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• W2075956940 abstract "The 5′ cap and 3′ poly(A) tail of classical eukaryotic mRNAs functionally communicate to synergistically enhance translation initiation. Synergy has been proposed to result in part from facilitated ribosome recapture on circularized mRNAs. Here, we demonstrate that this is not the case. In poly(A)-dependent, ribosome-depleted rabbit reticulocyte lysates, the addition of exogenous poly(A) chains of physiological length dramatically stimulated translation of a capped, nonpolyadenylated mRNA. When the poly(A):RNA ratio approached 1, exogenous poly(A) stimulated translation to the same extent as the presence of a poly(A) tail at the mRNA 3′ end. In addition, exogenous poly(A) significantly improved translation of capped mRNAs carrying short poly(A50) tails.Trans stimulation of translation by poly(A) required the eIF4G-poly(A)-binding protein interaction and resulted in increased affinity of eIF4E for the mRNA cap, exactly as we recently described for cap-poly(A) synergy. These results formally demonstrate that mRNA circularization per se is not the cause of cap-poly(A) synergy at least in vitro. The 5′ cap and 3′ poly(A) tail of classical eukaryotic mRNAs functionally communicate to synergistically enhance translation initiation. Synergy has been proposed to result in part from facilitated ribosome recapture on circularized mRNAs. Here, we demonstrate that this is not the case. In poly(A)-dependent, ribosome-depleted rabbit reticulocyte lysates, the addition of exogenous poly(A) chains of physiological length dramatically stimulated translation of a capped, nonpolyadenylated mRNA. When the poly(A):RNA ratio approached 1, exogenous poly(A) stimulated translation to the same extent as the presence of a poly(A) tail at the mRNA 3′ end. In addition, exogenous poly(A) significantly improved translation of capped mRNAs carrying short poly(A50) tails.Trans stimulation of translation by poly(A) required the eIF4G-poly(A)-binding protein interaction and resulted in increased affinity of eIF4E for the mRNA cap, exactly as we recently described for cap-poly(A) synergy. These results formally demonstrate that mRNA circularization per se is not the cause of cap-poly(A) synergy at least in vitro. eukaryotic initiation factor poly(A)-binding protein rabbit reticulocyte lysate untranslated region The 5′ and 3′ ends of the vast majority of eukaryotic mRNAs are modified post-transcriptionally by the addition, respectively, of a methylated m7GpppN cap structure and a poly(A) tail (1Banerjee A.K. Microbiol. Rev. 1980; 44: 175-205Crossref PubMed Google Scholar) (see Ref. 2Sachs A.B. Sarnow P. Hentze M.W. Cell. 1997; 89: 831-838Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar for review). The 5′ cap structure is recognized by the eukaryotic initiation factor (eIF)1 4E component of the eIF4F holoenzyme complex (see Ref. 3Morley S.J. Curtis P.S. Pain V.M. RNA. 1997; 3: 1085-1104PubMed Google Scholar for review). The eIF4F complex also comprises an ATP-dependent RNA helicase (eIF4A) and a central adapter protein (eIF4G), which binds both eIF4E and eIF4A. The currently accepted model of translation initiation posits that recruitment of the ribosomal 40 S subunit to the mRNA 5′ end requires recognition of the mRNA cap by eIF4E and the simultaneous interaction of both eIF4G and the 40 S subunit with the eIF3 multiprotein complex (reviewed in Ref. 4Hershey J.W.B. Merrick W.C. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 33-88Google Scholar). The mRNA 3′ poly(A) tail stimulates translation initiation both in vitro and ex vivo (5Munroe D. Jacobson A. Mol. Cell. Biol. 1990; 10: 3441-3455Crossref PubMed Scopus (279) Google Scholar, 6Tarun Jr., S.Z. Sachs A.B. Genes Dev. 1995; 9: 2997-3007Crossref PubMed Scopus (328) Google Scholar, 7Preiss T. Hentze M.W. Nature. 1998; 392: 516-520Crossref PubMed Scopus (215) Google Scholar, 8Iizuka N. Najita L. Franzusoff A. Sarnow P. Mol. Cell. Biol. 1994; 14: 7322-7330Crossref PubMed Scopus (240) Google Scholar) (reviewed in Ref. 9Jacobson A. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, NY1996: 451-480Google Scholar). Under appropriate competitive translation conditions, the 5′ cap and 3′ poly(A) tail synergistically promote translation initiation (6Tarun Jr., S.Z. Sachs A.B. Genes Dev. 1995; 9: 2997-3007Crossref PubMed Scopus (328) Google Scholar, 7Preiss T. Hentze M.W. Nature. 1998; 392: 516-520Crossref PubMed Scopus (215) Google Scholar, 8Iizuka N. Najita L. Franzusoff A. Sarnow P. Mol. Cell. Biol. 1994; 14: 7322-7330Crossref PubMed Scopus (240) Google Scholar, 9Jacobson A. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, NY1996: 451-480Google Scholar, 10Gallie D.R. Genes Dev. 1991; 5: 2108-2116Crossref PubMed Scopus (577) Google Scholar), in a process requiring the poly(A)-binding protein (PABP) (11Tarun Jr., S.Z. Sachs A.B. EMBO J. 1996; 15: 7168-7177Crossref PubMed Scopus (574) Google Scholar). The recent demonstrations that plant, yeast, and mammalian PABP can interact physically with the N-terminal part of eIF4G (11Tarun Jr., S.Z. Sachs A.B. EMBO J. 1996; 15: 7168-7177Crossref PubMed Scopus (574) Google Scholar, 12Le 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 (227) Google Scholar, 13Piron M. Vende P. Cohen J. Poncet D. EMBO J. 1998; 17: 5811-5821Crossref PubMed Scopus (299) Google Scholar) and that capped polyadenylated mRNAs can be physically circularized in vitro by the addition of purified eIF4E, eIF4G and PABP (14Wells S.E. Hillner P.E. Vale R.D. Sachs A.B. Mol. Cell. 1998; 2: 135-140Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar) suggest that cap-poly(A) cooperativity somehow results from the physical interactions between the mRNA 5′ and 3′ ends (9Jacobson A. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, NY1996: 451-480Google Scholar, 15Gallie D.R. Gene. 1998; 216: 1-11Crossref PubMed Scopus (239) Google Scholar). Indeed, disruption of the eIF4G-PABP interaction abolishes both the positive effects of poly(A) on uncapped mRNA translation in yeast (16Tarun Jr., S.Z. Wells S.E. Deardoff J.A. Sachs A.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9046-9051Crossref PubMed Scopus (262) Google Scholar) and abrogates cap-poly(A) synergy in cell-free mammalian extracts (17Michel Y.M. Poncet D. Piron M. Kean K.M. Borman A.M. J. Biol. Chem. 2000; 275: 32268-32276Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar).In mechanistic terms, the interaction of wheat germ PABP with eIF4F has been shown to increase the affinity of the eIF4F complex for cap analogue by some 40-fold (18Wei C-C. Balasta M.L. Ren J. Goss D.J. Biochemistry. 1998; 37: 1910-1916Crossref PubMed Scopus (112) Google Scholar). Similarly, the eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped 5′ end of polyadenylated mRNAs in mammalian extracts (19Borman A.M. Michel Y.M. Kean K.M. Nucleic Acids Res. 2000; 28: 4068-4075Crossref PubMed Google Scholar). Furthermore, the affinity of eIF4F-complexed plant PABP for poly(A) is significantly greater than that of free PABP (12Le 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 (227) Google Scholar). Thus, it seems probable that mRNA 5′–3′ end cross-talk enhances translation, at least in part, by stimulating the formation of initiation factor-mRNA complexes. Because this process only requires a series of RNA-protein and protein-protein interactions, it could occur, theoretically, in trans.Several further potential translational advantages could be envisaged to result from mRNA circularization. For instance, the PABP-eIF4G interaction would provide a means of tethering the eIF4F complex to a mRNA (albeit at the 3′ end), preventing its dissociation from the message after each initiation event and maintaining an elevated local concentration of eIF4F near the mRNA 5′ end. Noncovalent linking of mRNA ends could also facilitate the recapture of ribosomes that have terminated translation and dissociated from the RNA but remain in its proximity. Obviously, these additional potential advantages of mRNA functional circularization would require that the effects of PABP-bound poly(A) be exclusively intramolecular. In support ofcis-stimulation by poly(A), several studies have shown that the addition of exogenous poly(A) to in vitro translation extracts inhibited translation of all mRNAs, regardless of the nature of the 5′ and 3′ ends (20Gallie D.R. Tanguy R. J. Biol. Chem. 1994; 269: 17166-17173Abstract Full Text PDF PubMed Google Scholar, 21Jacobson A. Favreau M. Nucleic Acids Res. 1983; 11: 6353-6368Crossref PubMed Scopus (108) Google Scholar). In contrast, one study reported that whereas capped polyadenylated mRNA translation was inhibited by exogenous poly(A), that of capped nonpolyadenylated mRNA was slightly stimulated (∼1.5-fold) at certain poly(A) concentrations (5Munroe D. Jacobson A. Mol. Cell. Biol. 1990; 10: 3441-3455Crossref PubMed Scopus (279) Google Scholar). These previous analyses were performed in nuclease-treated rabbit reticulocyte lysates (RRL) or in wheat germ extracts, which are far from physiological in reproducing the positive effects of poly(A) on translation. Indeed, in such extracts, poly(A) dependence is rather low and the combined effects of cap and poly(A) on translation are at best additive (17Michel Y.M. Poncet D. Piron M. Kean K.M. Borman A.M. J. Biol. Chem. 2000; 275: 32268-32276Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar).We recently described a RRL-based system that faithfully reproduces the synergistic cooperation between the cap and poly(A) for translation initiation by mimicking the competitive environment of the intact cell. Because a competitive environment is assured by rendering translation components physically limiting, rather than by adding excess competitor mRNAs, this system allows the molecular dissection of the functional interactions between mRNA 5′ and 3′ ends (17Michel Y.M. Poncet D. Piron M. Kean K.M. Borman A.M. J. Biol. Chem. 2000; 275: 32268-32276Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 19Borman A.M. Michel Y.M. Kean K.M. Nucleic Acids Res. 2000; 28: 4068-4075Crossref PubMed Google Scholar,22Michel Y.M. Borman A.M. Paulous S. Kean K.M. Mol. Cell. Biol. 2001; 21: 4097-4109Crossref PubMed Scopus (84) Google Scholar). Here we describe the use of this system for a detailed study of the effect of exogenous poly(A) on translation of mRNAs carrying various combinations of 5′ cap and 3′ poly(A) tail. We demonstrate that poly(A) chains added in trans can dramatically enhance translation of either capped, nonpolyadenylated mRNAs or capped polyadenylated mRNAs carrying poly(A) tails of suboptimal length. As described previously for cap-poly(A) synergy,trans stimulation requires the integrity of the eIF4G-PABP interaction and results at least in part from an increased functional affinity of eIF4E for the 5′ ends of capped nonpolyadenylated mRNAs.RESULTSMany of the mechanisms proposed to underlie cap-poly(A) synergy require that the positive effects of poly(A) tails on translation are restricted to the mRNAs which carry these 3′ tails, that is that translation stimulation by poly(A) is exclusively a cisevent. The aim of the current study was to test this hypothesis. The recently described nuclease-treated, ribosome-depleted RRL translation system is ideal for such a study, because poly(A)-dependence results from physically limiting concentrations of ribosomes and ribosome-associated translation factors rather than the presence of intact, functional competitor mRNAs (19Borman A.M. Michel Y.M. Kean K.M. Nucleic Acids Res. 2000; 28: 4068-4075Crossref PubMed Google Scholar). Effectively, functional analyses of the mechanism of poly(A) action are hindered in competitor-based systems by the fact that poly(A)-dependence of the extract is lost as soon as translation conditions are altered (7Preiss T. Hentze M.W. Nature. 1998; 392: 516-520Crossref PubMed Scopus (215) Google Scholar, 17Michel Y.M. Poncet D. Piron M. Kean K.M. Borman A.M. J. Biol. Chem. 2000; 275: 32268-32276Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar,19Borman A.M. Michel Y.M. Kean K.M. Nucleic Acids Res. 2000; 28: 4068-4075Crossref PubMed Google Scholar). The pOp24 plasmids used for the present study have been described elsewhere (17Michel Y.M. Poncet D. Piron M. Kean K.M. Borman A.M. J. Biol. Chem. 2000; 275: 32268-32276Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Briefly, the plasmids contain a short oligonucleotide-derived 5′ UTR followed by the region coding for the human immunodeficiency virus (HIV-1Lai) p24 protein and the influenza virus NS 3′ UTR. Three versions of the plasmid differ only by the absence or presence of an A50 or A100 tract downstream of the 3′ UTR (see “Experimental Procedures”).As previously demonstrated (17Michel Y.M. Poncet D. Piron M. Kean K.M. Borman A.M. J. Biol. Chem. 2000; 275: 32268-32276Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 19Borman A.M. Michel Y.M. Kean K.M. Nucleic Acids Res. 2000; 28: 4068-4075Crossref PubMed Google Scholar), the effects of capping and polyadenylation on translation of mRNAs derived from the pOp24 plasmids in a standard RRL assay system are at best additive,i.e. the cumulative stimulatory effects of cap and poly(A) are not dramatically greater than the effects of the sum of each modification alone (Table I). However, when the same mRNAs are translated in a ribosome-depleted RRL extract under optimal conditions (Table I; Ref. 19Borman A.M. Michel Y.M. Kean K.M. Nucleic Acids Res. 2000; 28: 4068-4075Crossref PubMed Google Scholar), significant cap-poly(A) synergy is observed. This synergy is typically greater than 5-fold with poly(A) tails of 50 residues and exceeds 10-fold with poly(A100) tails (Table I). Similar poly(A) length-dependent increases in cap-poly(A) cooperativity have been reported in numerous in vitro translation systems (see for example Ref. 25Preiss T. Muckenthaler M. Hentze M.W. RNA. 1998; 4: 1321-1331Crossref PubMed Scopus (89) Google Scholar).Table IEffects of capping and polyadenylation on mRNA translation efficiency in standard (nuclease-treated) and ribosome-depleted rabbit reticulocyte lysatesCap/poly(A) context1-aRNAs were transcribed with or without cap /with or without a poly(A) tail of 50 or 100 A residues.Translation efficiency of pOp24-derived mRNAsStandard RRLDepleted RRL1-bCap-poly(A) synergy calculated as: stimulation upon capping and polyadenylation divided by [stimulation upon capping added to stimulation upon polyadenylation] is given in parentheses alongside each value for translation efficiency on +/+ mRNA in depleted RRL. For comparison, the same calculation is shown for translation in standard RRL. Final concentrations in mRNA were 5 μg/ml.−/−11-cTranslation efficiency of −/− mRNA in either translation system was arbitrarily taken as 1. Other values are given relative to the efficiency of −/− mRNA translation in each translation system.1+/−1411−/+501.82.8+/+5019 (1.2×)88 (6.4×)−/+1002.94.3+/+10023 (1.4×)166 (10.8×)1-a RNAs were transcribed with or without cap /with or without a poly(A) tail of 50 or 100 A residues.1-b Cap-poly(A) synergy calculated as: stimulation upon capping and polyadenylation divided by [stimulation upon capping added to stimulation upon polyadenylation] is given in parentheses alongside each value for translation efficiency on +/+ mRNA in depleted RRL. For comparison, the same calculation is shown for translation in standard RRL. Final concentrations in mRNA were 5 μg/ml.1-c Translation efficiency of −/− mRNA in either translation system was arbitrarily taken as 1. Other values are given relative to the efficiency of −/− mRNA translation in each translation system. Open table in a new tab Efficient Stimulation of Translation by Free Poly(A) in TransTo examine the potential of polyadenylic acid to stimulate translation efficiency in trans, we employed a commercial poly(A) preparation consisting of poly(A) chains in the approximate range of 15 to 600 residues, present in roughly equimolar proportions (Fig. 1 a, Load lane). This total poly(A) preparation was added at a range of concentrations to translation extracts programmed with Op24-derived mRNAs carrying a cap, a poly(A50) tail or both (Fig.1 b). Added poly(A) significantly inhibited translation of uncapped polyadenylated mRNA even at low concentrations (−/+ lanes). Similarly, translation of capped mRNA carrying an A50 tail was reduced 2–3-fold at low poly(A) concentrations (molar ratios of poly(A):mRNA <1:1) and then slightly but reproducibly stimulated at higher poly(A) to mRNA ratios to return to and even exceed the efficiency observed in the absence of poly(A) addition (Fig. 1 b, +/+ lanes). However, at the highest poly(A) concentration tested, translation efficiency was again suppressed with respect to the control lane (Fig. 1 b, +/+ lanes). Finally, although capped, nonpolyadenylated mRNA translation was unaffected by low concentrations of poly(A), it was dramatically stimulated even to exceed slightly that of +/+ mRNA translated in the absence of added poly(A) when the molar concentration of poly(A) to mRNA exceeded 1:1 (5–8-fold stimulation at poly(A):mRNA ratios of 2.5–10; Fig. 1 b, +/− lanes). Even when the molar ratio of poly(A) to +/− mRNA exceeded 10, although translation stimulation was significantly reduced, absolute efficiency still exceeded that observed in the absence of poly(A) addition.Thus, free poly(A) can dramatically stimulate capped, nonpolyadenylated mRNA translation and also moderately improve the translation of capped mRNAs, which carry short poly(A) tails. It should be noted that the order of addition of mRNA and poly(A) to extracts and the omission of extra Mg2+ ions to compensate for sequestering by poly(A) did not significantly alter the results (data not shown). Additionally, the observed effects were specific to poly(A), as evidenced by the inability of either poly(dA) or poly(U) to effect similar stimulation of translation on either +/− or +/+ mRNAs (Table II). Interestingly, high concentrations of poly(U) dramatically inhibited +/+ mRNA translation, presumably by interacting with the mRNA 3′ poly(A) tail and effectively sequestering this element. Finally, exogenous poly(A) had no discernible effect on mRNA stability in the depleted RRL system (data not shown), which is perhaps not surprising given that poly(A) tails acting in cis stimulate translation in this system without altering mRNA half-life (17Michel Y.M. Poncet D. Piron M. Kean K.M. Borman A.M. J. Biol. Chem. 2000; 275: 32268-32276Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 22Michel Y.M. Borman A.M. Paulous S. Kean K.M. Mol. Cell. Biol. 2001; 21: 4097-4109Crossref PubMed Scopus (84) Google Scholar).Table IIEffects of different homopolymers of equivalent length on translation efficiencyMolar ratio of polymer to mRNA+/− mRNA translation with:+/+ mRNA translation with:Poly(A)Poly(dA)Poly(U)Poly(A)Poly(dA)Poly(U)0.21.270.871.390.380.790.660.41.821.011.900.390.200.800.82.110.970.860.420.520.752.55.461.060.740.970.700.543.04.011.500.541.770.880.06Translation efficiency is expressed relative to that observed with the same mRNA in the absence of homopolymer addition, which was arbitrarily set as 1.0 for each mRNA. In absolute terms, translation of +/+ mRNA was 8-fold more efficient than that of +/− mRNA under the experimental conditions used (final mRNA concentrations of 5 μg/ml). Open table in a new tab Length Dependence of Poly(A) for Trans StimulationTo address the length requirements for this trans stimulation of translation by free poly(A), the original heterogeneous poly(A) preparation was separated electrophoretically, and three distinct populations of different sizes were excised from the gels and purified: 19–52 residues (Pop. 1), 50–180 residues (Pop.2), and 150–400 residues (Pop. 3) (Fig. 1 a; see “Experimental Procedures”). These fractionated poly(A) populations were then each compared at equivalent molar concentrations (with respect to the number of poly(A) chains) with the nonfractionated original preparation for their effects on capped (+/−) and capped poly(A50) (+/+) mRNA translation (Fig.2). Extracts were programmed with 2.5 rather than 5 μg/ml pOp24-derived mRNAs to facilitate the addition of molar excess poly(A). Under these conditions, stimulation of +/− and +/+ mRNA translation with the nonfractionated poly(A) preparation was even more dramatic (14- and 2.5-fold stimulation, respectively, with 2.5 μg/ml of +/− and +/+ mRNAs, as opposed to 5- and 1.5-fold using 5 μg/ml of the same RNAs; compare Fig.1 b and left-hand panel, Fig. 2).Figure 2Trans stimulation of translation by poly(A) is poly(A) chain length-dependent.Ribosome-depleted RRL translation reactions were programmed with 2.5 μg/ml (final RNA concentration) Op24 mRNAs synthesized to contain a cap (+/−) or cap and poly(A50) tail (+/+). Reactions were then supplemented with buffer (0 lanes) or the indicated molar ratios (with respect to programming RNA) of poly(A) pool (Total lanes) or purified poly(A) populations 1, 2, or 3 (see Fig. 1 A, Pop. lanes). MgCl2was added in a 1:1 molar ratio with respect to the added A residues. Analysis of translation products was exactly as described in the legend for Fig. 1 B; open squares and filled circles correspond to +/+ and +/− mRNAs, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Short poly(A) chains (19–52 residues) had no significant effect on the translation of +/− mRNAs but moderately inhibited +/+ mRNA translation at all of the poly(A) concentrations tested (Fig. 2,Pop 1). Conversely, the addition of poly(A) chains of between 50 and 180 residues affected the translation of both +/− and +/+ mRNAs in a manner similar to that observed with the nonfractionated poly(A) preparation (Pop 2 and Total lanes, Fig. 2), with the exception that +/− mRNA translation was stimulated less efficiently with population 2 poly(A) than with nonfractionated poly(A) at equivalent molar concentrations. Finally, poly(A) chains of 150–400 residues stimulated both +/− and +/+ mRNA translation, albeit less dramatically than the intermediate length chains (Pop 3 and Pop 2 lanes, Fig. 2). However, stimulation of +/− and +/+ mRNA translation was observed at poly(A) to mRNA ratios of less than 1, suggesting that these long poly(A) chains could simultaneously trans-activate the translation of several mRNA molecules. It should also be noted that although the estimated added molar excesses of poly(A) for the different populations are approximations (see “Experimental Procedures”), two independent series of fractionated poly(A) prepared from the same original poly(A) pool gave virtually identical results (data not shown).Effects of Exogenous Poly(A) Chains on the Dose Responses of mRNAs Translated in Depleted RRLOne of the features of translation in the ribosome-depleted RRL system is that the translation extracts are much more rapidly saturated by capped, polyadenylated than by capped, nonpolyadenylated mRNAs (Ref. 17Michel Y.M. Poncet D. Piron M. Kean K.M. Borman A.M. J. Biol. Chem. 2000; 275: 32268-32276Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar; Fig.3). Effectively, a poly(A) tail actingin cis causes a displacement in the dose response of a capped mRNA, without actually increasing the yield of translation product obtained from the RNA under saturating conditions. Thus, the effects of exogenous poly(A) chains on the dose responses of capped, nonpolyadenylated and capped, polyadenylated mRNAs were determined. Exogenous poly(A) chains did not increase the absolute yield of translation product from either +/− or +/+ mRNAs, but rather they behaved like a poly(A) tail acting in cis, provoking saturation of the translation extracts at significantly reduced mRNA concentrations (Fig. 3). Indeed, translation of +/+ mRNA at the highest RNA concentrations tested was actually inhibited by the addition of exogenous poly(A), presumably because the exogenous poly(A) was acting to render the concentration of mRNA saturating.Figure 3Effects of exogenous poly(A) on the dose responses of mRNAs translated in depleted RRL. Ribosome-depleted RRL translation reactions were programmed with (from left to right) 12, 6, 3, or 1.5 μg/ml of Op24 mRNAs synthesized to contain a cap (+/−) or cap and poly(A50) tail (+/+) as indicated. Reactions then received H100 buffer (− panels) or a constant 4-fold molar excess (with respect to mRNA) of population 2 poly(A) chains in H100 buffer (+ panels). MgCl2 was added in a 1:1 molar ratio with respect to the added A residues in the poly(A) population. Analysis of translation efficiency was exactly as described in the legend for Fig. 1; translation efficiency (in arbitrary units) is plotted beloweach panel. Open and filled circles (+/− mRNA) and squares (+/+ mRNA) correspond, respectively, to reactions without and with exogenous poly(A). The stimulation of +/− and +/+ mRNA translation by exogenous poly(A) is also shown at each mRNA concentration (triangles anddashed lines).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Trans Stimulation by Poly(A) Requires the eIF4G-PABP Interaction and Increases the Functional Affinity of eIF4E for CapWe previously demonstrated that 5′ cap-3′poly(A) cooperative translation stimulation required the integrity of the eIF4G-PABP interaction. Cap-poly(A) synergy was abolished when extracts were incubated with a fragment of the rotavirus nonstructural protein NSP3, which interacts with eIF4G and displaces PABP from the eIF4F complex (17Michel Y.M. Poncet D. Piron M. Kean K.M. Borman A.M. J. Biol. Chem. 2000; 275: 32268-32276Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Thus, we evaluated whether trans stimulation of translation by poly(A) also depended on the eIF4G-PABP interaction. To this end, translation extracts were pretreated with NSP3 fragment or with control buffer, programmed with +/− or +/+ poly(A50) mRNAs and then supplemented with different amounts of 50–180 residue poly(A) chains. Inclusion of NSP3 fragment at 10 μg/ml, a concentration previously determined to be sufficient to disrupt the eIF4G-PABP interaction (Ref. 17Michel Y.M. Poncet D. Piron M. Kean K.M. Borman A.M. J. Biol. Chem. 2000; 275: 32268-32276Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar; data not shown), abolished cap-poly(A) synergy on +/+ mRNA as expected and moreover totally inhibited the stimulatory effects of exogenous poly(A) on +/− and +/+ mRNA translation at all of the concentrations of poly(A) tested (Fig.4 A). However, NSP3 did not neutralize the inhibitory effects of low concentrations of poly(A) on +/+ mRNA translation.Figure 4Trans stimulation of translation by free poly(A) requires the eIF4G-PABP interaction. A, ribosome-depleted RRL translation reactions were preincubated for 15 min at 4 °C with NSP3 (10 μg/ml final concentration) or buffer before being programmed with +/− or +/+ pOp24-derived mRNAs (2.5 μg of RNA/ml) and supplemented with increasing molar concentrations of population 2 poly(A) as indicated. Analysis of translation efficiency was exactly as described in the legend to Fig. 1.Open and filled symbols correspond, respectively, to reactions pretreated with NSP3 or buffer and programmed with +/+ (squares) or +/− mRNAs (circles).B, ribosome-depleted RRL translation reactions were preincubated with NSP3 (10 μg/ml) or control buffer prior to being programmed with 2.5 μg/ml pOp24-derived capped, nonpolyadenylated mRNA. Reactions were then supplemented with a 2.5-fold molar excess (with respect to mRNA) of population 2 poly(A) chains in H100 buffer or H100 buffer alone. Finally, reactions received increasing concentrations of m7GpppG cap analogue. MgCl2was added in a 1:1 molar ratio with respect to the added cap analogue and A residues in the poly(A) population. Analysis of translation efficiency was exactly as described in the legend for Fig. 1. Translation efficiency for +/− mRNA translated in the presence of buffer alone is indicated (open circles); population 2 poly(A) alone (closed circles) or NSP3 plus population 2 poly(A) (filled squares) is plotted against cap analogue concentration.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It has also been demonstrated previously that the eIF4G-PABP interaction on capped, polyadenylated mRNAs increases the functional affinity of eIF4E for the capped mRNA 5′ end (18Wei C-C. Balasta M.L. Ren J. Goss D.J. Biochemistry. 1998; 37: 1910-1916Crossref PubMed Scopus (112) Google Scholar, 19Borman A.M. Michel Y.M. Kean K.M. Nucleic Acids Res. 2000; 28: 4068-4075Crossref PubMed Google Scholar). Indeed, 8–10-fold" @default.
• W2075956940 created "2016-06-24" @default.
• W2075956940 creator A5055423758 @default.
• W2075956940 creator A5055578001 @default.
• W2075956940 creator A5062892359 @default.
• W2075956940 creator A5077978563 @default.
• W2075956940 date "2002-09-01" @default.
• W2075956940 modified "2023-09-30" @default.
• W2075956940 title "Free Poly(A) Stimulates Capped mRNA Translation in Vitro through the eIF4G-Poly(A)-binding Protein Interaction" @default.
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