The 5' and 3' extremities of the satellite tobacco necrosis virus translational enhancer domain contribute differentially to stimulation of translation

The translational enhancer domain (TED) of satellite tobacco necrosis virus (STNV) RNA stimulates translation of uncapped RNAs autonomously. Here we set out to identify the 5' and 3' extremities of TED and features of these sequences with respect to translation. We found that both in wheat germ extract and in tobacco protoplasts, the 5' border is confined to 3 nt. Mutational analysis revealed that the autonomous function of TED is sensitive to 5' flanking sequences. At the 3' end of TED, 23 nt have a cumulative, quantitative effect on translation in wheat germ extract, whereas in tobacco protoplasts, the most 3' 14 nt of these 23 nt do not enhance translation. The 5' and 3' sequence requirements triggered the development of a new secondary structure model. In this model, TED folds into a phylogenetically conserved stem-loop structure in which the essential 5' nucleotides base-pair with the 3' nucleotides that stimulate translation both in vitro and in vivo. Importantly, the 14 3' nucleotides in TED that stimulate translation in the wheat germ extract only do not require the predicted base-pairing in order to function. The discrepancy between in vitro and in vivo sequence requirements thus correlates with potential base-pairing requirements, opening the possibility that TED contains two functional domains.     

A novel interaction of Cap-binding protein complexes eukaryotic initiation factor (eIF) 4F and eIF(iso)4F with a region in the 3'-untranslated region of satellite tobacco necrosis virus

Satellite tobacco necrosis virus (STNV) RNA is naturally uncapped at its 5' end and lacks polyadenylation at its 3' end. Despite lacking these two hallmarks of eukaryotic mRNAs, STNV-1 RNA is translated very efficiently. A approximately 130-nucleotide translational enhancer (TED), located 3' to the termination codon, is necessary for efficient cap-independent translation of STNV-1 RNA. The STNV-1 TED RNA fragment binds to the eukaryotic cap-binding complexes, initiation factor (eIF) 4F and eIF(iso)4F, as measured by nitrocellulose binding and fluorescence titration. STNV-1 TED is a potent inhibitor of in vitro translation when added in trans. This inhibition is reversed by the addition of eIF4F or eIF(iso)4F, and the subunits of eIF4F and eIF(iso)4F cross-link to STNV-1 TED, providing additional evidence that these factors interact directly with STNV-1 TED. Deletion mutagenesis of the STNV-1 TED indicates that a minimal region of approximately 100 nucleotides is necessary to promote cap-independent translation primarily through interaction with the cap binding subunits (eIF4E or eIF(iso)4E) of eIF4F or eIF(iso)4F.     

The 3'' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.     

Effect of capping upon the mRNA properties of satellite tobacco necrosis virus ribonucleic acid.

The mRNA guanyltransferase-mRNA methyltransferases of vaccinia virions can be used to introduce a 5'-terminal m7g(5')pp(5')Apm... capping group onto the RNA of satellite tobacco necrosis virus (STNV RNA) to yield intact capped STNV RNA. Studies with an in vitro system from wheat germ and limiting quantities of capped and uncapped STNV RNA show that the rates and extents of formation of initiation complexes of protein synthesis by intact capped and uncapped STNV RNA are identical, suggesting that 5'-terminal cap groups cannot function in the translation of STNV RNA. Also, the cap analogue pm7G equally inhibits the initiation and the translation of limiting quantities of both capped and uncapped STNV RNA. These contrasting observations suggest that the wheat germ system contains a pm7G sensitive protein and that STNV RNA has a tertiary structure that restricts the function of an added 5'-terminal capping group. This theory is supported by observations that fragmented capped STNV RNA is better at forming initiation complexes than is equally fragmented uncapped STNV RNA.     

Eucaryotic initiation factor 4B of wheat germ binds to the translation initiation region of a messenger ribonucleic acid

Purified preparations of eucaryotic initiation factor 4B (eIF4B) from wheat germ bind the monocistronic, uncapped, mRNA satellite tobacco necrosis virus RNA (STNV RNA) in nitrocellulose-mediated binding assays. This reaction is mRNA specific and yields dissociation constants (Kd) in the 10(-7)-10(-8) M range, depending upon the particular enzyme preparation tested. Purified wheat germ eIF4A, in the presence or absence of ATP, does not bind STNV RNA efficiently, but added eIF4A and ATP do enhance the efficiency of the eIF4B-dependent binding of STNV RNA. Wheat germ eIF4B binds the oligonucleotide containing the 5'-terminal 52 nucleotides of STNV RNA (designated 1-52) with the same affinity as intact STNV RNA. This binding affinity is less with the 1-44 oligonucleotide of STNV RNA and does not occur with the 1-33 oligonucleotide of STNV RNA that contains the 5'-terminal untranslated region and the initiator AUG codon at positions 30-32 of this mRNA. Wheat germ eIF4B therefore binds the translation initiation region of STNV RNA, and this binding requires up to 20 nucleotides on the 3' side of the initiator AUG codon of this mRNA. Wheat germ eIF4B also efficiently binds an oligonucleotide containing nucleotides from positions 13-52 in from the 5' terminus of STNV RNA, thereby establishing that the postulated 5'-terminal stem and loop secondary structure of STNV RNA [Leung, D. W., Browning, K. S., Heckman, J. E., RajBhandary, U. L., & Clark, J. M., Jr. (1979) Biochemistry 18, 1361-1366] is not functional or essential for this specific binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potyvirus genome-linked protein, VPg, directly affects wheat germ in vitro translation: interactions with translation initiation factors eIF4F and eIFiso4F

Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nm, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nm compared with 108 nm). Kinetic studies of eIF4F and eIFiso4F with VPg show approximately 2.6-fold faster association for eIFiso4F.VPg as compared with eIF4F.VPg. The dissociation rate was approximately 2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F.VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.     

Tobacco etch virus mRNA preferentially binds wheat germ eukaryotic initiation factor (eIF) 4G rather than eIFiso4G

The 5'-leader of tobacco etch virus (TEV) genomic RNA directs the efficient translation from the naturally uncapped viral RNA. The TEV 143-nt 5'-leader folds into a structure that contains two domains, each of which contains RNA pseudoknots. The 5'-proximal pseudoknot 1 (PK1) is necessary to promote cap-independent translation (Zeenko, V., and Gallie, D. R. (2005) J. Biol. Chem. 280, 26813-26824). During the translation initiation of cellular mRNAs, eIF4G functions as an adapter that recruits many of the factors involved in stimulating 40 S ribosomal subunit binding to an mRNA. Two related but highly distinct eIF4G proteins are expressed in plants, animals, and yeast. The two plant eIF4G isoforms, referred to as eIF4G and eIFiso4G, differ in size (165 and 86 kDa, respectively) and their functional differences are still unclear. Although eIF4G is required for the translation of TEV mRNA, it is not known if eIF4G binds directly to the TEV RNA itself or if other factors are required. To determine whether binding affinity and isoform preference correlates with translational efficiency, fluorescence spectroscopy was used to measure the binding of eIF4G, eIFiso4G, and their complexes (eIF4F and eIFiso4F, respectively) to the TEV 143-nt 5'-leader (TEV1-143) and a shorter RNA that contained PK1. A mutant (i.e. S1-3) in which the stem of PK1 was disrupted resulting in impaired cap-independent translation, was also tested. These studies demonstrate that eIF4G binds TEV1-143 and PK1 RNA with approximately 22-30-fold stronger affinity than eIFiso4G. eIF4G and eIF4F bind TEV1-143 with similar affinity, whereas eIFiso4F binds with approximately 6-fold higher affinity than eIFiso4G. The binding affinity of eIF4G, eIF4F, and eIFiso4G to S1-3 was reduced by 3-5-fold, consistent with the reduction in the ability of this mutant to promote cap-independent translation. Temperature-dependent binding studies revealed that binding of the TEV 5'-leader to these initiation factors has a large entropic contribution. Overall, these results demonstrate the first direct interaction of eIF4G with the TEV 5'-leader in the absence of other initiation factors. These data correlate well with the observed translational data and provide more detailed information on the translational strategy of potyviruses.     

Inhibition of Pokeweed Antiviral Protein (PAP) by Turnip Mosaic Virus Genome-linked Protein (VPg)

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.     

Evidence for the regulation of protein synthesis by a wheat germ phosphoprotein factor

A 48-kilodalton phosphoprotein, termed T-protein or pT, isolated from wheat germ and purified to homogeneity is found to inhibit the translation of tobacco mosaic virus (TMV) RNA in both wheat germ and reticulocyte lysates. The translation of TMV RNA in both systems was inhibited over 80% by 8 microM pT. There was no evidence to indicate that the reticulocyte lysate also contained a pT-like protein. pT was rapidly phosphorylated in the wheat germ and reticulocyte lysates. Although the relationship between pT phosphorylation and inhibition of protein synthesis is not known, there is evidence to indicate that complete phosphorylation of pT is not required for inhibition. Furthermore, no significant differences in the kinetics of inhibition of protein synthesis between prephosphorylated and unmodified pT were observed. Investigation of the mechanism of inhibition indicated that neither the aminoacylation of tRNA nor the elongation of nascent polypeptide chains was affected by pT. On the other hand, pT was found to prevent the formation of the 80S initiation complex. This action of pT was not due to the binding of pT to the ribosomes. However, the effect of pT was found to vary with the concentrations and types of mRNA used in the translational system. These results suggest that pT may interact with specific region(s) of the mRNA and prevent its translation. Alternatively, pT could block the translation of mRNA by binding to one or more of the initiation factors that interact with mRNA to facilitate mRNA binding to the 43S preinitiation complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turnip mosaic virus VPg interacts with Arabidopsis thaliana eIF(iso)4E and inhibits in vitro translation

The interaction between turnip mosaic virus (TuMV) viral protein linked to the genome (VPg) and Arabidopsis thaliana eukaryotic initiation factor (iso)4E (eIF(iso)4E) was investigated to address the influence of potyviral VPg on host cellular translational initiation. Affinity chromatographic analysis showed that the region comprising amino acids 62-70 of VPg is important for the interaction with eIF(iso)4E. In vitro translation analysis showed that the addition of VPg significantly inhibited translation of capped RNA in eIF(iso)4E-reconstituted wheat germ extract. This result indicates that VPg inhibits cap-dependent translational initiation via binding to eIF(iso)4E. The inhibition by VPg of in vitro translation of RNA with wheat germ extract did not depend on RNase activity. Our present results may indicate that excess VPg produced at the encapsidation stage shuts off cap-dependent translational initiation in host cells by inhibiting complex formation between eIF(iso)4E and cellular mRNAs.     

Translation of capped and uncapped vesicular stomatitis virus and reovirus mRNA'S. Sensitivity to m7GpppAm and ionic conditions.

In an attempt to elucidate the role of the 5'-terminal 7-methylguanosine residue in translation of mammalian mRNAs, vesicular stomatitis virus (VS virus), and reovirus mRNAs containing and lacking this residue, and also Qbeta RNA, were translated in cell-free extracts from reticulocytes and wheat germ under a variety of ionic conditions. Optimal translation of mRNAs lacking a 5' m7G occurred at concentrations of KOAc or KCl which were lower than those optimal for normal "capped" mRNAs. However, this salt dependence was much less marked in the mammalian reticulocyte extract and, at salt concentrations optimal for translation of normal capped mRNAs, reticulocyte lysates translated uncapped with mRNAs at 30 to 60% the normal efficiency. At low K+ concentrations, wheat germ ribosomes bound and translated appreciable amounts of uncapped VS virus mRNA; controls showed that no m7G residue is added to the 5' end of the bound RNA. Analogues of the 5' end, such as m7GpppAm, inhibited translation of both normal and uncapped VS virus RNAs in wheat germ extracts to about the same extent, but the efficiency of its action was reduced at low K+ concentrations. We conclude that there is a reduced importance of the 5' m7G residue in ribosome-mRNA recognition at low K+ concentrations, and that translation of mRNAs in reticulocyte extract is, under any reaction conditions, less dependent on the presence of a 5' "cap" than in wheat germ extracts.     

Features of the autonomous function of the translational enhancer domain of satellite tobacco necrosis virus.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a cap and a poly(A) tail. Translation of STNV RNA in vitro is promoted by a 120-nt translational enhancer domain (TED) in the 3'-untranslated region. TED also stimulates translation of heterologous mRNAs. In this study, we show that TED stimulates translation of a cat mRNA by increasing translation efficiency to the level of capped mRNA. This stimulatory activity is not impaired by translation through TED. TED stimulates translation efficiency from different positions within the mRNA, varying from the 5' end to 940 nt downstream of the coding region. Duplication of TED has an additive effect on translation stimulation only when located at both ends of the mRNA. On dicistronic RNAs, TED stimulates translation of both cistrons to the same extent. These data suggest that TED acts primarily by recruiting the translational machinery to the RNA.     

Conservation of RNA structures enables TNV and BYDV 5' and 3' elements to cooperate synergistically in cap-independent translation

The subgenomic RNA 2 of tobacco necrosis virus A (TNV sgRNA2) encodes the viral coat protein, is unpolyadenylated and presumably uncapped. Here, we show that TNV sgRNA2 is translated cap independently. This cap-independent translation requires the leader and a 140 nt element of the trailer both in wheat germ extract and in tobacco protoplasts. Similar to barley yellow dwarf virus (BYDV), the TNV 5′ and 3′ elements stimulate translation synergistically. Computer-aided phylogenetic analysis of the secondary structure of the TNV trailer revealed that the 3′ translation element is part of a major conserved stem–loop that contains similarities to structures in the BYDV 3′ translation element. These data suggest that the translation mechanisms of TNV sgRNA2 and BYDV RNA are related. To further characterize this relationship, we tested whether cooperativity exists between TNV sgRNA2 and BYDV 5′ and 3′ elements. We found that the TNV sgRNA2 5′ element stimulates translation synergistically with the BYDV 3′ element in vitro. This finding is the first evidence for conservation of structures that enable a 5′–3′ interaction stimulating cap-independent translation.     

Translational machinery and protein folding: evidence of conformational variants of the estrogen receptor alpha

As an approach to understand how translation may affect protein folding, we analyzed structural and functional properties of the human estrogen receptor alpha synthesized by different eukaryotic translation systems. A minimum of three conformations of the receptor were detected using limited proteolysis and a sterol ligand-binding assay. The receptor in vitro translated in rabbit reticulocyte lysate was rapidly degraded by protease, produced major bands of about 34 kDa and showed a high affinity for estradiol. In a wheat germ translation system, the receptor was more slowly digested. Two soluble co-existing conformations were evident by different degradation patterns and estradiol binding. Our data show that differences in the translation machinery may result in alternative conformations of the receptor with distinct sterol binding properties. These studies suggest that components of the cellular translation machinery itself might influence the protein folding pathways and the relative abundance of different receptor conformers.     

Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.     

A testis cytoplasmic RNA-binding protein that has the properties of a translational repressor.

Translation of the mouse protamine 1 (Prm-1) mRNA is repressed for several days during male germ cell differentiation. With the hope of cloning genes that regulate the translational repression of Prm-1, we screened male germ cell cDNA expression libraries with the 3' untranslated region of the Prm-1 RNA. From this screen we obtained two independent clones that encode Prbp, a Prm-1 RNA-binding protein. Prbp contains two copies of a double-stranded-RNA-binding domain. In vitro, the protein binds to a portion of the Prm-1 3' untranslated region previously shown to be sufficient for translational repression in transgenic mice, as well as to poly(I). poly(C). Prbp protein is present in multiple forms in cytoplasmic extracts prepared from wild-type mouse testes and is absent from testes of germ cell-deficient mouse mutants, suggesting that Prbp is restricted to the germ cells of the testis. Immunocytochemical localization confirmed that Prbp is present in the cytoplasmic compartment of late-stage meiotic cells and haploid round spermatids. Recombinant Prbp protein inhibits the translation of multiple mRNAs in a wheat germ lysate, suggesting that Prbp acts to repress translation in round spermatids. While this protein lacks complete specificity for Prm-1-containing RNAs in vitro, the properties of Prbp are consistent with it acting as a general repressor of translation.