eIF4G Functionally Differs from eIFiso4G in Promoting Internal Initiation, Cap-independent Translation, and Translation of Structured mRNAs

Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to an mRNA. Plants, animals, and yeast each express two eIF4G homologs, which share only 30, 46, and 53% identity, respectively. We have examined the functional differences between plant eIF4G proteins, referred to as eIF4G and eIFiso4G, when present as subunits of eIF4F and eIFiso4F, respectively. The degree to which a 5′-cap stimulated translation was inversely correlated with the concentration of eIF4F or eIFiso4F and required the poly(A)-binding protein for optimal function. Although eIF4F and eIFiso4F directed translation of unstructured mRNAs, eIF4F supported translation of an mRNA containing 5′-proximal secondary structure substantially better than did eIFiso4F. Moreover, eIF4F stimulated translation from uncapped monocistronic or dicistronic mRNAs to a greater extent than did eIFiso4F. These data suggest that at least some functions of plant eIFiso4F and eIF4F have diverged in that eIFiso4F promotes translation preferentially from unstructured mRNAs, whereas eIF4F can promote translation also from mRNAs that contain a structured 5′-leader and that are uncapped or contain multiple cistrons. This ability may also enable eIF4F to promote translation from standard mRNAs under cellular conditions in which cap-dependent translation is inhibited. Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to an mRNA. Plants, animals, and yeast each express two eIF4G homologs, which share only 30, 46, and 53% identity, respectively. We have examined the functional differences between plant eIF4G proteins, referred to as eIF4G and eIFiso4G, when present as subunits of eIF4F and eIFiso4F, respectively. The degree to which a 5′-cap stimulated translation was inversely correlated with the concentration of eIF4F or eIFiso4F and required the poly(A)-binding protein for optimal function. Although eIF4F and eIFiso4F directed translation of unstructured mRNAs, eIF4F supported translation of an mRNA containing 5′-proximal secondary structure substantially better than did eIFiso4F. Moreover, eIF4F stimulated translation from uncapped monocistronic or dicistronic mRNAs to a greater extent than did eIFiso4F. These data suggest that at least some functions of plant eIFiso4F and eIF4F have diverged in that eIFiso4F promotes translation preferentially from unstructured mRNAs, whereas eIF4F can promote translation also from mRNAs that contain a structured 5′-leader and that are uncapped or contain multiple cistrons. This ability may also enable eIF4F to promote translation from standard mRNAs under cellular conditions in which cap-dependent translation is inhibited. eukaryotic initiation factors poly(A)-binding protein mitogen-activated protein kinase eukaryotic elongation factor Protein synthesis requires the participation of numerous eukaryotic initiation factors (eIFs)1 that assist the binding of 40 S ribosomal subunits to an mRNA and the assembly of the 80 S ribosome at the correct initiation codon. The 5′-cap structure (m7GpppN, where N represents any nucleotide) serves as the binding site for the cap-binding protein eIF4E, the small subunit of eIF4F. eIF4G, the large subunit of eIF4F, interacts with several proteins in addition to eIF4E, including eIF4A (which is required to remove secondary structure within the 5′-leader sequence that would otherwise inhibit scanning of the 40 S ribosomal subunit), eIF3 (which promotes 40 S ribosomal subunit binding to the mRNA), and the poly(A)-binding protein (PABP; which stabilizes eIF4F binding to the 5′-cap) (1Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 2Metz A.M. Browning K.S. J. Biol. Chem. 1996; 271: 31033-31036Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 3Neff C.L. Sachs A.B. Mol. Cell. Biol. 1999; 19: 5557-5564Crossref PubMed Scopus (53) Google Scholar, 4Wei C.-C. Balasta M.L. Ren J. Goss D.J. Biochemistry. 1998; 37: 1910-1916Crossref PubMed Scopus (112) Google Scholar, 5Korneeva N.L. Lamphear B.J. Hennigan F.L. Rhoads R.E. J. Biol. Chem. 2000; 275: 41369-41376Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The N-terminal domain of eIF4G is responsible for binding eIF4E and PABP; the middle domain binds eIF3 and eIF4A; and in mammalian eIF4G, the C-terminal domain binds a second molecule of eIF4A as well as Mnk1, a MAPK-activated protein kinase responsible for phosphorylating eIF4E (6Imataka H. Sonenberg N. Mol. Cell. Biol. 1997; 17: 6940-6947Crossref PubMed Scopus (240) Google Scholar, 7Imataka H. Gradi A. Sonenberg N. EMBO J. 1998; 17: 7480-7489Crossref PubMed Scopus (471) Google Scholar, 8Pyronnet S. Imataka H. Gingras A.C. Fukunaga R. Hunter T. Sonenberg N. EMBO J. 1999; 18: 270-279Crossref PubMed Scopus (537) Google Scholar). Consequently, eIF4G functions as a scaffold protein that recruits many of the factors involved in stimulating 40 S ribosomal subunit binding to an mRNA. Two related but highly distinct eIF4G proteins were first identified in plants (9Browning K.S. Webster C. Roberts J.K. Ravel J.M. J. Biol. Chem. 1992; 267: 10096-10100Abstract Full Text PDF PubMed Google Scholar). The two plant eIF4G proteins, referred to as eIF4G and eIFiso4G, differ in size (165 and 86 kDa, respectively). Two forms of eIF4G are also observed in yeast and mammals (10Goyer C. Altmann M. Lee H.S. Blanc A. Deshmukh M. Woolford J.L. Trachsel H. Sonenberg N. Mol. Cell. Biol. 1993; 13: 4860-4874Crossref PubMed Scopus (144) Google Scholar, 11Gradi A. Imataka H. Svitkin Y.V. Rom E. Raught B. Morino S. Sonenberg N. Mol. Cell. Biol. 1998; 18: 334-342Crossref PubMed Scopus (249) Google Scholar), but do not differ substantially in molecular mass and are more conserved. Mammalian eIF4GI and eIF4GII are 46% identical (11Gradi A. Imataka H. Svitkin Y.V. Rom E. Raught B. Morino S. Sonenberg N. Mol. Cell. Biol. 1998; 18: 334-342Crossref PubMed Scopus (249) Google Scholar), and yeast eIF4G1 and eIF4G2 are 53% identical (10Goyer C. Altmann M. Lee H.S. Blanc A. Deshmukh M. Woolford J.L. Trachsel H. Sonenberg N. Mol. Cell. Biol. 1993; 13: 4860-4874Crossref PubMed Scopus (144) Google Scholar), in contrast to plant eIF4G and eIFiso4G, which are only 30% identical. 2K. S. Browning, unpublished data. Mammalian eIF4GII functionally complements eIF4GI to a significant extent (11Gradi A. Imataka H. Svitkin Y.V. Rom E. Raught B. Morino S. Sonenberg N. Mol. Cell. Biol. 1998; 18: 334-342Crossref PubMed Scopus (249) Google Scholar), and yeast can tolerate the deletion of either gene encoding eIF4G, although at least one gene is required for viability (10Goyer C. Altmann M. Lee H.S. Blanc A. Deshmukh M. Woolford J.L. Trachsel H. Sonenberg N. Mol. Cell. Biol. 1993; 13: 4860-4874Crossref PubMed Scopus (144) Google Scholar). Although these studies suggest that both eIF4G proteins in eukaryotic species are largely functionally similar, differences also have been reported. For example, deletion of the gene encoding yeast eIF4G1 leads to a synthetic lethal interaction with cdc33-1, an eIF4E temperature-sensitive mutant, whereas deletion of the gene encoding eIF4G2 does not (12Tarun S.Z. Wells S.E. Deardorff J.A. Sachs A.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9046-9051Crossref PubMed Scopus (266) Google Scholar). Moreover, yeast eIF4G2 supports translation of uncapped polyadenylated mRNA to a greater extent than does eIF4G1 (12Tarun S.Z. Wells S.E. Deardorff J.A. Sachs A.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9046-9051Crossref PubMed Scopus (266) Google Scholar). Whether this is due to functional differences or to the in vivo levels of each eIF4G is unknown. Cleavage of mammalian eIF4G occurs following infection with poliovirus or human rhinovirus, resulting in the inhibition of protein synthesis (13Gradi A. Svitkin Y.V. Imataka H. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11089-11094Crossref PubMed Scopus (272) Google Scholar, 14Svitkin Y.V. Gradi A. Imataka H. Morino S. Sonenberg N. J. Virol. 1999; 73: 3467-3472Crossref PubMed Google Scholar). For both viruses, cleavage of eIF4GI occurs prior to that of eIF4GII, and the cleavage of the latter correlates with the loss of protein synthesis following viral infection (13Gradi A. Svitkin Y.V. Imataka H. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11089-11094Crossref PubMed Scopus (272) Google Scholar, 14Svitkin Y.V. Gradi A. Imataka H. Morino S. Sonenberg N. J. Virol. 1999; 73: 3467-3472Crossref PubMed Google Scholar, 15Goldstaub D. Gradi A. Bercovitch Z. Grosmann Z. Nophar Y. Luria S. Sonenberg N. Kahana C. Mol. Cell. Biol. 2000; 20: 1271-1277Crossref PubMed Scopus (107) Google Scholar). In contrast, the cleavage of eIF4GI and eIF4GII that occurs during apoptosis is temporally similar (16Bushell M. Wood W. Clemens M.J. Morley S.J. Eur. J. Biochem. 2000; 267: 1083-1091Crossref PubMed Scopus (77) Google Scholar). Although wheat eIF4F and eIFiso4F support translation in vitro and exhibit RNA-dependent ATP hydrolysis activity and ATP-dependent RNA unwinding activity (17Abramson R.D. Browning K.S. Dever T.E. Lawson T.G. Thach R.E. Ravel J.M. Merrick W.C. J. Biol. Chem. 1988; 263: 5462-5467Abstract Full Text PDF PubMed Google Scholar, 18Browning K.S. Lax S.R. Ravel J.M. J. Biol. Chem. 1987; 262: 11228-11232Abstract Full Text PDF PubMed Google Scholar, 19Lax S. Fritz W. Browning K. Ravel J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 330-333Crossref PubMed Scopus (60) Google Scholar, 20Lax S. Browning K.S. Maia D.M. Ravel J.M. J. Biol. Chem. 1986; 261: 15632-15636Abstract Full Text PDF PubMed Google Scholar), the affinity of eIF4F for hypermethylated cap structures is lower than that of eIFiso4F (21Carberry S.E. Darzynkiewicz E. Goss D.J. Biochemistry. 1991; 30: 1624-1627Crossref PubMed Scopus (53) Google Scholar), and the ATPase activity of eIF4F is greater than that of eIFiso4F when mRNA is used to stimulate the RNA-dependent activity (20Lax S. Browning K.S. Maia D.M. Ravel J.M. J. Biol. Chem. 1986; 261: 15632-15636Abstract Full Text PDF PubMed Google Scholar). Moreover, binding studies with oligonucleotides suggest that eIF4F binding is sensitive to the presence of secondary structure and that eIFiso4F exhibits a binding preference for linear structures (22Carberry S.E. Goss D.J. Biochemistry. 1991; 30: 4542-4545Crossref PubMed Scopus (33) Google Scholar). Whether the two types of eIF4G present in eukaryotic cells exhibit specialization in determining which mRNAs are translated or whether they differ in the efficiency in which they support translation has not been investigated for any species. In this study, the functional differences of plant eIF4G and eIFiso4G were investigated during the translation of capped or uncapped mRNAs, mRNAs containing a structured 5′-leader, or dicistronic mRNAs. The addition of eIF4F or eIFiso4F (in which eIF4G and eIFiso4G are present as subunits, respectively) to lysates depleted of eIF4F and eIFiso4F supported the translation of an unstructured mRNA; however, only eIF4F significantly supported translation from an mRNA with a structured 5′-leader. eIF4F increased the translation of an uncapped mRNA and stimulated the translation from the second cistron of a dicistronic mRNA to a greater extent than did eIFiso4F, suggesting that eIF4F has evolved to promote translation from nonstandard mRNAs,i.e. those that lack a cap, contain a structured 5′-leader, or contain multiple cistrons, or has evolved to promote translation from standard mRNAs under cellular conditions in which cap-dependent translation is inhibited, whereas eIFiso4F may be largely limited to facilitating translation from standard mRNAs. Additionally, the concentration of eIF4F, eIFiso4F, and PABP determined the extent to which the cap stimulated translation: PABP was required for the cap to stimulate translation efficiently; however, increased levels of PABP, eIF4F, or eIFiso4F substantially reduced the competitive advantage that a cap conferred to an mRNA. These observations suggest that eIF4F and eIFiso4F have undergone functional specialization that allows them to discriminate between mRNAs. Moreover, these observations suggest that developmental changes in the cellular concentration of eIF4F, eIFiso4F, or PABP may influence the extent of cap-dependent translation. The T7-based monocistronic and dicistronic luciferase constructs have been described previously (23Gallie D.R. Ling J. Niepel M. Morley S.J. Pain V.M. Nucleic Acids Res. 2000; 28: 2943-2953Crossref PubMed Scopus (34) Google Scholar). DNA concentration was quantitated spectrophotometrically following linearization and brought to 0.5 mg/ml. In vitro transcription was carried out as described previously (24Yisraeli J.K. Melton D.A. Methods Enzymol. 1989; 180: 42-50Crossref PubMed Scopus (125) Google Scholar) using 40 mm Tris-HCl (pH 7.5), 6 mm MgCl2, 100 μg/ml bovine serum albumin, 0.5 mm ATP, 0.5 mm CTP, 0.5 mm UTP, 0.5 mm GTP, 10 mm dithiothreitol, 0.3 units/μl RNasin (Promega), and 0.5 units/μl T7 RNA polymerase. The constructs used terminated in a A50 tail. Capped RNAs were synthesized using 3 μg of template in the same reaction mixture as described above, except that GTP was used at 160 μm, and 1 mm m7GpppG was included. Under these conditions, >95% of the mRNA is capped. The free energy of secondary structures used in this study was calculated at a temperature of 37 °C using MFOLD of GCG Software Package Version 10, which is based on the Zuker algorithm for determining multiple optimal and suboptimal secondary structures (25Zuker M. Science. 1989; 244: 48-52Crossref PubMed Scopus (1728) Google Scholar) with the folding parameters as described (26Jaeger J.A. Turner D.H. Zuker M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7706-7710Crossref PubMed Scopus (780) Google Scholar). Wheat PABP (27Le H. Chang S.-C. Tanguay R.L. Gallie D.R. Eur. J. Biochem. 1997; 243: 350-357Crossref PubMed Scopus (25) Google Scholar), eIF4F and eIFiso4F (9Browning K.S. Webster C. Roberts J.K. Ravel J.M. J. Biol. Chem. 1992; 267: 10096-10100Abstract Full Text PDF PubMed Google Scholar), eIF4B (28Browning K.S. Maia D.M. Lax S.R. Ravel J.M. J. Biol. Chem. 1987; 262: 538-541Abstract Full Text PDF PubMed Google Scholar), eIF4A (29Lax S.R. Lauer S.J. Browning K.S. Ravel J.M. Methods Enzymol. 1986; 118: 109-128Crossref PubMed Scopus (91) Google Scholar), and recombinant eIFiso4G and eIFiso4E (30van Heerden A. Browning K.S. J. Biol. Chem. 1994; 269: 17454-17457Abstract Full Text PDF PubMed Google Scholar) were purified as described. The purification of eIF4G and eIF4E will be described elsewhere. Proteins from control and depleted wheat germ lysates were resolved using standard SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to 0.22 μm nitrocellulose membrane by electroblotting. Following transfer, the nitrocellulose membranes were blocked in 5% milk and 0.01% thimerosal in TPBS (0.1% Tween 20, 13.7 mm NaCl, 0.27 mm KCl, 1 mmNa2HPO4, and 0.14 mmKH2PO4), followed by incubation with primary antibodies diluted typically 1:1000 to 1:2000 in TPBS with 1% milk for 1.5 h. The blots were then washed twice with TPBS and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies (Southern Biotechnology Associates, Inc.) diluted to 1:10,000 for 1 h. The blots were washed twice with TPBS, and the signal was detected typically between 1 and 15 min using chemiluminescence (Amersham Pharmacia Biotech). 200 μl of wheat germ extract (Promega) was added to 300 μl of m7GTP-Sepharose (Amersham Pharmacia Biotech) or 100 μl of poly(A)-agarose (Sigma) and incubated with rotation at 4 °C for 30 min. The lysate was collected by centrifugation (800 × g for 1 min) through a spin column (Promega) and used immediately. The extent of depletion of eIF4G, eIF4E, eIFiso4G, eIFiso4E, eIF4A, eIF4B, eIF3, eEF2, PABP, or Hsp101 was determined by Western analysis following resolution of the extract by SDS-polyacrylamide gel electrophoresis. mRNA constructs were translated using complete or depleted wheat germ lysate as described by the manufacturer, except that all amino acids were unlabeled. The lysates were supplemented with recombinant initiation factors or factors purified from wheat germ extract as indicated. In wheat germ lysate, eIF4A is present in a >30-fold molar excess relative to eIF4G (31Browning K.S. Humphreys J. Hobbs W. Smith G.B. Ravel J.M. J. Biol. Chem. 1990; 265: 17967-17973Abstract Full Text PDF PubMed Google Scholar). Consequently, a similar ratio was used when lysates were supplemented with eIF4A. The ratio of eIF4B to eIF4G has not been measured; and therefore, the ratio used for supplementation was determined empirically. The reactions were incubated for 3 h, and 2-μl aliquots were assayed in a MonoLight 2010 luminometer for luciferase activity. Each mRNA construct was translated in triplicate, and the mean ± S.D. for each construct is reported. An alignment of eIF4G from wheat, human, and Saccharomyces cerevisiaerevealed that plant eIF4G and eIFiso4G are most conserved with eIF4G of other eukaryotes in the region responsible for interaction with eIF4A and eIF3 (Fig. 1) (1Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). A second conserved region is the eIF4E-binding domain (1Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 32Mader S. Lee H. Pause A. Sonenberg N. Mol. Cell. Biol. 1995; 15: 4990-4997Crossref PubMed Google Scholar). The Mnk1-binding domain and a second eIF4A-binding site have been mapped to the C-terminal region of human eIF4G (6Imataka H. Sonenberg N. Mol. Cell. Biol. 1997; 17: 6940-6947Crossref PubMed Scopus (240) Google Scholar, 8Pyronnet S. Imataka H. Gingras A.C. Fukunaga R. Hunter T. Sonenberg N. EMBO J. 1999; 18: 270-279Crossref PubMed Scopus (537) Google Scholar), but are not present in yeast and plant eIF4F proteins. However, plant eIF4G and eIFiso4G do contain a domain near their C terminus that shares limited conservation with the human eIF4G proteins and is absent from the yeast orthologs. Plant eIFiso4G differs most from eIF4G in that it lacks an ∼700-amino acid-long N-terminal region present in eIF4G. In this respect, plant eIF4G is more similar to human eIF4G than is eIFiso4G. The yeast eIF4G proteins also contain an N-terminal region, although it is shorter than that present in plant eIF4G or in either mammalian eIF4G protein. Although the domain responsible for interaction with PABP is not conserved among eIF4G proteins, it is located within this N-terminal region of human and yeast eIF4G proteins. The PABP interaction domain within plant eIF4G has not been identified precisely, but the putative site in eIFiso4G has been mapped to its N-terminal region (33Le H. Tanguay R.L. Balasta M.L. Wei C.-C. Browning K.S. Metz A.M. Goss D.J. Gallie D.R. J. Biol. Chem. 1997; 272: 16247-16255Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). To examine the function of eIF4G or eIFiso4G in vitro, it was necessary to generate an eIF4G- and eIFiso4G-dependent lysate. This was accomplished by depleting wheat germ lysate of eIF4F (composed of eIF4G and eIF4E) and eIFiso4F (composed of eIFiso4G and eIFiso4E) through their binding to m7GTP-Sepharose. Western analysis confirmed that the level of eIF4E and eIFiso4E was reduced by 90–95%, as was that of eIF4G and eIFiso4G (Fig.2). To examine whether the depleted lysate was eIF4F- or eIFiso4F-dependent, cappedluc-A50 mRNA was translated in a fractionated lysate supplemented with increasing amounts of purified eIF4F or eIFiso4F. The extent to which the reporter mRNA was translated was determined by measuring luciferase activity. A reduction in the level of eIF4F and eIFiso4F reduced translation by >95% (compare translation in complete and fractionated lysates) (Fig.3), as would be expected following a reduction in those initiation factors that are normally required for efficient translation. Residual translational activity of the fractionated lysate may be the result of the low level of either eIF4G and eIFiso4G remaining in the lysate (Fig. 2) or a result of PABP (see below). Supplementation with 16 nm eIF4F increased translation from the reporter mRNA nearly 10-fold in the fractionated lysate, but did not affect translation of the same mRNA in the unfractionated lysate (Fig. 3). Translation in the fractionated lysate was also dependent on eIFiso4F, whereas supplementation of the unfractionated lysate with eIFiso4F did not affect translation (Fig. 3). A comparison of their relative effects on translation reveals that eIF4F was more stimulatory than was eIFiso4F. Native eIF4F and recombinant eIF4F are equally active in supporting translation in vitro, as are native eIFiso4F and recombinant eIFiso4F (30van Heerden A. Browning K.S. J. Biol. Chem. 1994; 269: 17454-17457Abstract Full Text PDF PubMed Google Scholar), 3K. S. Browning, unpublished data. suggesting that there is no significant difference in the fraction of each purified factor that is active. The stimulation afforded by eIFiso4F was more nonlinear at the highest concentration used for this factor than that observed for eIF4F, suggesting that even higher concentrations of eIFiso4F would not yield a level of translation comparable to that observed for eIF4F. The nonlinearity of the activity of these factors has been reported previously (34Fletcher L. Corbin S.D. Browning K.S. Ravel J.M. J. Biol. Chem. 1990; 265: 19582-19587Abstract Full Text PDF PubMed Google Scholar). The greater nonlinearity of eIF4F at lower concentrations may indicate its higher affinity for RNA compared with eIFiso4F, a possibility that is supported by the observation that the eIFiso4F dose-response curve is slightly sigmoidal (Fig. 3; see Fig. 5C). These data suggest that the endogenous level of eIF4F and eIFiso4F in the unfractionated lysate is necessary for maximum translational activity and that their removal from the lysate renders the fractionated lysate dependent upon the addition of either eIF4F or eIFiso4F.Figure 3Reduction in eIF4F and eIFiso4F results in an eIF4F- and eIFiso4F-dependent lysate. Cappedluc-A50 mRNA (3.4 ng/μl) was translated in the unfractionated lysate (left panel) or in the eIF4F/eIFiso4F-dependent lysate (right panel). Each lysate was supplemented with the indicated amounts of eIF4F or eIFiso4F, and the amount of luciferase synthesized was measured in a luminometer. eIF4A was included at a ratio of 1:70 (for eIF4F assays) or 1:30 (for eIFiso4F assays). Luciferase expression is indicated as light units from 2 μl of each reaction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5eIF4F (but not eIFiso4F) supports the translation of an mRNA with 5′-proximal secondary structure.The eIF4F/eIFiso4F-dependent lysate was programmed with 7.5 ng/μl luc-A50 (A and C) or SL13-luc-A50 (B andD) mRNA and supplemented with the indicated amounts of eIF4F (A and B) or eIFiso4F (C andD). eIF4A was included at a 1:70 ratio of eIF4A to eIF4F or at a 1:30 ratio of eIF4A to eIFiso4F. Each mRNA construct was translated in triplicate, and the mean ± S.D. for each construct is reported. The -fold stimulation provided by eIF4F or eIFiso4F is indicated above each histogram. Luc, luciferase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine whether eIF4F and eIFiso4F differ in the extent to which they support translation of an mRNA with a structured 5′-leader, it was first necessary to determine the translational characteristics of mRNAs with or without secondary structure in the eIF4F/eIFiso4F-dependent lysate. A stable stem-loop structure containing a 24-base pair stem of ΔG = −42.9 kcal/mol was introduced 4 nucleotides downstream of the 5′ terminus of the luc reporter mRNA (referred to as SL24-luc-A50). In addition, deletions were made within the 24-base pair stem-loop to generate less stable structures with 19-, 13-, and 7-base pair stems of ΔG = −31.8, −21.3, and −4.5 kcal/mol, respectively (referred to as SL19-luc-A50, SL13-luc-A50, and SL7-luc-A50, respectively). The presence of these structures was shown previously to inhibit translation in unfractionated lysate as a function of their stability (35Niepel M. Ling J. Gallie D.R. FEBS Lett. 1999; 462: 79-84Crossref PubMed Scopus (30) Google Scholar). To determine the effect of the stem-loop on translation, each construct was synthesized in vitro as a capped mRNA containing a A50 tail and translated at three different concentrations in a lysate reduced in eIF4F and eIFiso4F. When the eIF4F/eIFiso4F-dependent lysate was programmed with a 9.6 ng/μl concentration of each construct, the presence of a 24-, 19-, 13-, or 7-base pair stem-loop (i.e.SL24-luc-A50, SL19-luc-A50, SL13-luc-A50, and SL7-luc-A50, respectively) inhibited translation to 15.7, 12.3, 16.3, and 61.5% of that of the control mRNA with an unstructured leader (Fig.4). As expected, decreasing the RNA concentration reduced the amount of luciferase produced (compare the absolute levels of expression in Fig. 4 and note the difference in scale on the x axis). However, the presence of the same stem-loop structures was progressively less inhibitory as the concentration of the input mRNA decreased (see the relative values to the right of each histogram in Fig. 4, which are relative to the expression from the control luc-A50 mRNA), and translation from the SL7-luc-A50and SL13-luc-A50 mRNAs was actually higher than that from the control mRNA at the lowest RNA concentration tested. Consequently, secondary structure of lower stability (e.g.SL7-luc-A50 and SL13-luc-A50 mRNAs) was inhibitory only at a high RNA concentration, whereas more stable secondary structure (e.g.SL19-luc-A50 and SL24-luc-A50 mRNAs) was inhibitory at all RNA concentrations tested, albeit less inhibitory at low RNA concentrations. These data suggest that the translational machinery, such as eIF4A, which functions as an RNA helicase and as a subunit of eIF4F (or eIFiso4F), is required during translation in direct proportion to the degree of secondary structure present in the 5′-leader of an mRNA (36Svitkin Y.V. Pause A. Haghighat A. Pyronnet S. Witherell G. Belsham G.J. Sonenberg N. RNA. 2001; 7: 382-394Crossref PubMed Scopus (338) Google Scholar), is sufficient to unwind moderately stable secondary structure when an mRNA is present at a low concentration. However, higher concentrations of mRNA may act to titrate the RNA helicase activity such that the structure cannot be removed from all of the input RNA, resulting in the inhibition of translation by secondary structure of even moderate stability. To examine the effect of eIF4F or eIFiso4F on the translation of a structured mRNA, the SL13-luc-A50 mRNA construct was selected as an mRNA with moderately stable secondary structure. This mRNA also exhibited the greatest potential range in expression as exemplified by the degree to which it was translated at high or low RNA concentrations (Fig. 4). Supplementation of the eIF4F/eIFiso4F-dependent lysate programmed with eIF4F increased translation from SL13-luc-A50 mRNA up to 14.7-fold (Fig. 5B), whereas supplementation with eIFiso4F did not increase translation from the same mRNA (Fig. 5D). In contrast, translation from the control (i.e. unstructured) mRNA increased when the lysate was supplemented with either eIF4F (Fig. 5A) or eIFiso4F (Fig. 5C). For the structured mRNA (Fig.5B), eIF4F was less stimulatory at high concentrations than at lower concentrations. This effect is also observed for unstructured mRNAs if eIF4F is added to the lysate to a level higher than was used for these studies (data not shown). An excess of eIF4F (i.e. a level of eIF4F that is in excess of the binding capacity of the input mRNA) may compete with the bound eIF4F for other factors needed for translation. The secondary structure present in the construct of Fig. 5B may reduce the amount of eIF4F that can bind the mRNA, which would result in a condition of excess eIF4F at a lower concentration than normally observed for an unstructured mRNA. Once in excess, free eIF4F may compete with the bound eIF4F for other necessary factors, resulting in an apparent lower level of stimulation. As eIF4F is considered to be present in limiting amounts in vivo in eukaryotes, such in vitroconditions of excess eIF4F are unlikely to be present in vivo. These data suggest that eIF4F and eIFiso4F differ considerably in their ability to promote translation from an mRNA containing a structured 5′-leader. The introduction of each stem-loop increases the length of the 5′-leader relative to the construct lacking the secondary structure, which could have an effect on translation efficiency. For example, introduction of SL24, SL19, SL13, or SL7 results in a leader length of 64, 53, 41, or 29 nucleotides, respectively. To determine whether the length of the 5′-leader influences the degree to which eIF4F or eIFiso4F increases translation, capped luc-A50 mRNAs containing a 17-, 72-, or 144-nucleotide unstructured 5′-leader were translated in the eIF4F/eIFiso4F-dependent lysate and supplemented with eIF4F or eIFiso4F. Expression was affected only to a small extent as a function of the 5′-leader length, with a 2-fold increase in translation as a function