Removal of beta subunit of the eukaryotic polypeptide chain initiation factor 2 by limited proteolysis

It is generally considered that the eukaryotic polypeptide chain initiation factor 2 (eIF-2) from rabbit reticulocytes consists of three nonidentical subunits termed alpha, beta, and gamma, in order of increasing molecular weight. However, a recent report [Stringer, E. A., Chaudhuri, A., Valenzuela, D. & Maitra, U. (1980) Proc. Natl. Acad. Sci. USA 77, 3356-3359] suggested that this factor is made up of only two subunits. In this paper we show that limited proteolysis of rabbit reticulocyte eIF-2 leads to loss of the beta subunit. This modified eIF-2 has the same activity as the native factor in promoting ternary (eIF-2.GTP.Met-tRNAi) and 40S (eIF-2.GTP.Met-tRNAi.40S ribosome) initiation complex formation. Like native eIF-2, the protease-treated factor can restore translation in heme-deficient lysates. On the other hand, the treated factor is less stable than the native protein.     

Adherence to the first-AUG rule when a second AUG codon follows closely upon the first

The rule that eukaryotic ribosomes initiate translation exclusively at the 5' proximal AUG codon is abrogated under rare conditions. One circumstance that has been suggested to allow dual initiation is close apposition of a second AUG codon. A possible mechanism might be that the scanning 40S ribosomal subunit flutters back and forth instead of stopping cleanly at the first AUG. This hypothesis seems to be ruled out by evidence presented herein that in certain mRNAs, the first of two close AUG codons is recognized uniquely. To achieve this, the 5' proximal AUG has to be provided with the full consensus sequence; even small departures allow a second nearby AUG codon to be reached by leaky scanning. This context-dependent leaky scanning unexpectedly fails when the second AUG codon is moved some distance from the first. A likely explanation, based on analyzing the accessibility of a far-downstream AUG codon under conditions of initiation versus elongation, is that 80S elongating ribosomes advancing from the 5' proximal start site can mask potential downstream start sites.     

Rpg1, the Saccharomyces cerevisiae homologue of the largest subunit of mammalian translation initiation factor 3, is required for translational activity

Eukaryotic initiation factor 3 (eIF3) consists of at least eight subunits and plays a key role in the formation of the 43 S preinitiation complex by dissociating 40 and 60 S ribosomal subunits, stabilizing the ternary complex, and promoting mRNA binding to 40 S ribosomal subunits. The product of the Saccharomyces cerevisiae RPG1 gene has been described as encoding a protein required for passage through the G1 phase of the cell cycle and exhibiting significant sequence similarity to the largest subunit of human eIF3. Here we show that under nondenaturing conditions, Rpg1p copurifies with a known yeast eIF3 subunit, Prt1p. An anti-Rpg1p antibody co-immunoprecipitates Prt1p, and an antibody directed against the Myc tag of a tagged version of Prt1p co-immunoprecipitates Rpg1p, demonstrating that both proteins are present in the same complex. A cell-free translation system derived from the temperature-sensitive rpg1-1 mutant strain becomes inactivated by incubation at 37 degreesC, and its activity can be restored by the addition of the Rpg1-containing protein complex. Finally, the rpg1-1 temperature-sensitive mutant strain shows a dramatic reduction of the polysome/monosome ratio upon shift to the restrictive temperature. These data show that Rpg1p is an authentic eIF3 subunit and plays an important role in the initiation step of translation.     

Mutations in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) that overcome the inhibitory effect of eIF-2 alpha phosphorylation on translation initiation

Phosphorylation of eIF-2 alpha in Saccharomyces cerevisiae by the protein kinase GCN2 leads to inhibition of general translation initiation and a specific increase in translation of GCN4 mRNA. We isolated mutations in the eIF-2 alpha structural gene that do not affect the growth rate of wild-type yeast but which suppress the toxic effects of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2. These eIF-2 alpha mutations also impair translational derepression of GCN4 in strains expressing wild-type GCN2 protein. All four mutations alter single amino acids within 40 residues of the phosphorylation site in eIF-2 alpha; however, three alleles do not decrease the level of eIF-2 alpha phosphorylation. We propose that these mutations alter the interaction between eIF-2 and its recycling factor eukaryotic translation initiation factor 2B (eIF-2B) in a way that diminishes the inhibitory effect of phosphorylated eIF-2 on the essential function of eIF-2B in translation initiation. These mutations may identify a region in eIF-2 alpha that participates directly in a physical interaction with the GCN3 subunit of eIF-2B.     

Reduced dosage of genes encoding ribosomal protein S18 suppresses a mitochondrial initiation codon mutation in Saccharomyces cerevisiae

A yeast mitochondrial translation initiation codon mutation affecting the gene for cytochrome oxidase subunit III (COX3) was partially suppressed by a spontaneous nuclear mutation. The suppressor mutation also caused cold-sensitive fermentative growth on glucose medium. Suppression and cold sensitivity resulted from inactivation of the gene product of RPS18A, one of two unlinked genes that code the essential cytoplasmic small subunit ribosomal protein termed S18 in yeast. The two S18 genes differ only by 21 silent substitutions in their exons; both are interrupted by a single intron after the 15th codon. Yeast S18 is homologous to the human S11 (70% identical) and the Escherichia coli S17 (35% identical) ribosomal proteins. This highly conserved family of ribosomal proteins has been implicated in maintenance of translational accuracy and is essential for assembly of the small ribosomal subunit. Characterization of the original rps18a-1 missense mutant and rps18a delta and rps18b delta null mutants revealed that levels of suppression, cold sensitivity and paromomycin sensitivity all varied directly with a limitation of small ribosomal subunits. The rps18a-1 mutant was most affected, followed by rps18a delta then rps18b delta. Mitochondrial mutations that decreased COX3 expression without altering the initiation codon were not suppressed. This allele specificity implicates mitochondrial translation in the mechanism of suppression. We could not detect an epitope-tagged variant of S18 in mitochondria. Thus, it appears that suppression of the mitochondrial translation initiation defect is caused indirectly by reduced levels of cytoplasmic small ribosomal subunits, leading to changes in either cytoplasmic translational accuracy or the relative levels of cytoplasmic translation products.     

The alpha-subunit of the mammalian guanine nucleotide-exchange factor eIF-2B is essential for catalytic activity in vitro

Eukaryotic initiation factor (eIF)-2B, the guanine nucleotide exchange factor for eIF-2, consists of five distinct subunits in both mammals and the yeast Saccharomyces cerevisiae. The exchange reaction mediated by eIF-2B can be regulated by phosphorylation of eIF-2 on its alpha-subunit. This represents a key control point in the initiation of translation. The functions of the individual subunits of the eIF-2B complex remain unclear. Mutational analysis in Saccharomyces cerevisiae suggested that the smallest subunit (the alpha) is dispensable for exchange, but required for the inhibition of eIF-2B by eIF-2(alphaP). Here we present evidence that, in mammalian cells, eIF-2Balpha is essential for the activity of the complex, since preparations of eIF-2B lacking this subunit are not active in nucleotide exchange in vitro, although the complex still contains the beta, gamma, delta and epsilon subunits.     

Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro

AUG-unrelated translation initiation was found in an insect picorna-like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3'-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3' boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5' terminus of the capsid-coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5' part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.     

Expression of a phosphorylation-resistant eukaryotic initiation factor 2 alpha-subunit mitigates heat shock inhibition of protein synthesis

Protein synthesis is dramatically reduced upon exposure of cells to elevated temperature. Concordant with this inhibition, multiple phosphorylation and dephosphorylation reactions occur on specific eukaryotic initiation factors that are required for protein synthesis. Most notably, phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF-2 alpha) on serine residue 51 occurs. To identify the importance of phosphorylation in control of protein synthesis, we have evaluated the effects of expression of a mutant eIF-2 alpha which is resistant to phosphorylation. Expression of a serine to alanine mutant at residue 51 of eIF-2 alpha partially protected cells from the inhibition of protein synthesis in response to heat treatment. The overexpressed serine to alanine 51 mutant subunit was incorporated into the eIF-2 heterotrimer and was resistant to phosphorylation. These results are consistent with the hypothesis that heat shock inhibition of translation is mediated in part through phosphorylation of eIF-2 alpha. Expression of the wild type or mutant eIF-2 alpha did not affect cell survival or induction of hsp70 mRNA upon heat shock, indicating that although eIF-2 alpha is a heat shock-induced protein, its increased synthesis during heat shock does not alter the heat-shock response.     

Cell transformation results in the loss of the density-dependent translational regulation of the expression of fibroblast growth factor 2 isoforms

Alternative initiation of translation at three CUG and one AUG start codons leads to the synthesis of four isoforms of fibroblast growth factor 2 (FGF-2) that have distinct intracellular localizations and affect the cell phenotype differently. We show here that the expression of FGF-2 CUG-initiated isoforms decreases in a cell-density-dependent manner in normal human skin fibroblasts (HSFs) concomitantly with the FGF-2 mRNA level. In contrast, CUG-initiated FGF-2 expression is constitutive in SK-HEP-1 cells and in HSFs transformed with SV40 large T antigen. Cell transfection using a plasmid containing the FGF-2 mRNA leader fused to chloramphenicol acetyl transferase demonstrated that up-regulation of the CUG codons depends on cis-elements located in this leader. Furthermore, UV cross-linking experiments revealed a correlation between CUG codons utilization and the binding of several proteins to the mRNA leader. On the basis of the presence of an internal ribosome entry site (IRES) in the FGF-2 mRNA, we used bicistronic vectors to transfect normal and transformed cells. The density-dependent regulation in normal HSFs was cap-dependent, whereas the constitutive CUG-initiated FGF-2 expression in transformed cells occurred essentially by an IRES-dependent mechanism. Unexpectedly, the use of the AUG start codon occurred exclusively by internal entry, which suggests the presence of a second independent IRES in the FGF-2 mRNA that would be constitutive. A study of the eIF-4E levels and of the 4E-BP1 phosphorylation state at increasing cell densities showed a decrease of the eIF-4E level, concomitant with 4E-BP1 dephosphorylation in normal cells but not in transformed cells. These data point out a complex mechanism for the regulation of FGF-2 isoforms expression involving both the cap-dependent and the cap-independent initiation of translation and favor a positive role of CUG-initiated FGF-2 in cellular proliferation and transformation.     

Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families

As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.     

Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit

Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion. Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type strains and strains harboring mutations in the eIF-2gamma structural gene (GCD11) predicted to alter ligand binding by eIF-2. The alteration of tyrosine 142 in yeast eIF-2gamma, corresponding to histidine 66 in Escherichia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNAi(Met) without affecting the k(off) value for guanine nucleotides. In contrast, non-lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring-binding loop increased the off-rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF-2B, without altering the apparent dissociation constant for Met-tRNAi(Met). For eIF-2[gamma-K250R], the increased off-rate also seen for GTP was masked by the presence of Met-tRNAi(Met) in vitro. In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow-growth phenotype and reduced GCN4 expression in gcd11-K250R and gcd11-Y142H strains. These studies indicate that the gamma-subunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 complex, and further suggest that the level of Met-tRNAi(Met) is critical for maintaining wild-type rates of initiation in vivo.     

Prognostic significance of cyclin D1 and retinoblastoma expression in combination with p53 abnormalities in primary, resected non-small cell lung cancers

Accumulating evidence shows that the repertoire of major histocompatibility complex class I-restricted epitopes extends beyond conventional translation reading frames. Previously, we reported that scanthrough translation, where the initiating AUG of a primary open reading frame is bypassed, is most likely to account for the presentation of cryptic epitopes from alternative reading frames within the influenza A PR/8/34 nucleoprotein gene. Here, we confirm and extend these findings using an epitope cassette construct that features two well-defined CD8(+) T cell (TCD8+) epitopes in alternative reading frames, each preceded by a single start codon. Expression of one epitope depends on scanning of the ribosome over the first AUG with translation initiation occurring at the second AUG. We find that scanthrough translation has great potency in our system, with its impact being modulated, as predicted, by the base composition surrounding the first initiation codon, the number of start codons preceding the point of alternate reading frame initiation, and the efficiency with which the epitope itself is generated. Additionally, we investigated the efficiency of eukaryotic translation termination codons, to assess codon readthrough as a mechanism for cryptic epitope expression from 3' untranslated regions. In contrast with initiation codons, eukaryotic stop codons appear to be highly efficient at preventing expression of epitopes encoded in 3' untranslated regions, suggesting that 3' untranslated regions are not a common source of cryptic epitope substrate. We conclude that scanthrough is a powerful mechanism for the expression of epitopes encoded in upstream alternative open reading frames that may contribute significantly to TCD8+ responses and to tolerance induction.     

Universally conserved translation initiation factors

The process by which translation is initiated has long been considered similar in Bacteria and Eukarya but accomplished by a different unrelated set of factors in the two cases. This not only implies separate evolutionary histories for the two but also implies that at the universal ancestor stage, a translation initiation mechanism either did not exist or was of a different nature than the extant processes. We demonstrate herein that (i) the "analogous" translation initiation factors IF-1 and eIF-1A are actually related in sequence, (ii) the "eukaryotic" translation factor SUI1 is universal in distribution, and (iii) the eukaryotic/archaeal translation factor eIF-5A is homologous to the bacterial translation factor EF-P. Thus, the rudiments of translation initiation would seem to have been present in the universal ancestor stage. However, significant development and refinement subsequently occurred independently on both the bacterial lineage and on the archaeal/eukaryotic line.     

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.     

A difference in the rate of ribosomal elongation balances the synthesis of eukaryotic translation initiation factor (eIF)-2 alpha and eIF-2 beta

Eukaryotic translation initiation factor 2 (eIF-2) is a heterotrimer composed of three subunits designated alpha, beta, and gamma. These proteins exist in equimolar amounts in the cell and have not been detected as isolated subunits. Our research examines the basis of their balanced synthesis. Northern analysis of K562 cell mRNA revealed that eIF-2 beta was five times more abundant than eIF-2 alpha. However, immunoprecipitation of pulse-labeled K562 cells showed an equimolar rate of synthesis of eIF-2 alpha and -beta despite the 5-fold difference in the size of their mRNA pools. Addition of equal amounts of synthetic capped mRNA for eIF-2 alpha and eIF-2 beta to an in vitro translation reaction produced five times more eIF-2 alpha protein than eIF-2 beta. Determination of the polysome profile for alpha and beta mRNA in K562 cells indicated eIF-2 alpha was translated more efficiently than eIF-2 beta. Substitution of either the initiation codon context or the leader of the beta mRNA for that of alpha had only a minor effect on the translational efficiency of beta. Comparison of the rate of ribosomal elongation for the two mRNAs indicated that ribosomes associated with the beta mRNA elongate at a rate 4-fold less than that of eIF-2 alpha. Thus, the balanced translation of alpha and beta mRNA is primarily the result of a 4-fold difference in the rate of ribosomal elongation.     

Functional interactions between yeast mitochondrial ribosomes and mRNA 5' untranslated leaders

Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5' untranslated leaders (5'-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5'-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5'-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5'-UTLs which, in conjunction with mRNA-specific translational activation, is required for organellar translation initiation.