Eucaryotic initiation factor 4B controls eIF3-mediated ribosomal entry of viral reinitiation factor

The cauliflower mosaic virus reinitiation factor TAV interacts with host translation initiation factor 3 (eIF3) and the 60S ribosomal subunit to accomplish translation of polycistronic mRNAs. Interaction between TAV and eIF3g is critical for the reinitiation process. Here, we show that eIF4B can preclude formation of the TAV/eIF3 complex via competition with TAV for eIF3g binding; indeed, the eIF4B- and TAV-binding sites on eIF3g overlap. Our data indicate that eIF4B interferes with TAV/eIF3/40S ribosome complex formation during the first initiation event. Consequently, overexpression of TAV in plant protoplasts affects only second initiation events. Transient overexpression of eIF4B in plant protoplasts specifically inhibits TAV-mediated reinitiation of a second ORF. These data suggest that TAV enters the host translation machinery at the eIF4B removal step to stabilize eIF3 on the translating ribosome, thereby allowing translation of polycistronic viral RNA.     

A new plant protein interacts with eIF3 and 60S to enhance virus‐activated translation re‐initiation

The plant viral re‐initiation factor transactivator viroplasmin (TAV) activates translation of polycistronic mRNA by a re‐initiation mechanism involving translation initiation factor 3 (eIF3) and the 60S ribosomal subunit (60S). QJ;Here, we report a new plant factor—re‐initiation supporting protein (RISP)—that enhances TAV function in re‐initiation. RISP interacts physically with TAV in vitro and in vivo. Mutants defective in interaction are less active, or inactive, in transactivation and viral amplification. RISP alone can serve as a scaffold protein, which is able to interact with eIF3 subunits a/c and 60S, apparently through the C‐terminus of ribosomal protein L24. RISP pre‐bound to eIF3 binds 40S, suggesting that RISP enters the translational machinery at the 43S formation step. RISP, TAV and 60S co‐localize in epidermal cells of infected plants, and eIF3–TAV–RISP–L24 complex formation can be shown in vitro. These results suggest that RISP and TAV bridge interactions between eIF3‐bound 40S and L24 of 60S after translation termination to ensure 60S recruitment during repetitive initiation events on polycistronic mRNA; RISP can thus be considered as a new component of the cell translation machinery.     

Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator-viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV-TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP--a novel and specific target of TOR/S6K1--in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes.     

Phosphorylation of a reinitiation supporting protein,RISP, determines its function in translation reinitiation

Reinitiation supporting protein, RISP, interacts with 60S (60S ribosomal subunit) and eIF3 (eukaryotic initiation factor 3) in plants. TOR (target-of-rapamycin) mediates RISP phosphorylation at residue Ser267, favoring its binding to eL24 (60S ribosomal protein L24). In a viral context, RISP, when phosphorylated, binds the CaMV transactivator/ viroplasmin, TAV, to assist in an exceptional mechanism of reinitiation after long ORF translation. Moreover, we show here that RISP interacts with eIF2 via eIF2β and TOR downstream target 40S ribosomal protein eS6. A RISP phosphorylation knockout, RISP-S267A, binds preferentially eIF2β, and both form a ternary complex with eIF3a in vitro. Accordingly, transient overexpression in plant protoplasts of RISP-S267A, but not a RISP phosphorylation mimic, RISP-S267D, favors translation initiation. In contrast, RISP-S267D preferentially binds eS6, and, when bound to the C-terminus of eS6, can capture 60S in a highly specific manner in vitro, suggesting that it mediates 60S loading during reinitiation. Indeed, eS6-deficient plants are highly resistant to CaMV due to their reduced reinitiation capacity. Strikingly, an eS6 phosphomimic, when stably expressed in eS6-deficient plants, can fully restore the reinitiation deficiency of these plants in cellular and viral contexts. These results suggest that RISP function in translation (re)initiation is regulated by phosphorylation at Ser267.     

TOR and S6K1 promote translation reinitiation of uORF‐containing mRNAs via phosphorylation of eIF3h

Mammalian target‐of‐rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF‐mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin‐1. Torin‐1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1—eIF3h—is phosphorylated and detected in polysomes in response to auxin. In TOR‐deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF‐mRNAs and eIF3h was impaired. Transient expression of eIF3h‐S178D in plant protoplasts specifically upregulates uORF‐mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.     

Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex.

We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S.eIF3. Met-tRNAf.eIF2.GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf.eIF2.GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.     

eIF3: a versatile scaffold for translation initiation complexes

Translation initiation in eukaryotes depends on many eukaryotic initiation factors (eIFs) that stimulate both recruitment of the initiator tRNA, Met-tRNA(i)(Met), and mRNA to the 40S ribosomal subunit and subsequent scanning of the mRNA for the AUG start codon. The largest of these initiation factors, the eIF3 complex, organizes a web of interactions among several eIFs that assemble on the 40S subunit and participate in the different reactions involved in translation. Structural analysis suggests that eIF3 performs this scaffolding function by binding to the 40S subunit on its solvent-exposed surface rather than on its interface with the 60S subunit, where the decoding sites exist. This location of eIF3 seems ideally suited for its other proposed regulatory functions, including reinitiating translation on polycistronic mRNAs and acting as a receptor for protein kinases that control protein synthesis.     

The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M_r 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.     

Final Pre-40S Maturation Depends on the Functional Integrity of the 60S Subunit Ribosomal Protein L3

Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles.     

Functions of eIF3 downstream of 48S assembly impact AUG recognition and GCN4 translational control

The binding of eIF2-GTP-tRNA(i)(Met) ternary complex (TC) to 40S subunits is impaired in yeast prt1-1 (eIF3b) mutant extracts, but evidence is lacking that TC recruitment is a critical function of eIF3 in vivo. If TC binding was rate-limiting in prt1-1 cells, overexpressing TC should suppress the temperature-sensitive phenotype and GCN4 translation should be strongly derepressed in this mutant, but neither was observed. Rather, GCN4 translation is noninducible in prt1-1 cells, and genetic analysis indicates defective ribosomal scanning between the upstream open reading frames that mediate translational control. prt1-1 cells also show reduced utilization of a near-cognate start codon, implicating eIF3 in AUG selection. Using in vivo cross-linking, we observed accumulation of TC and mRNA/eIF4G on 40S subunits and a 48S 'halfmer' in prt1-1 cells. Genetic evidence suggests that 40S-60S subunit joining is not rate-limiting in the prt1-1 mutant. Thus, eIF3b functions between 48S assembly and subunit joining to influence AUG recognition and reinitiation on GCN4 mRNA. Other mutations that disrupt eIF2-eIF3 contacts in the multifactor complex (MFC) diminished 40S-bound TC, indicating that MFC formation enhances 43S assembly in vivo.     

Depletion and deletion analyses of eucaryotic translation initiation factor 1A in Saccharomyces cerevisiae

Translation initiation factor eIF1A is a highly conserved, small, acidic protein that is required for cell growth in yeast. Biochemical studies in vitro implicate eIF1A in dissociating ribosomes, promoting methionyl-tRNA(i) binding to 40S ribosomal subunits, scanning of mRNAs and recognizing the AUG initiation codon. To elucidate the pleiotropic functions of eIF1A in vivo, the factor was depleted by placing its gene behind the repressible GAL1 promoter. After Saccharomyces cerevisiae cells were shifted to glucose medium, depletion of eIF1A was seen after 3-4 generations, corresponding with cessation of cell growth. Polysome profiles of the depleted strain showed ribosome run-off from mRNAs, indicating that eIF1A is involved in the initiation phase of translation. A decrease in free 40S ribosomes and an apparent increase in free 60S ribosomes were attributed to the formation of 40S subunit dimers. The result suggests that one of the functions of eIF1A is to prevent formation of 40S dimers. Mutant forms of eIF1A lacking either the positively charged N-terminal region or the negatively charged C-terminal region were constructed and tested for their ability to confer cell growth as the sole source of eIF1A. Either deletion supports cell growth, albeit at a slower rate, and causes a reduction in polysomes, although eIF1A lacking the N-terminal region is more deleterious. Therefore the charged terminal regions contribute to, but are not absolutely essential for, eIF1A function.     

Reprogramming mRNA translation during stress

The survival of mammalian cells exposed to adverse environmental conditions requires a radical reprogramming of protein translation. Stress-activated kinases target components of the initiation machinery (e.g. eIF2alpha, eIF4E-BP, eIF4B, and ribosomal protein S6) to inhibit the translation of 'housekeeping' proteins and promote the translation of repair enzymes. Accumulating untranslated mRNA is concentrated at stress granules where it is sorted and triaged to sites of storage, reinitiation, or decay. At the same time, the translation of mRNAs encoding repair enzymes is selectively preserved by both internal ribosome entry site-dependent and -independent mechanisms. In combination, these stress-activated processes coordinately reprogram mRNA translation and decay in a way that conserves anabolic energy, preserves essential mRNAs, and promotes the repair of stress-induced molecular damage.     

Control of Paip1-Eukayrotic Translation Initiation Factor 3 Interaction by Amino Acids through S6 Kinase

The simultaneous interaction of poly(A)-binding protein (PABP) with eukaryotic translation initiation factor 4G (eIF4G) and the mRNA 3′ poly(A) tail promotes translation initiation. We previously showed that the interaction of PABP-interacting protein 1 (Paip1) with PABP and eukaryotic translation initiation factor 3 (eIF3; via the eIF3g subunit) further stimulates translation. Here, we demonstrate that the interaction of eIF3 with Paip1 is regulated by amino acids through the mTORC1 signaling pathway. The Paip1-eIF3 interaction is impaired by the mTORC1 inhibitors, rapamycin and PP242. We show that ribosomal protein S6 kinases 1 and 2 (S6K1/2) promote the interaction of eIF3 with Paip1. The enhancement of Paip1-eIF3 interaction by amino acids is abrogated by an S6K inhibitor or shRNA against S6K1/2. S6K1 interacts with eIF3f and, in vitro, phosphorylates eIF3. Finally, we show that S6K inhibition leads to a reduction in translation by Paip1. We propose that S6K1/2 phosphorylate eIF3 to stimulate Paip1-eIF3 interaction and consequent translation initiation. Taken together, these data demonstrate that eIF3 is a new translation target of the mTOR/S6K pathway.     

Expression of truncated eukaryotic initiation factor 3e (eIF3e) resulting from integration of mouse mammary tumor virus (MMTV) causes a shift from cap-dependent to cap-independent translation

Integration of mouse mammary tumor virus (MMTV) at the common integration site Int6 occurs in the gene encoding eIF3e, the p48 subunit of translation initiation factor eIF3. Integration is at any of several introns of the Eif3e gene and causes the expression of truncated Eif3e mRNAs. Ectopic expression of the truncated eIF3e protein resulting from integration at intron 5 (3e5) induces malignant transformation, but by an unknown mechanism. Because eIF3e makes up at least part of the binding site for eIF4G, we examined the effects of 3e5 expression on protein synthesis. We developed an NIH3T3 cell line that contains a single copy of the 3e5 sequence at a predetermined genomic site. Co-immunoprecipitation indicated diminished binding of eIF3 to eIF4G, signifying a reduction in recruitment of the mRNA-unwinding machinery to the 43 S preinitiation complex. Cell growth and overall protein synthesis were decreased. Translation driven by the eIF4G-independent hepatitis C virus internal ribosome entry sequence (HCV IRES) in a bicistronic mRNA was increased relative to cap-dependent translation. Endogenous mRNAs encoding XIAP, c-Myc, CYR61, and Pim-1, which are translated in a cap-independent manner, were shifted to heavier polysomes whereas mRNAs encoding GAPDH, actin, L32, and L34, which are translated in a cap-dependent manner, were shifted to lighter polysomes. We propose that expression of 3e5 diminishes eIF4G interaction with eIF3 and causes abnormal gene expression at the translational level. The correlation between up-regulation of cap-independent translation and MMTV-induced tumorigenesis contrasts with the well established model for malignant transformation involving up-regulation of highly cap-dependent translation.     

Evidence for a Negative Cooperativity between eIF5A and eEF2 on Binding to the Ribosome

eIF5A is the only protein known to contain the essential and unique amino acid residue hypusine. eIF5A functions in both translation initiation due to its stimulation of methionyl-puromycin synthesis and translation elongation, being highly required for peptide-bound formation of specific ribosome stalling sequences such as poly-proline. The functional interaction between eIF5A, tRNA, and eEF2 on the surface of the ribosome is further clarified herein. Fluorescence anisotropy assays were performed to determine the affinity of eIF5A to different ribosomal complexes and reveal its interaction exclusively and directly with the 60S ribosomal subunit in a hypusine-dependent manner (K_i^{60S-eIF5A-Hyp} = 16 nM, K_i^{60S-eIF5A-Lys} = 385 nM). A 3-fold increase in eIF5A affinity to the 80S is observed upon charged-tRNA_i^Met binding, indicating positive cooperativity between P-site tRNA binding and eIF5A binding to the ribosome. Previously identified conditional mutants of yeast eIF5A, eIF5A^Q22H/L93F and eIF5A^K56A, display a significant decrease in ribosome binding affinity. Binding affinity between ribosome and eIF5A-wild type or mutants eIF5A^K56A, but not eIF5A^Q22H/L93F, is impaired in the presence of eEF2 by 4-fold, consistent with negative cooperativity between eEF2 and eIF5A binding to the ribosome. Interestingly, high-copy eEF2 is toxic only to eIF5A^Q22H/L93F and causes translation elongation defects in this mutant. These results suggest that binding of eEF2 to the ribosome alters its conformation, resulting in a weakened affinity of eIF5A and impairment of this interplay compromises cell growth due to translation elongation defects.     

Up-regulation of PDCD4 in senescent human diploid fibroblasts

Programmed cell death 4 (PDCD4) has a common MI domain sharing with death associated protein 5 (DAP5) and a component of eukaryotic translation initiation factor (eIF4G) complex and it might also work as a tumor suppressor. We could find that the message and product of Pdcd4 gene were up-regulated in senescent human diploid fibroblasts. In yeast two hybrid analysis, the C-terminal region of PDCD4 interacted with ribosomal protein S13 (RPS13), ribosomal protein L5 (RPL5), and TI-227H. In in vitro binding assay, RPS13, a component of 40S ribosome was stably bound to PDCD4. We also found that PDCD4 was localized to polysome fractions. We could pull out eIF4G with GST-PDCD4, but eIF4E did not interact with PDCD4. From these results, we could assume that PDCD4 might regulate the eIF4G-dependent translation through direct interactions with eIF4G and RPS13 in senescent fibroblasts.     

20S proteasome differentially alters translation of different mRNAs via the cleavage of eIF4F and eIF3

The molecular basis for coordinated regulation of protein synthesis and degradation is not understood. Here we report that the 20S proteasome endoproteolytically cleaves the translation initiation factors eIF4G, a subunit of eIF4F, and eIF3a, a subunit of eIF3. The cleavage of eIF4G or eIF3a differentially affects the assembly of ribosomal preinitiation complexes on different cellular and viral mRNAs in an in vitro system containing pure components. Inhibition of proteolytic activity of the 20S proteasome with specific inhibitors prevents cleavage of both factors in vitro and in vivo, restores assembly of ribosomal complexes in vitro, and differentially affects translation of different mRNAs in vivo. These studies demonstrate the importance of the endoproteolytic activity of proteasomes in regulation of cellular processes and suggest a link between protein synthesis and degradation.