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A viral sequence in the 3′-untranslated region mimics a 5′ cap in facilitating translation of uncapped mRNA

1997; Springer Nature; Volume: 16; Issue: 13 Linguagem: Inglês

10.1093/emboj/16.13.4107

1460-2075

Shanping Wang, Karen Browning, W. Allen Miller,

RNA and protein synthesis mechanisms

Article1 July 1997free access A viral sequence in the 3′-untranslated region mimics a 5′ cap in facilitating translation of uncapped mRNA Shanping Wang Shanping Wang Molecular, Cellular and Developmental Biology Program, 351 Bessey Hall, Iowa State University, Ames, IA, 50011 USA Present address: Laboratory of Tumor Biology, Massachusetts General Hospital Cancer Center, Building 149, Harvard University, Charlestown, MA, 02129 USA Search for more papers by this author Karen S. Browning Karen S. Browning Department of Chemistry and Biochemistry, University of Texas, Austin, TX, 78712 USA Search for more papers by this author W.Allen Miller Corresponding Author W.Allen Miller Molecular, Cellular and Developmental Biology Program, 351 Bessey Hall, Iowa State University, Ames, IA, 50011 USA Departments of Plant Pathology, and Biochemistry & Biophysics, 351 Bessey Hall, Iowa State University, Ames, IA, 50011 USA Search for more papers by this author Shanping Wang Shanping Wang Molecular, Cellular and Developmental Biology Program, 351 Bessey Hall, Iowa State University, Ames, IA, 50011 USA Present address: Laboratory of Tumor Biology, Massachusetts General Hospital Cancer Center, Building 149, Harvard University, Charlestown, MA, 02129 USA Search for more papers by this author Karen S. Browning Karen S. Browning Department of Chemistry and Biochemistry, University of Texas, Austin, TX, 78712 USA Search for more papers by this author W.Allen Miller Corresponding Author W.Allen Miller Molecular, Cellular and Developmental Biology Program, 351 Bessey Hall, Iowa State University, Ames, IA, 50011 USA Departments of Plant Pathology, and Biochemistry & Biophysics, 351 Bessey Hall, Iowa State University, Ames, IA, 50011 USA Search for more papers by this author Author Information Shanping Wang1,2, Karen S. Browning3 and W.Allen Miller 1,4 1Molecular, Cellular and Developmental Biology Program, 351 Bessey Hall, Iowa State University, Ames, IA, 50011 USA 2Present address: Laboratory of Tumor Biology, Massachusetts General Hospital Cancer Center, Building 149, Harvard University, Charlestown, MA, 02129 USA 3Department of Chemistry and Biochemistry, University of Texas, Austin, TX, 78712 USA 4Departments of Plant Pathology, and Biochemistry & Biophysics, 351 Bessey Hall, Iowa State University, Ames, IA, 50011 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:4107-4116https://doi.org/10.1093/emboj/16.13.4107 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info For recognition by the translational machinery, most eukaryotic cellular mRNAs have a 5′ cap structure [e.g. m7G(5′)ppp(5′)N]. We describe a translation enhancer sequence (3′TE) located in the 3′-untranslated region (UTR) of the genome of the PAV barley yellow dwarf virus (BYDV-PAV) which stimulates translation from uncapped mRNA by 30- to 100-fold in vitro and in vivo to a level equal to that of efficient capped mRNAs. A four base duplication within the 3′TE destroyed the stimulatory activity. Efficient translation was recovered by addition of a 5′ cap to this mRNA. Translation of both uncapped mRNA containing the 3′TE in cis and capped mRNA lacking any BYDV-PAV sequence was inhibited specifically by added 3′TE RNA in trans. This inhibition was reversed by adding initiation factor 4F (eIF4F), suggesting that the 3′TE, like the 5′ cap, mediates eIF4F-dependent translation initiation. The BYDV-PAV 5′UTR was necessary for the 3′TE to function, except when the 3′TE itself was moved to the 5′UTR. Thus, the 3′TE is sufficient for recruiting the translation factors and ribosomes, while the viral 5′UTR may serve only for the long distance 3′–5′ communication. Models are proposed to explain this novel mechanism of cap-independent translation initiation facilitated by the 3′UTR. Introduction Almost all eukaryotic cellular mRNAs contain a 5′ m7G(5′)ppp(5′)N cap structure which is required for efficient initiation of translation. According to the ribosome scanning model of eukaryotic translation initiation, initiation factor eIF4F specifically recognizes the 5′ cap structure and, with the help of other initiation factors such as eIF3, recruits the 43S ribosomal subunit initiation complex that then scans 5′ to 3′ along the mRNA (Kozak, 1989; Merrick and Hershey, 1996). When the first (or second in the case of leaky scanning) AUG codon is reached, the 60S ribosomal subunit joins and peptide elongation ensues. Although the ribosome scanning model explains the mechanisms of various translational regulatory elements in the 5′-untranslated regions (UTRs) of mRNAs, numerous examples exist of translational control elements in the 3′UTRs of mRNAs. The 3′ poly(A) tail, found on most eukaryotic cellular mRNAs, stimulates translation initiation and stabilizes mRNA (Jacobson, 1996). The 5′ cap and poly(A) tail act synergistically to stimulate initiation in vivo (Gallie, 1991) and in a yeast in vitro translation system (Tarun and Sachs, 1995). In the case of viral RNAs that lack a poly(A) tail, sequences in the 3′UTR, such as the pseudoknot-rich domain of tobacco mosaic virus (TMV), appear to substitute functionally for a poly(A) tail to stimulate translation in conjunction with the 5′ cap (Gallie and Walbot, 1990; Gallie, 1991). Other cis-acting elements in 3′UTRs control translation initiation either by modulating poly(A) tail length during embryo development (Sheets et al., 1995) or via binding of a specific regulatory protein that inhibits initiation (Standart and Jackson, 1994; Curtis et al., 1995; Dubnau and Struhl, 1996). In all these examples, the mRNA must have a 5′ cap in order for the 3′ element to function. Thus, in previously described mRNAs, cap recognition is likely to be an essential component in the communication between 3′ and 5′ ends (Tarun and Sachs, 1995; Hentze, 1997). Mammalian eIF4F complex is comprised of eIF4E (the cap-binding subunit), eIF4G (p220) and the more loosely associated eIF4A (helicase) (Merrick, 1994). Plant cells contain two forms of the 4F complex: eIF4F, consisting of 26 and 220 kDa subunits (homologous to eIF4E and eIF4G, respectively), and eIFiso4F, consisting of 28 and 86 kDa subunits (Browning, 1996). eIF4A probably has the same function in plants but does not co-purify with eIF4F, so it is not considered a subunit of this complex (Browning, 1996). In defined cell-free systems, capped mRNAs have a reduced requirement for eIF4F compared with uncapped mRNAs (Fletcher et al., 1990). Many viral mRNAs lack a 5′ cap or a poly(A) tail, or both. They have evolved ways of ensuring efficient translation of their genes from uncapped mRNAs (Carrington and Freed, 1990; Tsukiyamakohara et al., 1992; Sarnow, 1995), often at the expense of the host cell. The most well-documented example is the translation of picornavirus RNAs (Jackson and Kaminski, 1995). Picornaviral RNAs lack a 5′ cap structure and have an extremely long, highly structured 5′UTR, including many AUG codons upstream of the start codon of the main open reading frame (ORF). Rather than scanning from the 5′ end, ribosomes bind internally in this long leader at the internal ribosomal entry site (IRES) just upstream of the start codon, in a cap-independent manner (Pelletier and Sonenberg, 1988; Sarnow, 1995). Although IRES-mediated cap-independent translation does not conform to the rule of 5′ cap–eIF4E recognition, it still employs the other canonical initiation factors including the eIF4G subunit of eIF4F (Pestova et al., 1996), and follows the scanning concept of ribosome binding to the 5′UTR followed by scanning in a 3′ direction until the appropriate start codon is reached for initiation of translation. In contrast to the above examples, we showed previously that the genomic RNA of the PAV barley yellow dwarf virus (BYDV-PAV) harbors a sequence in the 3′UTR that confers cap-independent translation initiation at the 5′ AUG of the mRNA in wheat germ extracts (Wang and Miller, 1995). The BYDVs are ubiquitous, economically important viruses of small grains (D'Arcy and Burnett, 1995). BYDV-PAV is a subgroup I luteovirus, whereas some other BYDVs are members of the very divergent subgroup II (Miller et al., 1995; Miller and Rasochova, 1997). The mechanism of translation initiation of BYDV-PAV is remarkable because the 3′ translation enhancer (3′TE) that confers cap-independent translation is located 5 kb downstream, and is separated by several large untranslated ORFs, from the initiation codon. Furthermore, unlike other translational control elements in the 3′UTR, such as poly(A) tails, the 3′TE controls initiation at the 5′ AUG in the absence of a 5′ cap. The only other mRNA known to resemble this, in function, is satellite tobacco necrosis virus (STNV) RNA (Danthinne et al., 1993; Timmer et al., 1993). This RNA differs from BYDV-PAV in that its 3′ stimulatory element is located immediately 3′ of the only ORF on the RNA, and 600 nucleotides downstream of the initiator AUG. The stimulatory elements of BYDV-PAV and STNV RNAs share no obvious sequence homology. For both RNAs, cap-independent translation has been reported only in cell-free extracts. Like capped mRNAs, STNV RNA translation depends on eIF4F or eIFiso4F, but the amount of factor needed for maximal translation is one-tenth of that needed for capped mRNAs (Timmer et al., 1993). Here, we (i) localize the 3′TE to a smaller region in the BYDV-PAV genome than reported previously, (ii) show that it functions in vivo but requires more of the 3′UTR than in wheat germ extracts, (iii) provide evidence that the 3′TE facilitates initiation in an eIF4F-dependent manner and (iv) show that the translation initiation and 3′–5′ communication functions can be uncoupled. These data shed light on possible new mechanisms for a 3′UTR in mediating translation initiation. Results Localization of minimal 3′TE sequence Previously, we showed that a 500 base region in the genomic RNA of BYDV-PAV facilitated translation of uncapped mRNA in vitro (Wang and Miller, 1995). Here, we show the ability of a smaller portion of this region to stimulate translation of uncapped mRNA. The mRNAs are in vitro transcripts containing the Escherichia coli uidA (GUS) reporter gene flanked by various 5′ and 3′UTRs (Figure 1A). Uncapped GUS mRNA that contains the 109 nucleotide sequence spanning the intergenic region between BYDV-PAV ORFs 5 and 6 in its 3′UTR (bases 4817–4925 of the BYDV-PAV genome) was translated with the same efficiency in vitro as transcripts that contain the larger (500 nt) 3′TE sequence (Figure 1B, lanes 3–5). This construct yielded >50-fold more GUS protein than transcripts lacking the intercistronic sequence (Figure 1B, compare lanes 2 and 3). Deletion of bases upstream of nucleotide 4837 (Wang and Miller, 1995) or downstream of nucleotide 4873 (data not shown) within the 3′TE abolished stimulatory activity. Therefore, a subset (bases 4817–4925) of the previously reported 500 base fragment is sufficient to confer translation enhancement of uncapped mRNA in a wheat germ extract. We refer to this 109 nucleotide sequence as the in vitro-defined, or 109 nt, 3′TE. Figure 1.(A) Maps of transcripts. The genome organization of BYDV-PAV RNA with numbered ORFs (Miller et al., 1988) is shown at the top. A bold black line beneath the ORFs (numbered boxes) indicates the 5677 nt genomic RNA. Maps below the genome depict transcripts coding for GUS (ORF not to scale) containing viral sequence (bold lines), vector or Ω sequence (thin lines) or poly(A) tails (A30) in their UTRs. The 109 nt 3′TE is the intergenic region (between dashed lines) between ORFs 5 and 6. BF transcripts are identical to those shown, except that they contain the four base duplication at the filled and re-ligated BamHI site (B) within the 109 nt 3′TE. Abbreviations: Sc, ScaI4513; B, BamHI4837; P, PstI5009; Sm, SmaI5677 (numbered as in Miller et al., 1988); R1, EcoRI; ICR1, EcoICRI; X1, XbaI. (B and C) Wheat germ translation products of the indicated transcripts. Each uncapped mRNA (0.2 pmol) was translated and their products analyzed electrophoretically as in Wang and Miller (1995). The relative radioactivity in the GUS product, as determined with a Phosphorimager, is indicated below each lane. (B) Molecular weights (in kDa) of translation products of brome mosaic virus (BMV) RNA (lane 1) and GUS (68 kDa) are at the left. The translation efficiency in lane 5 was defined arbitrarily as 100%. Templates used in lanes 2–5 were uncapped RNAs prepared by run-off transcription from plasmids linearized with the indicated restriction enzymes. (C) All transcripts were from EcoRI-cut plasmids, except for lanes 1 and 2, which were from EcoICRI-cut plasmid. Transcripts with names ending in A+ are polyadenylated. C, capped transcript; U, uncapped transcript. Download figure Download PowerPoint To confirm the specificity of the stimulation by the 3′TE, a mutation was introduced by inserting a four base duplication (GAUC) within the BamHI4837 site in the 3′TE (PGUS109BF; all clones with the BF designation contain this BamHI fill-in mutation). This duplication abolished the stimulatory activity of the 3′TE (Figure 1C, compare lanes 4 and 6). Addition of a 5′ cap rescued translation of these mutant transcripts to the level observed for uncapped, 3′TE-containing PGUS109 (Figure 1C, lanes 4 and 5). The presence of a poly(A) tail had little effect on translation of any transcripts in wheat germ extracts (Figure 1C), which is consistent with previously reported observations (Gallie, 1991; Wang and Miller, 1995). Activity of the 3′TE in vivo To assess the activity of the 3′TE in vivo, GUS-encoding transcripts with various 5′ and 3′ UTRs were electroporated into oat protoplasts, and translational efficiency was measured by assaying for GUS activity. An uncapped control transcript, containing a plasmid-derived 5′UTR and a 30 nt poly(A) tail yielded no significant GUS activity (Figure 2A, VecGUSA+). When capped, this mRNA expressed GUS at 50-fold above background, consistent with the essential role for the 5′ cap. Replacement of the plasmid-derived 5′UTR with that from BYDV-PAV stimulated GUS expression from capped mRNA 3-fold further (Figure 2A, PGUSA+). Addition of the 109 nt 3′TE was not sufficient for translation of mRNA in vivo (Figure 2A, compare PGUS109 and PGUS). The presence of a poly(A) tail in addition to the 109 nt 3′TE resulted in a 10-fold increase above background in GUS expression from uncapped mRNA (Figure 2A, compare uncapped PGUS109A+ with uncapped PGUS109). However, addition of a 5′ cap to this transcript stimulated GUS expression by another order of magnitude (Figure 2, compare capped and uncapped PGUS109A+). Construct PGUS109BFA+, which contains the four base GAUC duplication, yielded no GUS activity when lacking a cap, but was fully active when capped (Figure 2A). These results show that the 109 nt 3′TE only partially, but significantly, facilitated cap-independent translation in vivo. Figure 2.(A and B) GUS expression in protoplasts electroporated with transcripts containing various 5′ and 3′ UTRs (see Figure 1A). GUS activity is measured in nmoles (nm) of methylumbelliferone (MU) produced per μg of cellular protein per minute in a 2 h reaction. Transcripts containing a poly(A) tail (A+) are from EcoRI-linearized plasmids; others are from plasmids linearized with SmaI. Transcript PGUS is from EcoICRI-linearized pPGUS109. In (A), data represent averages (± SD) from three separate experiments, each of which was performed in duplicate. In (B), data represent averages from two separate experiments, each of which was performed in duplicate. (C) Wheat germ translation products of transcripts used in (B) were analyzed and quantified as in Figure 1. C, capped; U, uncapped transcript. Download figure Download PowerPoint We tested the effect of additional viral sequence in the 3′UTR on 3′TE function in vivo. PAV bases 4515–5677 (the 3′-terminal 1162 bases of the genome) were placed in the 3′UTR of the GUS reporter construct. Uncapped transcript from this plasmid gave activity >100 times background (Figure 2A and B, PGUS1162). Addition of a cap stimulated expression no more than 2-fold (Figure 2A and B, compare capped and uncapped PGUS1162). The four base duplication in the BamHI4837 site abolished expression from uncapped transcript both in vitro (Figure 2C, lanes 2 and 4) and in vivo (Figure 2B, compare uncapped PGUS1162BF with uncapped PGUS1162), verifying the specificity of the 3′TE effect. Furthermore, the four base duplication had no effect on expression of the capped transcript (Figure 2B, compare capped PGUS1162BF with capped PGUS1162). Interestingly, the stimulatory activity of the 1162 nt 3′ end of BYDV-PAV sequence did not require a poly(A) tail (Figure 2B, compare PGUS1162 and PGUS1162A+). Thus, sequence(s) different from the 3′TE functionally substitutes for a poly(A) tail. In addition, full stimulation of cap-independent translation requires more viral sequence in vivo than in vitro. Because of this difference, rather than define the 3′TE as a precise sequence, we functionally define it as the sequence that stimulates cap-independent translation from the 3′UTR and is rendered non-functional by mutation at the BamHI site. All the results above were obtained from RNA transcripts harboring the 5′UTR from BYDV-PAV (indicated by the 'P' preceding 'GUS' in the transcript name). The requirement for the 5′UTR from BYDV-PAV RNA was examined by replacing it with the 5′UTR (Ω sequence) of TMV RNA. This sequence stimulates translation in vitro (with or without a cap) and in vivo in a highly cap-dependent manner (Gallie, 1991; Sleat and Wilson, 1992). Substitution of the BYDV-PAV 5′UTR with Ω permitted a low rate of translation of uncapped mRNA in vitro (Figure 2C, lane 8). However, Ω in place of the BYDV-PAV 5′UTR abolished translation of uncapped mRNA in vivo, even in the presence of the 1162 nt virus-derived 3′UTR (Figure 2B, ΩGUS1162), but permitted efficient translation of capped ΩGUS1162 RNA. Thus, Ω facilitates translation in a cap-dependent manner only, and does not cooperate with the 3′TE to promote cap-independent translation. Previously, we showed that a vector-derived 5′UTR also failed to support 3′TE activity in vitro (Wang and Miller, 1995). Thus, the 3′TE probably requires at least a portion of the BYDV-PAV 5′UTR for cap-independent translation in these contexts, indicating specific interactions between the 3′ and 5′UTR. The fact that expression of the capped, poly(A)+, Ω-containing mRNA which is considered to be an optimal plant message is no higher than that of uncapped mRNA containing the full BYDV-PAV 5′ and 3′UTRs (Figure 2B, compare uncapped PGUS1162 and capped ΩGUS1162A+), demonstrates the very high levels of expression that are conferred by this cap-independent translation signal. The 3′TE mimics a 5′ cap in its effect on RNA stability It is possible that the 3′TE stimulates gene expression in vivo, at least in part, by increasing RNA stability. However, we showed previously that the 109 nt 3′TE did not affect RNA stability in wheat germ extracts (Wang and Miller, 1995). To determine the effect of the larger, in vivo-defined 3′TE on RNA stability in vivo, RNA transcripts with a wild-type or mutant (the four base duplication) 1162 nt 3′UTR were electroporated into oat protoplasts under the same conditions as the GUS gene expression experiments, and RNA degradation was monitored by Northern blot hybridization. The RNA transcripts with either the wild-type or the defective 3′TE did not differ significantly in degradation rate (Figure 3A). Figure 3.Relative stability of mRNAs electroporated in oat protoplasts. (A) Northern blot hybridization detecting uncapped transcripts isolated from oat protoplasts at the indicated times after electroporation. The blot was hybridized with 32P-labeled antisense GUS transcript as described in Materials and methods. (B) Kinetics of GUS enzymatic activity accumulation at the indicated times after electroporation of oat protoplasts. The GUS assay was performed as in Figure 2. Download figure Download PowerPoint Northern blot hybridization detects only the physical stability of total cellular GUS mRNAs, and cannot discriminate between translatable and untranslatable RNAs. As a more accurate assay of RNA stability, the functional RNA half-life was measured by the kinetics of GUS synthesis. The activity of GUS (a very stable enzyme) should stop increasing sooner for a functionally unstable mRNA than for a stable one. Of the three mRNAs for which GUS activity was detectable, none leveled off until at least 30 h after electroporation (Figure 3B). This is beyond the 20 h timepoint used in Figure 2. Uncapped PGUS1162 mRNA appeared to be slightly less stable than its capped counterpart, but showed a very similar stability (shape of curve) to the capped P1162BF mutant. Thus, the capped mRNA with the defective 3′TE (PGUS1162BF) and uncapped mRNA containing active 3′TE (PGUS1162) have similar 'functional' stabilities, suggesting that the 3′TE either has no role in stabilizing mRNA or that it contributes a similar amount to stability as does the presence of a 5′ cap. The similar physical stability of uncapped PGUS1162 and uncapped PGUS1162BF (Figure 3A), but dramatic differences in GUS gene expression (Figure 3B), indicate that the 3′TE functions at the translational level. The 3′TE decreases the requirement for eIF4F for efficient translation of uncapped mRNA One possible role for the 3′TE in enhancing translation of uncapped mRNA is to efficiently recruit essential initiation factor(s) that facilitate(s) binding of the ribosomal small subunit, in a manner analogous to that of the 5′ cap of mRNAs. The binding of eIF4F to the 5′ cap structure confers the selective translation of capped, as opposed to uncapped, mRNA (Browning, 1996). The translation-enhancing sequence from STNV RNA dramatically decreased the amount of the rate-limiting initiation factor eIF4F, required for maximal translation efficiency in vitro (Timmer et al., 1993). The eIF4F requirements of mRNAs containing or lacking the 3′TE were compared. The already efficient translation of capped 3′TE-lacking mRNA and of uncapped mRNA containing the 109 nt 3′TE was not stimulated by exogenous eIF4F (Figure 4, lanes 1–10). Consequently, eIF4F was not rate-limiting for these mRNAs. In contrast, the endogenous eIF4F levels were rate-limiting for uncapped mRNA with the defective 3′TE, because translation efficiency of this mRNA increased correspondingly with the addition of exogenous eIF4F (Figure 4, lanes 11–15). As a control, bovine serum albumin (BSA) was added instead of eIF4F, and was observed to have no effect on translation of any mRNAs (data not shown). Thus, the 109 nt 3′TE appears to reduce the amount of eIF4F required for efficient translation, a property that is normally conferred by a 5′ cap (Browning, 1996). Figure 4.Effect of added eIF4F on translation of capped and uncapped mRNAs containing or lacking the functional 109 nt 3′TE in wheat germ extract. PGUS transcript was prepared by in vitro transcription from pPGUS109 that had been linearized with EcoICRI, which removes the 3′TE. PGUS109 was prepared from the same plasmid linearized with EcoRI. Transcript PGUS109BF is from the BamHI-filled mutant of pPGUS109 (pPGUS109BF), linearized with EcoRI. Translation reactions were performed as for Figure 1. Amount of added initiation factor: lanes 1, 6 and 11, 0; lanes 2, 7 and 12, 0.25 μg; lanes 3, 8 and 13, 0.5 μg; lanes 4, 9 and 14, 1.0 μg; lanes 5, 10 and 15, 2.0 μg. The translation efficiency of each mRNA with no exogenous translation factor was defined as 1. Download figure Download PowerPoint The 109 nt 3′TE inhibits translation of mRNAs in trans by a mechanism that is reversed by exogenous eIF4F If the 3′TE enhances translation by efficiently recruiting eIF4F, either directly or via an unidentified trans-acting factor, we predict that 3′TE-dependent or cap-dependent translation would be inhibited by the 3′TE in trans. The free 3′TE RNA should act as a competitor for eIF4F or other factors that mediate 3′TE stimulation in cis. To test this, a 109 nt transcript comprising only the in vitro-defined 3′TE RNA was added in excess to wheat germ extracts containing various mRNAs. Indeed, translation of uncapped 3′TE-plus mRNA (PGUS109) was inhibited by the addition of 3′TE RNA (Figure 5A, lanes 1–4). Addition of the defective 3′TE, which differs only by having the GAUC duplication (3′TEBF), had no inhibitory effect (Figure 5A, lanes 5–7). This shows that the inhibition by wild-type 109 nt 3′TE was not simply a non-specific effect of adding RNA to the wheat germ extract. Addition of eIF4F to the reaction reversed the trans inhibition by the 3′TE (Figure 5A, lanes 8–12). Thus, either the 3′TE-mediated cap-independent translation requires eIF4F or the mechanism is normally eIF4F-independent and added eIF4F allows translation of PGUS109 to bypass the 3′TE-mediated mechanism. To distinguish between these possibilities, the effect of the 3′TE on translation of capped mRNA lacking any BYDV-PAV sequence was examined. Excess added wild-type 3′TE RNA inhibited translation of capped mRNA containing Ω as its 5′UTR (Figure 5B, lanes 1–5). Again, the defective 3′TEBF RNA had no effect on the translation of this capped mRNA (Figure 5B, lanes 6–9), and addition of eIF4F restored translation of ΩGUS mRNA in the presence of inhibitory levels of 3′TE (Figure 5B, lanes 10–14). Thus, the 109 nt 3′TE competes with capped mRNA for factor(s) required for cap-dependent translation and does not need to interact with the BYDV-PAV 5′UTR to do so. The restoration of translatability of both mRNAs in the presence of inhibitory levels of 3′TE by eIF4F strongly implicates a role, either direct or indirect, for eIF4F in cap-independent translation mediated by the 3′TE in cis. Figure 5.Inhibition of translation by the 109 nt 3′TE in trans, and restoration by eIF4F. Uncapped PGUS109 (A) or capped ΩGUS (B) mRNAs (0.1 pmol) were translated in wheat germ extracts as in Figure 1, with the indicated amounts of added transcript comprising the 109 nt 3′TE (3′TE) or mutant 3′TE with the GAUC duplication at the BamHI site (3′TEBF) as competitor RNA. Amount of eIF4F added: (A) lanes 1–8, none; lanes 9–12, 0.5, 1.0, 2.0 and 4.0 μg, respectively; (B) lanes 1–10, none; lanes 11–14, 0.25, 0.5, 1.0 and 2.0 μg, respectively. The 109 nt 3′TE RNA and 113 nt 3′TEBF RNA were synthesized in uncapped form from SmaI-linearized p3′TE and p3′TEBF, respectively (Figure 1A). Translation was performed in wheat germ extracts as described in Materials and methods. Download figure Download PowerPoint The 109 nt 3′TE functions in the 5′UTR So far, by all tests, the 3′TE functionally mimics a 5′ cap. If this is the case, like the cap, the 3′TE should function in the 5′UTR. Hence, the 109 nt 3′TE was moved to the 5′ end of the GUS gene in place of the BYDV-PAV 5′UTR (construct 109GUSA, Figure 1A). This mRNA was translated cap-independently (Figure 6A, lanes 3 and 4). In contrast, the nearly identical construct, 109BFGUS, differing only by the GAUC duplication, was not translated in a cap-independent manner (Figure 6A, compare lanes 6 and 4). Capped mRNAs, even with the non-functional 3′TE at the 5′UTR, were still translated efficiently (Figure 6A, compare lanes 5 and 3), similar to the results seen when the mutant 3′TE was located at the 3′UTR (Figure 1C). Furthermore, in trans, free 109 nt 3′TE (but not the defective mutant form) inhibited translation of uncapped mRNA that contained the 109 nt 3′TE at the 5′UTR (Figure 6B). Figure 6.Translation of transcripts having the 109 nt 3′TE in place of the viral 5′UTR upstream of the GUS ORF. (A) In vitro translation in wheat germ extracts. Prior to transcription, templates for transcripts in lanes 3–6 were linearized with XbaI, and templates for transcripts in lanes 7–10 were linearized with EcoRI, resulting in the poly(A) tail indicated by A+. (B) Effect of addition of the 109 nt 3′TE or 113 nt 3′TEBF transcripts in trans on translation of uncapped 109GUS in wheat germ extracts. 109GUS RNA was transcribed from XbaI-cut p109GUSA. (C) GUS activity from the indicated transcripts 20 h after electroporation into oat protoplasts, assayed as described in Materials and methods and Figure 2. Download figure Download PowerPoint To test the function of the 5′-located 3′TE in vivo, polyadenylated transcripts containing wild-type or defective 3′TE in the 5′UTR were electroporated into oat protoplasts. The wild-type 109 nt 3′TE, but not the mutant 3′TE, stimulated GUS expression to a level 30-fold greater than background (Figure 6C, compare uncapped 109BFGUSA+ with uncapped 109GUSA+). Addition of a 5′ cap stimulated translation another 7-fold, similar to what was observed when the 109 nt 3′TE was located in the 3′UTR (Figure 2A, PGUS109A+). As a control, a construct also lacking the BYDV-PAV 5′UTR but with the 109 nt 3′TE in the 3′UTR (ΩGUS109A+) gave no GUS expression unless capped (Figure 6C). This is not surprising because, as mentioned previously (Figure 2B), a similar construct but with the full 1162 nt 3′UTR from BYDV-PAV (ΩGUS1162) also showed no cap-independent translation. The 1162 nt 3′UTR (full, in vivo-defined 3′TE) could not be tested in the 5′UTR owing to the presence of numerous AUG codons. Interestingly, the 5′-located 109 nt 3′TE stimulated translation of uncapped mRNA to a level that was nearly one-half that of uncapped PGUS1162 in proto