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At least 199 records · Page 11Linked to original sources

PubMed1988

The transcription and translation in vitro of individual cereal storage-protein genes from wheat (Triticum aestivum, cv. Chinese Spring). Evidence for translocation of the translation products and disulphide-bond formation.

Genes coding for the high-Mr ['high-molecular-weight' (HMW)] glutenin subunit 12 and for a gamma-gliadin from wheat (Triticum aestivum, cv. Chinese Spring) were subcloned into transcription-translation vectors. In each case transcription in vitro yielded a RNA transcript which when added to a rabbit reticulocyte cell-free translation system directed the synthesis of a polypeptide of appropriate Mr by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE). When dog pancreatic microsomal vesicles were added to the translation system, translocation of the newly synthesized polypeptides occurred, as judged by protection from proteolysis. When translation and translocation of the gamma-gliadin was carried out under conditions favouring the formation of disulphide bonds, a polypeptide was synthesized which had a faster mobility on SDS/PAGE carried out under non-reducing conditions than under reducing conditions. This suggests that the processed and translocated gamma-gliadin forms an intramolecular disulphide bond or bonds during synthesis in vitro.

Cell-Free System↗

PubMed1997

Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation.

The yeast translation factor eIF4G associates with both the cap-binding protein eIF4E and the poly(A)-binding protein Pab1p. Here we report that the two yeast eIF4G homologs, Tif4631p and Tif4632p, share a conserved Pab1p-binding site. This site is required for Pab1p and poly(A) tails to stimulate the in vitro translation of uncapped polyadenylylated mRNA, and the region encompassing it is required for the cap and the poly(A) tail to synergistically stimulate translation. This region on Tif4631p becomes essential for cell growth when the eIF4E binding site on Tif4631p is mutated. Pab1p mutations also show synthetic lethal interactions with eIF4E mutations. These data suggest that eIF4G mediates poly(A) tail stimulated translation in vitro, and that Pab1p and the domain encompassing the Pab1p-binding site on eIF4G can compensate for partial loss of eIF4E function in vivo.

Amino Acid Sequence↗

PubMed1996

Interplay of methionine tRNAs with translation elongation factor Tu and translation initiation factor 2 in Escherichia coli.

According to their role in translation, tRNAs specifically interact either with elongation factor Tu (EFTu) or with initiation factor 2 (IF2). We here describe the effects of overproducing EFTu and IF2 on the elongator versus initiator activities of various mutant tRNAMet species in vivo. The data obtained indicate that the selection of a tRNA through one or the other pathway of translation depends on the relative amounts of the translational factors. A moderate overexpression of EFTu is enough to lead to a misappropriation of initiator tRNA in the elongation process, whereas overproduced IF2 allows the initiation of translation to occur with unformylated tRNA species. In addition, we report that a strain devoid of formylase activity can be cured by the overproduction of tRNAMetf. The present study brings additional evidence for the importance of formylation in defining tRNAMetf initiator identity, as well as a possible explanation for the residual growth of bacterial strains lacking a functional formylase gene such as observed in Guillon, J. M., Mechulam, Y., Schmitter, J.-M., Blanquet, S., and Fayat, G. (1992) J. Bacteriol. 174, 4294-4301.

Cloning, Molecular↗

PubMed1997

The position dependence of translational regulation via RNA-RNA and RNA-protein interactions in the 5'-untranslated region of eukaryotic mRNA is a function of the thermodynamic competence of 40 S ribosomes in translational initiation.

Cap proximity is a requirement to enable secondary structures and RNA-binding proteins to repress translational initiation via the 5'-untranslated region (5'-UTR) of mammalian mRNAs. We show that in Saccharomyces cerevisiae, unlike mammalian cells, the in vitro translational repressive effect of the mammalian iron regulatory protein 1 (IRP1) is independent of the site of its target in the 5'-UTR, the iron-responsive element (IRE). In vitro studies demonstrate that the binding affinity of IRP1 is also unaffected by the position of the IRE. Using IRE loop mutants, we observe an almost complete loss of IRP1-dependent repression in yeast concomitant with a 150-fold reduction in binding affinity for the IRE target. This mirrors the natural quantitative range of iron-induced adjustment of IRE/IRP1 affinity in mammalian cells. By enhancing the stability of the IRE stem-loop, we also show that its intrinsic folding energy acts together with the binding energy of IRP1 to give an additive capacity to restrict translational initiation. An IRE.IRP1 complex in a cap-distal position in yeast blocks scanning 40 S ribosomes on the 5'-UTR. It follows that the position effect of mammalian site-specific translational repression is dictated by the competence of the mammalian preinitiation complex to destabilize inhibitory structures at different steps of the initiation process.

Base Sequence↗

PubMed2002

Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2alpha.

Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m(7)G-cap and then moves along the mRNA until an initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5'-untranslated regions, which allow initiation independently of the 5'-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation initiation factor, eIF2alpha, by activating specific kinases: (i) amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER kinase, PERK kinase; and (iii) double-stranded RNA, which activates double-stranded RNA-dependent protein kinase (PKR) by mimicking viral infection. Amino acid starvation and ER stress caused transient phosphorylation of eIF2alpha during the first hour of treatment, whereas double-stranded RNA caused a sustained phosphorylation of eIF2alpha after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the protein kinase Pim-1. In contrast, translation mediated by the IRESs from the cationic amino acid transporter, cat-1, and of the cricket paralysis virus intergenic region, were stimulated 3- to 10-fold by all three treatments. eIF2alpha phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2alpha. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5'-untranslated regions.

Animals↗

PubMed2005

Heme-regulated inhibitor kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure.

Exposure to arsenite inhibits protein synthesis and activates multiple stress signaling pathways. Although arsenite has diverse effects on cell metabolism, we demonstrated that phosphorylation of eukaryotic translation initiation factor 2 at Ser-51 on the alpha subunit was necessary to inhibit protein synthesis initiation in arsenite-treated cells and was essential for stress granule formation. Of the four protein kinases known to phosphorylate eukaryotic translation initiation factor 2alpha, only the heme-regulated inhibitor kinase (HRI) was required for the translational inhibition in response to arsenite treatment in mouse embryonic fibroblasts. In addition, HRI expression was required for stress granule formation and cellular survival after arsenite treatment. In vivo studies elucidated a fundamental requirement for HRI in murine survival upon acute arsenite exposure. The results demonstrated an essential role for HRI in mediating arsenite stress-induced phosphorylation of eukaryotic translation initiation factor 2alpha, inhibition of protein synthesis, stress granule formation, and survival.

Animals↗

PubMed1990

A genetic link between an mRNA-specific translational activator and the translation system in yeast mitochondria.

Translation of the Saccharomyces cerevisiae mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the products of at least three nuclear genes, PET122, PET494 and PET54. pet122 mutations that remove 24-67 amino acid residues from the carboxyterminus of the gene product were found to be suppressed by unlinked nuclear mutations. These unlinked suppressors fail to suppress both a pet122 missense mutation and a complete pet122 deletion. One of the suppressor mutations causes a heat-sensitive nonrespiratory growth phenotype in an otherwise wild-type strain and reduces translation of all mitochondrial gene products in cells grown at high temperature. This suppressor maps to a newly identified gene on chromosome XV termed PET123. The sequence of a DNA fragment carrying PET123 contains one major open reading frame encoding a basic protein of 318 amino acids. Inactivation of the chromosomal copy of PET123 by interruption of this open reading frame causes cells to become rho- (sustain large deletions in their mtDNA). This phenotype is characteristic for null alleles of genes whose products are essential for general mitochondrial protein synthesis. Thus our data strongly suggest that the PET123 protein is a component of the mitochondrial translation apparatus that interacts directly with the coxIII-mRNA-specific translational activator PET122.

Alleles↗

PubMed1997

In vivo analysis of Saccharomyces cerevisiae COX2 mRNA 5'-untranslated leader functions in mitochondrial translation initiation and translational activation.

We have used mutational and revertant analysis to study the elements of the 54-nucleotide COX2 5'-untranslated leader involved in translation initiation in yeast mitochondria and in activation by the COX2 translational activator. Pet111p. We generated a collection of mutants with substitutions spanning the entire COX2 5'-UTL by in vitro mutagenesis followed by mitochondrial transformation and gene replacement. The phenotypes of these mutants delimit a 31-nucleotide segment, from -16 to -46, that contains several short sequence elements necessary for COX2 5'-UTL function in translation. The sequences from -16 to -47 were shown to be partially sufficient to promote translation in a foreign context. Analysis of revertants of both the series of linker-scanning alleles and two short deletion/ insertion alleles has refined the positions of several possible functional elements of the COX2 5'-untranslated leader, including a putative RNA stem-loop structure that functionally interacts with Pet111p and an octanucleotide sequence present in all S. cerevisiae mitochondrial mRNA 5'-UTLs that is a potential rRNA binding site.

Base Sequence↗

PubMed2006

Rapamycin inhibits liver growth during refeeding in rats via control of ribosomal protein translation but not cap-dependent translation initiation.

We examined the role of the mammalian target of rapamycin (mTOR) in hepatic cell growth. To dissociate cell growth from cell proliferation, we employed an in vivo model of nonproliferative liver growth in rats, refeeding after 48 h of food deprivation. Starvation resulted in a decrease in liver mass, liver protein, and cell size, all of which were largely restored after 24 h of refeeding. Administration of the mTOR inhibitor, rapamycin, before the refeeding period partially inhibited the restoration of liver protein content. Refeeding was also associated with an increase in ribosomal protein S6 phosphorylation and phosphorylation of the eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1). 4E-BP1 phosphorylation was accompanied by a decrease in the abundance of the complex containing 4E-BP1 with eIF4E. These changes were prevented by rapamycin administration. However, association of eIF4E and eIF4G and eIF2alpha phosphorylation, both of which are stimulated by refeeding, were insensitive to rapamycin. The functional importance of these observations was confirmed by polysome fractionation, which showed that translation initiation of 5' oligopyrimidine tract-containing mRNAs, which encode ribosomal proteins, was inhibited by rapamycin, whereas translation of signal transducer and activator of transcription 1 (STAT1), a cap-dependent mRNA, was unaffected. The abundance of ribosomal proteins paralleled total protein content during refeeding in both control and rapamycin-injected rats. We conclude that accretion of liver protein during refeeding is dependent on mTOR-mediated activation of the translation of ribosomal proteins but not dependent on mTOR-mediated activation of cap-dependent translation initiation.

Animals↗

PubMed1995

GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3.

GCN4 mRNA is translated by a reinitiation mechanism involving four short upstream open reading frames (uORFs) in its leader sequence. Decreasing the activity of eukaryotic initiation factor-2 (eIF-2) by phosphorylation inhibits general translation in yeast but stimulates GCN4 expression by allowing ribosomes to scan past the uORFs and reinitiate at GCN4 instead. GCD10 was first identified genetically as a translational repressor of GCN4. We show here that GCD10 is an essential protein of 54.6 kD that is required in vivo for the initiation of total protein synthesis. GCD10 binds RNA in vitro and we present strong biochemical evidence that it is identical to the RNA-binding subunit of yeast initiation factor-3 (eIF-3). eIF-3 is a multisubunit complex that stimulates translation initiation in vitro at several different steps. We suggest that gcd10 mutations decrease the ability of eIF-3 to stimulate binding of eIF-2/GTP/Met-tRNA(iMet) ternary complexes to small ribosomal subunits in vivo. This would explain why mutations in eIF-3 mimic eIF-2 alpha phosphorylation in allowing ribosomes to bypass the uORFs and reinitiate at GCN4. Our results indicate that GCN4 expression provides a sensitive in vivo assay for the function of eIF-3 in initiation complex formation.

Amino Acid Sequence↗

PubMed1987

Plastid Development in Pisum sativum Leaves during Greening : II. Post-Translational Uptake by Plastids as an Indicator System to Monitor Changes in Translatable mRNA for Nuclear-Encoded Plastid Polypeptides.

When isolated pea plastids are incubated with translation products of poly(A(+)) mRNA they specifically import precursor molecules of plastid polypeptides. Etioplasts and chloroplasts import the same polypeptides from identical translation products, and, the imported polypeptides can be well resolved by two-dimensional gel electrophoresis. Therefore, the posttranslational uptake system using isolated chloroplasts can monitor changes in the abundance of translatable plastid-targeted messages. Poly(A(+)) mRNA was isolated from peas at various times during greening and analyzed by this technique. (a) After 48 hours of illumination of dark-grown plants, the relative portion of nuclear encoded messages for plastid targeted proteins had increased by a factor of 2. The percentage of polypeptides recovered in the stroma fraction increased from about 50 to 65%. (b) More than 140 imported polypeptide species could be detected in fluorograms of two-dimensional gels, most of which could be identified throughout the time course of greening. At least 37 imported polypeptides decreased and 36 increased in relative abundance during greening of darkgreen plants. (c) In most cases, where differences in translatable messages were seen between dark- and light-grown plants, they were accompanied by parallel changes in polypeptide abundance.

Journal Article↗

PubMed1981

Encephalomyocarditis viral RNA can be translated under conditions of poliovirus-induced translation shutoff in vivo.

Superinfection with poliovirus of HeLa cells already infected with encephalomyocarditis (EMC) virus does not inhibit translation of EMC viral mRNA, whereas residual host translation is completely inhibited. This result indicates that the cap recognition factors inactivated by poliovirus are not required for translation of EMC viral mRNA in vivo, in agreement with previous in vitro experiments. This raises the question of why EMC virus has evolved a capindependent translation mechanism.

Carrier Proteins↗

PubMed1998

A small yeast RNA blocks hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation and inhibits replication of a chimeric poliovirus under translational control of the HCV IRES element.

Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to development of hepatocellular carcinoma. We previously identified a small yeast RNA (IRNA) capable of specifically inhibiting poliovirus (PV) internal ribosome entry site (IRES)-mediated translation. Here we report that IRNA specifically inhibits HCV IRES-mediated translation both in vivo and in vitro. A number of human hepatoma (Huh-7) cell lines expressing IRNA were prepared and characterized. Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell lines. Additionally, Huh-7 cells constitutively expressing IRNA became refractory to infection by a PV-HCV chimera in which the PV IRES is replaced by the HCV IRES. In contrast, replication of a PV-encephalomyocarditis virus (EMCV) chimera containing the EMCV IRES element was not affected significantly in the IRNA-producing cell line. Finally, the binding of the La autoantigen to the HCV IRES element was specifically and efficiently competed by IRNA. These results provide a basis for development of novel drugs effective against HCV infection.

Autoantigens↗

PubMed2006

Specific isoforms of translation initiation factor 4GI show differences in translational activity.

The eukaryotic initiation factor (eIF) 4GI gene locus (eIF4GI) contains three identified promoters, generating alternately spliced mRNAs, yielding a total of five eIF4GI protein isoforms. Although eIF4GI plays a critical role in mRNA recruitment to the ribosomes, little is known about the functions of the different isoforms, their partner binding capacities, or the role of the homolog, eIF4GII, in translation initiation. To directly address this, we have used short interfering RNAs (siRNAs) expressed from DNA vectors to silence the expression of eIF4GI in HeLa cells. Here we show that reduced levels of specific mRNA and eIF4GI isoforms in HeLa cells promoted aberrant morphology and a partial inhibition of translation. The latter reflected dephosphorylation of 4E-BP1 and decreased eIF4F complex levels, with no change in eIF2alpha phosphorylation. Expression of siRNA-resistant Myc-tagged eIF4GI isoforms has allowed us to show that the different isoforms exhibit significant differences in their ability to restore translation rates. Here we quantify the efficiency of eIF4GI promoter usage in mammalian cells and demonstrate that even though the longest isoform of eIF4GI (eIF4GIf) was relatively poorly expressed when reintroduced, it was more efficient at promoting the translation of cellular mRNAs than the more highly expressed shorter isoforms used in previous functional studies.

Amino Acid Sequence↗

PubMed2000

Eukaryotic translation initiation factor 4E (eIF4E) binding site and the middle one-third of eIF4GI constitute the core domain for cap-dependent translation, and the C-terminal one-third functions as a modulatory region.

The mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three roughly equal regions; an amino-terminal one-third (amino acids [aa] 1 to 634), which contains the poly(A) binding protein (PABP) and eIF4E binding sites; a middle third (aa 635 to 1039), which binds eIF4A and eIF3; and a carboxy-terminal third (aa 1040 to 1560), which harbors a second eIF4A binding site and a docking sequence for the Ser/Thr kinase Mnk1. Previous reports demonstrated that the middle one-third of eIF4GI is sufficient for cap-independent translation. To delineate the eIF4GI core sequence required for cap-dependent translation, various truncated versions of eIF4GI were examined in an in vitro ribosome binding assay with beta-globin mRNA. A sequence of 540 aa encompassing aa 550 to 1090, which contains the eIF4E binding site and the middle region of eIF4GI, is the minimal sequence required for cap-dependent translation. In agreement with this, a point mutation in eIF4GI which abolished eIF4A binding in the middle region completely inhibited ribosomal binding. However, the eIF4GI C-terminal third region, which does not have a counterpart in yeast, modulates the activity of the core sequence. When the eIF4A binding site in the C-terminal region of eIF4GI was mutated, ribosome binding was decreased three- to fourfold. These data indicate that the interaction of eIF4A with the middle region of eIF4GI is necessary for translation, whereas the interaction of eIF4A with the C-terminal region plays a modulatory role.

Amino Acid Sequence↗

PubMed2004

Translational repression mediates activation of nuclear factor kappa B by phosphorylated translation initiation factor 2.

Numerous stressful conditions activate kinases that phosphorylate the alpha subunit of translation initiation factor 2 (eIF2alpha), thus attenuating mRNA translation and activating a gene expression program known as the integrated stress response. It has been noted that conditions associated with eIF2alpha phosphorylation, notably accumulation of unfolded proteins in the endoplasmic reticulum (ER), or ER stress, are also associated with activation of nuclear factor kappa B (NF-kappaB) and that eIF2alpha phosphorylation is required for NF-kappaB activation by ER stress. We have used a pharmacologically activable version of pancreatic ER kinase (PERK, an ER stress-responsive eIF2alpha kinase) to uncouple eIF2alpha phosphorylation from stress and found that phosphorylation of eIF2alpha is both necessary and sufficient to activate both NF-kappaB DNA binding and an NF-kappaB reporter gene. eIF2alpha phosphorylation-dependent NF-kappaB activation correlated with decreased levels of the inhibitor IkappaBalpha protein. Unlike canonical signaling pathways that promote IkappaBalpha phosphorylation and degradation, eIF2alpha phosphorylation did not increase phosphorylated IkappaBalpha levels or affect the stability of the protein. Pulse-chase labeling experiments indicate instead that repression of IkappaBalpha translation plays an important role in NF-kappaB activation in cells experiencing high levels of eIF2alpha phosphorylation. These studies suggest a direct role for eIF2alpha phosphorylation-dependent translational control in activating NF-kappaB during ER stress.

Animals↗

PubMed2004

A novel function of the MA-3 domains in transformation and translation suppressor Pdcd4 is essential for its binding to eukaryotic translation initiation factor 4A.

An alpha-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4(E249K), Pdcd4(D253A), Pdcd4(D414K), Pdcd4(D418A), Pdcd4(E249K,D414K), and Pdcd4(D253A,D418A)) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4(D253A) and Pdcd4(D253A,D418A) do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4(D253A), Pdcd4(D418A), or Pdcd4(D235A,D418A). Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.

5' Untranslated Regions↗

PubMed2005

Translation of a small subset of Caenorhabditis elegans mRNAs is dependent on a specific eukaryotic translation initiation factor 4E isoform.

The mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) participates in protein synthesis initiation, translational repression of specific mRNAs, and nucleocytoplasmic shuttling. Multiple isoforms of eIF4E are expressed in a variety of organisms, but their specific roles are poorly understood. We investigated one Caenorhabditis elegans isoform, IFE-4, which has homologues in plants and mammals. IFE-4::green fluorescent protein (GFP) was expressed in pharyngeal and tail neurons, body wall muscle, spermatheca, and vulva. Knockout of ife-4 by RNA interference (RNAi) or a null mutation produced a pleiotropic phenotype that included egg-laying defects. Sedimentation analysis demonstrated that IFE-4, but not IFE-1, was present in 48S initiation complexes, indicating that it participates in protein synthesis initiation. mRNAs affected by ife-4 knockout were determined by DNA microarray analysis of polysomal distribution. Polysome shifts, in the absence of total mRNA changes, were observed for only 33 of the 18,967 C. elegans mRNAs tested, of which a disproportionate number were related to egg laying and were expressed in neurons and/or muscle. Translational regulation was confirmed by reduced levels of DAF-12, EGL-15, and KIN-29. The functions of these proteins can explain some phenotypes observed in ife-4 knockout mutants. These results indicate that translation of a limited subset of mRNAs is dependent on a specific isoform of eIF4E.

Alleles↗