. Clearly further studies with a variety of mRNAs and translation systems will be required before a complete picture of the relative importance of m'G, in translation is obtained. These studies and previous work suggest that both the source of the translation components and the mRNA in question will be important factors (22). Some progress toward identifying proteins which may interact with 5'-terminal m7G during or prior to protein synthesis initiation has been made (19 and 35). An interaction between VSV mRNA and IF-M3 has been reported to be inhibited by pm7G at concentrations which also inhibit VSV mRNA binding to ribosomes from a fractionated reticulocyte system (19). Because the effects of pm'G in this fractionated reticulocyte system are far greater than those which we observe in the reticulocyte lysate, it is possible that an artifactual dependence on m'G-directed recognition of VSV mRNA is generated in the fractionated system, perhaps because other factors have become limiting. In addition to this possible artifact, we would like to suggest that in such studies a control RNA other than encephalomyocarditis virion RNA, whose binding to IF-M3 was not inhibited by pm'G, should be used because it is now known that poliovirus (a close relative of encephalomyocarditis) contains virion RNA in which the 5' end is covalently linked to a protein.* Although a 5'-terminal m7G is very important in translation of VSV mRNA in the wheat germ system, it may not be important for all mRNAs in this plant system since it is known that satellite tobacco necrosis virus (STNV, 5'-end pppAp or ppAp) and bacteriophage Qp RNA can be translated by this system (18,36). However, it has not been determined if m7G is added on to a fraction of these RNAs, and little information is available on the efficiency of translation of these RNAs. A study which is relevant to this point is that of Shih et al. who have shown that the removal of m7G from the coat protein mRNA of brome grass mosaic virus RNA (5' end m7G5'ppp5'G) results in a 5-to g-fold reduction in translation (231, an effect which seems significantly less than that which we find for VSV mRNAs in this system.