The Absence of a m’G Cap on @-Globin mRNA and Alfalfa Mosaic Virus RNA 4 Increases the Amounts of Initiation Factor 4F Required for Translation*

beta-Globin mRNA and alfalfa mosaic virus (AMV) RNA 4, two naturally capped mRNAs, and satellite tobacco necrosis virus (STNV) RNA, a naturally uncapped mRNA, were prepared by in vitro transcription with and without a 5' m7G cap structure (m7G(5')ppp(5')N). The translation of the capped and uncapped forms of these mRNAs was measured in a crude S30 system and a partially purified system from wheat germ. In the S30 system the uncapped forms of beta-globin mRNA and AMV RNA 4 are much less active (greater than or equal to 10%) than their capped forms, whereas the uncapped and capped forms of STNV RNA are equally active. The low activity of uncapped beta-globin mRNA and AMV RNA 4 in the S30 system is due, in part, to inactivation of the uncapped mRNAs in this system. Additional studies, carried out in the partially purified system in which very little inactivation of the mRNAs occurs, show that the uncapped and capped forms of beta-globin mRNA or AMV RNA 4 differ markedly with respect to the amount of eukaryotic initiation factor (eIF)-4F required for translation. For beta-globin mRNA the absence of the 5' cap structure increases the concentration of eIF-4F required for half-maximal translation about 6-fold (from 10 to 60 nM) and for AMV RNA 4 it increases the concentration of eIF-4F about 12-fold (from 5 to 60 nM). The concentrations of eIF-3, eIF-4A, and eIF-4B required for half-maximal translation of the uncapped forms of beta-globin mRNA and AMV RNA 4 are either the same or only slightly higher (1.5- to 2-fold) than the concentrations required for the capped forms. With STNV RNA the concentration of eIF-4F required for half-maximal translation of either uncapped or capped STNV RNA is 3 nM, and the concentrations of eIF-3, eIF-4A, and eIF-4B required for the two forms are also the same. The translation of the capped and uncapped forms of beta-globin mRNA and AMV RNA 4 is inhibited strongly by low concentrations of m7GTP in the partially purified system containing low concentrations of eIF-4F. Under the same conditions, the translation of capped or uncapped STNV RNA is inhibited only slightly by m7GTP. These findings suggest the possibility that the mechanism by which eIF-4F interacts and initiates translation with naturally uncapped mRNAs may not be identical to the mechanism by which eIF-4F interacts and initiates translation of naturally capped mRNAs.

&Globin mRNA and alfalfa mosaic virus (AMV) RNA 4, two naturally capped mRNAs, and satellite tobacco necrosis virus (STNV) RNA, a naturally uncapped mRNA, were prepared by in vitro transcription with and without a 5' m'G cap structure (m7G(5')ppp (5')N). The translation of the capped and uncapped forms of these mRNAs was measured in a crude S30 system and a partially purified system from wheat germ. In the S30 system the uncapped forms of &globin mRNA and AMV RNA 4 are much less active (~10%) than their capped forms, whereas the uncapped and capped forms of STNV RNA are equally active. The low activity of uncapped &globin mRNA and AMV RNA 4 in the S30 system is due, in part, to inactivation of the uncapped mRNAs in this system. Additional studies, carried out in the partially purified system in which very little inactivation of the mRNAs occurs, show that the uncapped and capped forms of &globin mRNA or AMV RNA 4 differ markedly with respect to the amount of eukaryotic initiation factor (eIF)-4F required for translation.
For &globin mRNA the absence of the 5' cap structure increases the concentration of eIF-4F required for half-maximal translation about 6-fold (from 10 to 60 nM) and for AMV RNA 4 it increases the concentration of eIF-4F about 12-fold (from 5 to 60 nM). The concentrations of eIF-3, eIF-4A, and eIF-4B required for half-maximal translation of the uncapped forms of B-globin mRNA and AMV RNA 4 are either the same or only slightly higher (1.5-to 2-fold) than the concentrations required for the capped forms. With STNV RNA the concentration of eIF-4F required for half-maximal translation of either uncapped or capped STNV RNA is 3 nM, and the concentrations of eIF-3, eIF-4A, and eIF-4B required for the two forms are also the same. The translation of the capped and uncapped forms of &globin mRNA and AMV RNA 4 is inhibited strongly by low concentrations of m'GTP in the partially purified system containing low concentrations of eIF-4F.
Under the same conditions, the translation of capped or uncapped STNV RNA is inhibited only slightly by m7GTP. These findings suggest the possibility that the mechanism by which eIF-4F interacts and initiates translation with naturally uncapped mRNAs may not be identical to the mechanism by Previous work has shown that the absence of a 5' cap structure (m7G(5')ppp(5')N (1)) on a naturally capped mRNA such as reovirus RNA, VSV' RNA, or globin mRNA greatly decreases its ability to be translated (2), whereas capping of a naturally uncapped mRNA such as STNV RNA does not enhance its ability to be translated (3). There have been several reports (4)(5)(6) indicating that the translation of AMV RNA 4 is less sensitive than other naturally capped mRNAs to inhibition by m7G cap analogs (4,5) or by antibody to the 24-kDa cap binding protein (4). Also, AMV RNA 4 is translated in extracts from poliovirus-infected cells (7) and in eIF-4E-deficient extracts from a temperature-sensitive yeast mutant (8).
On the basis of these observations, it has been concluded the translation of AMV RNA 4 is less dependent upon the 5' cap structure than other capped mRNAs.
In this investigation we have prepared capped and uncapped AMV RNA 4, as well as capped and uncapped @-globin mRNA and STNV RNA, by in d-o transcription and have measured the abilities of these mRNAs to be translated in a crude S30 system and a partially purified system from wheat germ. We find that translation of AMV RNA 4 is dependent upon the cap structure. In the wheat germ S30 system, the uncapped forms of AMV RNA 4 and p-globin mRNA are inactivated more rapidly than the capped forms of these mRNAs. Further work carried out in the partially purified system in which very little inactivation of the capped or uncapped forms of these mRNAs occurs showed that the absence of a 5' cap on AMV RNA 4 or p-globin mRNA increases the amount of eIF-4F required for translation 6-to 12-fold. In addition, we find that m7GTP strongly inhibits the translation of both the capped and uncapped forms of AMV RNA 4 and @-globin mRNA in the partially purified system containing low concentrations of eIF-4F. In contrast, the uncapped form of STNV RNA is as stable as the capped form in the S30 system. In addition, the amounts of eIF-4F required for translation of the capped and uncapped forms in the partially purified system are the same, and m7GTP has very little effect on the translation of STNV RNA, either capped or uncapped. '  'RNA (pSTNVrzzg) was constructed as described previously (13). This construct contains ten extra bases at the 5' end of the transcribed mRNA (see Fig. 1). Rabbit globin mRNA was isolated as described previously (14); the cDNA was prepared and the subcloning was carried out as described previously (13 pg of eIF-4A, 2 pg of eIF-4B, and 4 +g of eIF-4F.

RESULTS
Capped and uncapped b-globin mRNA, AMV RNA 4, and STNV RNA were prepared by in u&o transcription of the plasmid constructs described under "Experimental Procedures." The nucleotide sequences of the 5' ends of the RNA transcripts are shown in Fig. 1. The abilities of these mRNAs to direct polypeptide synthesis in a crude S30 system from wheat germ are shown in Fig. 2. In this system polypeptide synthesis directed by capped /3-globin mRNA increased linearly with increasing amounts of capped p-globin mRNA and increased linearly with time up to about 45 min. Under the same conditions the amount of polypeptide synthesis obtained with uncapped fl-globin mRNA was less than 10% of that obtained with capped /3-globin mRNA. Similar results were obtained with AMV RNA 4. The amount of polypeptide synthesized in the presence of uncapped AMV RNA 4 was only lo-15% of that obtained in the presence of capped AMV RNA 4. In contrast to the results obtained with @globin mRNA and AMV RNA 4, the amount of polypeptide synthesis obtained with uncapped STNV RNA was the same as that obtained with capped STNV RNA, and the rates of incorporation were the same.
When the abilities of the capped and uncapped mRNAs to direct polypeptide synthesis were measured in a partially purified system from wheat germ, the results shown in Fig. 3 were obtained. The fractionated system contained purified eIF-3, eIF-4A, eIF-4B, eIF-4F, eIF-4C, and a small amount of 40-70% ammonium sulfate fraction sufficient to provide aminoacyl-tRNA synthetases, eIF-2, eIF-5, and elongation factors. In this system uncapped @globin mRNA was about 40% as active as capped fl-globin mRNA. Uncapped AMV RNA 4 was 40-50% as active as capped AMV RNA 4 and uncapped and capped STNV RNA were equally active. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products synthesized in the partially purified system (labeled with [?S]Met instead of [14C]Leu) showed that the polypeptides synthesized in the presence of the uncapped mRNAs were the same size as those synthesized in the presence of the capped mRNAs (data not shown).
The results described above suggested that uncapped bglobin mRNA and uncapped AMV RNA 4 were being inactivated in the S30 system. When the "P-labeled mRNAs were incubated for 10 min in the S30 system lacking amino acids and then precipitated with cold trichloroacetic acid, 80-90% of the "P was recovered in the precipitate (data not shown). Also when the mRNAs were incubated in the S30 system, In the panels on the Left, the S30 system described under "Experimental Procedures" was supplemented with capped (O---0) or uncapped (0---0) mRNAs, in the amounts indicated. In the panels on the right, the S30 system was supplemented with 10 pmol of B-globin mRNA, 5 pmol AMV RNA 4, or 5 pmol of STNV RNA and was incubated for the times indicated. recovered by phenol extraction, and analyzed by electrophoresis on a 4% polyacrylamide, 6 M urea gel, the mRNAs appeared to be intact, indicating that gross degradation had not occurred. However, when the mRNAs treated in this manner were tested for their ability to support polypeptide synthesis in the partially purified system, the results shown in Table I were obtained. Treated capped @-globin mRNA was about 90% as active as untreated capped P-globin mRNA, whereas treated uncapped ,&globin mRNA was only 20% as active as untreated uncapped @-globin mRNA. Similarly, treated capped AMV RNA 4 was 90% as active as the untreated capped AMV RNA 4, and treated uncapped AMV RNA 4 was only 27% as active as the untreated uncapped AMV RNA 4. Treated and untreated uncapped STNV RNA were equally active. These data showed that inactivation of the uncapped forms of @-globin mRNA and AMV RNA 4 occurred when the mRNAs were incubated in the S30 system. It is not known whether inactivation was due to removal of a small number of bases which would not be detected by the procedures described above or to some other modification of the mRNAs.
A comparison was made of the abilities of the capped and uncapped mRNAs to bind to 40 S ribosomal subunits in a system containing [35S]Met-tRNA and highly purified initia- In the panels on the k$& the partially purified system described under "Experimental Procedures" was supplemented with capped (O---0) or uncapped (O---0) mRNA, in the amounts indicated. In the panels on the rig/& the partially purified system was supplemented with 10 pmol of fi-glohin mRNA, 5 pmol of AMV RNA 4, or 5 pmol of STNV RNA and was incubated for the times indicated. tion factors, eIF-2, eIF-3, eIF-4A, eIF-4B, and eIF-4F. The results given in Table II show that uncapped @-globin mRNA was 50-60% as active as capped /3-globin mRNA. Uncapped AMV RNA 4 was about 70% as active as capped AMV 4 RNA, and capped and uncapped STNV RNA were equally active. These data indicate that the difference in the ability of capped and uncapped @-globin mRNA or AMV RNA 4 to support polypeptide synthesis in the partially purified translation system is due, primarily, to a difference in ability to bind to 40 S ribosomal subunits.
The amounts of eIF-3, eIF-4A, eIF-4B, and eIF-4F required for translation of the capped and uncapped mRNAs were determined in the partially purified polymerization system, and the results obtained are shown in Figs. 4-6. The concentrations of the factors required for half-maximal translation are given in Table III. These values were obtained from double-reciprocal plots of the data in Figs. 4-6. The doublereciprocal plots of the responses to eIF-4F are shown in Fig.  7. In the case of STNV RNA the concentrations of eIF-3, eIF-4A, eIF-4B, and eIF-4F required for half-maximal translation were the same for the uncapped and capped forms of this RNA. Also, there were no significant differences in the concentrations of eIF-3 required for half-maximal translation of n Each of the mRNAs (-100 pmol) were incubated for 10 min at 27 "C in 0.5 ml of the S30 incubation mixture lacking amino acids. The reaction mixture was extracted with phenol and the RNA was precipitated with ethanol, washed, dried, and suspended in 50 ~1 of sterile water. The concentration of mRNA was calculated from the amount of "P recovered. The mRNAs were then assayed for the ahility to support polypeptide synthesis in the partially purified system. The values given are picomoles of leucine incorporated into polypeptide per pmol of mRNA.
forms of AMV RNA 4 were about the same.
The most striking differences were in the concentrations of eIF-4F required for half-maximal translation of capped and uncapped @-globin mRNA and AMV RNA 4. The concentration of eIF-4F required for translation of uncapped @-globin mRNA was approximately 6-fold higher (60 versus 10 nM) than the concentration required for capped @-globin mRNA, and the concentration of eIF-4F required for uncapped AMV RNA 4 was approximately 12-fold higher (60 uersm 5 nM) than the concentration required for capped AMV RNA 4. From the plots given in Fig. '7, it can be seen that at infinitely high concentrations of eIF-4F the maximal velocities attainable with the capped and uncapped forms of fi-globin mRNA and AMV RNA 4 were the same. The recinrocals were calculated f&m %e values in Fiii 4, 5, and 6, assumini a molecular weight of 330,000 for eIF-4F (9).
The effects of m7GTP on the translation of the uncapped and capped forms of @-globin mRNA, AMV RNA 4, and STNV RNA were determined in the partially purified system, and the results are shown in Fig. 8. The effects of m7GTP on the translation of the capped forms of these mRNAs carried at different concentrations of eIF-4F are shown in the panels on the Left. At a low concentration of eIF-4F (5 nM), the concentration of m7GTP required to inhibit translation 50% was 5 PM for p-globin mRNA and 10 pM for AMV RNA 4. At a 4-fold higher concentration of eIF-4F (20 nM), the concentration of m7GTP required to obtain 50% inhibition increased to approximately 20 pM for capped @-globin mRNA and to approximately 100 pM for capped AMV RNA 4. Increasing the concentration of eIF-4F not only increased the concentration of m7GTP required for 50% inhibition, it also decreased the maximal inhibition obtained at saturating amounts of m7GTP. With AMV RNA 4 increasing the concentration of eIF-4F from 5 to 20 nM decreased the maximum inhibition from 90 to 50%. When the concentration of eIF-4F was increased to 40 nM, the maximal inhibition obtained was only about 40%. The data in the panels on the right show that the translation of the uncapped forms of @globin mRNA and AMV RNA 4 was inhibited by m7GTP. Inhibition of the translation of the uncapped forms was overcome by higher concentrations of eIF-4F (data not shown). As shown above ( Fig. 7 and Table III  in a crude S30 system from wheat germ, whereas capped and uncapped STNV RNA are equally active. The low activity of uncapped @globin mRNA and AMV RNA 4 in the S30 system is due, at least in part, to rapid inactivation of these mRNAs. The uncapped forms of @-globin mRNA and AMV RNA 4 are also less active than their capped forms when translation is carried out in a partially purified system from wheat germ in which very little inactivation of the uncapped mRNAs occurs. In contrast to the results obtained with the naturally capped mRNAs, the naturally uncapped mRNA, STNV RNA, is not rapidly inactivated in the crude S30 system, and the capped and uncapped forms of this mRNA are equally active in the crude and purified systems. Additional studies carried out in a partially purified system show that the difference in the activities of the capped and uncapped forms of p-globin mRNA and AMV RNA 4 is due primarily to the amounts of eIF-4F required. For @-globin mRNA the absence of the 5' cap structure increases the concentration of eIF-4F required for half-maximal translation about 6-fold (from 10 to 60 nM), and for AMV RNA 4 it increases the concentration of eIF-4F required about 12-fold (from 5 to 60 IIM). At infinitely high concentrations of eIF-4F the maximal translational velocities of the uncapped forms approach those of the capped forms. The concentrations of eIF-3, eIF-4A, and eIF-4B required for half-maximal translation of uncapped forms of fi-globin mRNA and AMV RNA 4 are either the same or only slightly higher (1.5-to 2-fold) than the concentrations required for the capped forms. With STNV RNA the concentrations of eIF-4F, as well as eIF-3, eIF-4A, and eIF-4B, required for translation are the same for the capped and uncapped forms. These data show that the 5' cap of a naturally capped mRNA not only protects the mRNA from inactivation but also enhances the efficiency of translation by lowering the concentration of eIF-4F required. Our finding that the translation of both the capped and uncapped forms of @-globin mRNA is inhibited by low concentrations of m7GTP agrees with the results obtained previously for chemically decapped @-globin mRNA (22) and capped and uncapped VSV RNA (23). In addition we find that at low concentrations of eIF-4F the translation of capped and uncapped AMV RNA 4 is inhibited by low concentrations of m7GTP. Increasing the concentration of eIF-4F not only increases the concentration of m7GTP required for half-maximal inhibition it also decreases the maximal inhibition obtained with saturating amounts of m7GTP. Reports by van Vloten-Doting e.t al. (24) and Herson et al. (25) showed that increasing the concentration of AMV RNA 4 also decreased the maximal inhibition obtained with saturating amounts of cap analog. Other reports, indicating that the translation of AMV RNA 4 is less sensitive than other mRNAs to inhibition by m7G analogs (4,5) or antibody to cap binding protein (4), can be explained on the basis of the low concentration of eIF-4F required for translation of AMV RNA 4 and the decrease in the maximal inhibition obtained with cap analogs when the amount of eIF-4F and/or mRNA is increased.
Previous work has shown that eIF-4F binds to the 5' m7G of capped mRNAs and in conjunction with eIF-4A and eIF-4B catalyzes the ATP-dependent unwinding of the 5' end of the mRNA (6,26). Although STNV RNA does not naturally have a 5' cap, it still requires eIF-4F for translation, presumably to prepare the 5' end of the mRNA for binding to the 40 S ribosome. The concentration of eIF-4F required for halfmaximal translation of STNV RNA (3 nM) is close to that required for AMV RNA 4 (5 nM). However, at the same low concentrations of eIF-4F (5 nM) and at the same concentrations of RNA (50 nM), concentrations of m7GTP that strongly inhibit the translation of AMV RNA 4 have very little effect on the translation of STNV RNA. This finding indicates that binding of m7GTP to the 26-kDa cap binding protein of eIF-4F does not interfere appreciably with the ability of eIF-4F to interact with the 5' end of STNV RNA and suggests the possibility that the mechanism by which eIF-4F interacts and initiates translation with naturally uncapped mRNAs may not be identical to the mechanism by which eIF-4F interacts and initiates translation of naturally capped mRNAs.
AcImo~~e~gmerzrs-We wish to thank Gayle Smith for preparation of the wheat germ initiation factors and Pam Pate for the preparation of the manuscript.