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At least 145 records · Page 8Linked to original sources

PubMed2006

Role of the RNA2 3' non-translated region of Blackcurrant reversion nepovirus in translational regulation.

The 3' non-translated regions (NTRs) of mRNAs of eukaryotes and their viruses often contain translational enhancers (TEs). Blackcurrant reversion nepovirus (BRV) has a genome composed of two uncapped polyadenylated RNAs with very long 3' NTRs, nucleotide sequences of which are very conserved between different BRV isolates. In this work, we studied a role of the RNA2 3' NTR in translation, using mutagenesis of the firefly luciferase reporter mRNA, in protoplasts of Nicotiana benthamiana. The RNA2 3' NTR was found to contain a cap-independent TE (3' CITE), which must base pair with the 5' NTR to facilitate translation. The BRV 3' CITE and poly(A) tail provided a major contribution to translational efficiency, with less input from other 3' NTR parts. The BRV 3' CITE does not share similarity in nucleotide sequence and secondary structure with other viruses and thus represents a new class of 3' CITE.

3' Untranslated Regions↗

PubMed1990

Translated translational operator in Escherichia coli. Auto-regulation in the infC-rpmI-rplT operon.

The genes coding for translation initiation factor IF3 (infC) and for the ribosomal proteins L35 (rpmI) and L20 (rplT) are transcribed in that order from a promoter in front of infC. The last two cistrons of the operon (rpmI and rplT) can be transcribed from a weak secondary promoter situated within the first cistron (infC). Previous experiments have shown that the expression of infC, the first cistron of the operon, is negatively autoregulated at the translational level and that the abnormal AUU initiation codon of infC is responsible for the control. We show that the expression of the last cistron (rplT) is also autoregulated at the posttranscriptional level. The L20 concentration regulates the level of rplT expression by acting in trans at a site located within the first cistron (infC) and thus different from that at which IF3 is known to act. This regulatory site, several hundred nucleotides upstream from the target gene (rplT), was identified through deletions, insertions and a point mutation. Thus, the expression of the operon is controlled in trans by the products of two different cistrons acting at two different sites. The localization within an open reading frame (infC) of a regulatory site acting in cis on the translation of a downstream gene (rplT) is new and was unforeseen since ribosomes translating through the regulatory site might be expected to impair either the binding of L20 or the mRNA secondary structure change caused by the binding. The possible competition between translation of the regions acting in cis and the regulation of the expression of the target gene is discussed.

Bacterial Proteins↗

PubMed1996

Translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and ribosomal proteins L20 and L35.

The Escherichia coli IF3-L35-L20 operon encodes translation initiation factor IF3 and the ribosomal proteins L35 and L20, respectively. The expression of the genes encoding the two ribosomal proteins is negatively regulated at the translational level by L20, which acts at an operator located within the IF3 gene and just upstream of the L35 gene. We have previously shown that L20 directly represses the expression of the L35 gene, and indirectly that of the L20 gene, via translational coupling. On the basis of mutational analysis and in vitro RNA structure probing experiments, we proposed that a large secondary structure in which the translation initiation site of the L20 gene is sequestered by base-pairing, is responsible for coupling. The ribosome binding site of the L20 gene becomes available when the secondary structure is melted by ribosomes translating the L35 mRNA. Here we describe that this secondary structure forms in vivo by showing that single mutations in either strand reduce coupling and that compensatory mutations that re-establish pairing also re-establish coupling. In vitro 'toeprinting' analysis enabled us to show that the wild-type inhibitory secondary structure directly blocks ribosome binding to the ribosome binding site of rpIT.

Bacterial Proteins↗

PubMed1999

In vitro-translated diphtheria toxin A chain inhibits translation in wheat germ extracts: analysis of biologically active, caspase-3-resistant diphtheria toxin mutants.

The diphtheria toxin A chain (DTA) is a potent cytocidal agent that inactivates elongation factor 2. This activity of DTA inhibits protein synthesis and rapidly leads to cell death through apoptosis. In this paper, we have developed a simple in vitro assay for DTA activity in which in vitro-translated DTA is used to inhibit the translation of proteins in wheat germ extracts. Inhibition of translation by DTA is dependent on cofactor NAD+, and the analysis of an attenuated DTA mutant indicates that this in vitro assay accurately reflects the in vivo activity of DTA. We have also identified aspartic acid at residue 8 (Asp-8) of DTA as a site of cleavage by the cell-death protease caspase-3. Cleavage of DTA by caspase-3 inactivates its ability to inhibit translation in wheat germ extracts. Conservative mutations at Asp-8 render DTA resistant to cleavage by caspase-3, but only slightly affect the ability of DTA to inhibit translation in vitro. Moreover, caspase-3-resistant DTA mutants are toxic in cells in tissue culture. The in vitro assay that we describe here will be useful for the rapid analysis of DTA activity and the development of DTA mutants with altered biological properties that may be of therapeutic value. Lastly, these studies serve as a prototype for the creation of caspase-resistant effector molecules.

Amino Acid Sequence↗

PubMed2000

Cleavage of eukaryotic translation initiation factor 4GII correlates with translation inhibition during apoptosis.

Eukaryotic translation initiation factor 4G (eIF4G), which has two homologs known as eIF4GI and eIF4GII, functions in a complex (eIF4F) which binds to the 5' cap structure of cellular mRNAs and facilitates binding of capped mRNA to 40S ribosomal subunits. Disruption of this complex in enterovirus-infected cells through eIF4G cleavage is known to block this step of translation initiation, thus leading to a drastic inhibition of cap-dependent translation. Here, we show that like eIF4GI, the newly identified homolog eIF4GII is cleaved during apoptosis in HeLa cells and can serve as a substrate for caspase 3. Proteolysis of both eIF4GI and eIF4GII occurs with similar kinetics and coincides with the profound translation inhibition observed in cisplatin-treated HeLa cells. Both eIF4GI and eIF4GII can be cleaved by caspase 3 with similar efficiency in vitro, however, eIF4GII is processed into additional fragments which destroy its core central domain and likely contributes to the shutoff of translation observed in apoptosis. Cell Death and Differentiation (2000) 7, 1234 - 1243.

Animals↗

PubMed2006

A novel mechanism for Bcr-Abl action: Bcr-Abl-mediated induction of the eIF4F translation initiation complex and mRNA translation.

The oncogenic kinase Bcr-Abl is thought to cause chronic myelogenous leukemia (CML) by altering the transcription of specific genes with growth- and survival-promoting functions. Recently, Bcr-Abl has also been shown to activate an important regulator of protein synthesis, the mammalian target of rapamycin (mTOR), which suggests that dysregulated translation may also contribute to CML pathogenesis. In this study, we found that both Bcr-Abl and the rapamycin-sensitive mTORC1 complex contribute to the phosphorylation (inactivation) of 4E-BP1, an inhibitor of the eIF4E translation initiation factor. Experiments with rapamycin and the Bcr-Abl inhibitor, imatinib mesylate, in Bcr-Abl-expressing cell lines and primary CML cells indicated that Bcr-Abl and mTORC1 induced formation of the translation initiation complex, eIF4F. This was characterized by reduced 4E-BP1 binding and increased eIF4G binding to eIF4E, two events that lead to the assembly of eIF4F. One target transcript is cyclin D3, which is regulated in Bcr-Abl-expressing cells by both Bcr-Abl and mTORC1 in a translational manner. In addition, the combination of imatinib and rapamycin was found to act synergistically against committed CML progenitors from chronic and blast phase patients. These experiments establish a novel mechanism of action for Bcr-Abl, and they provide insights into the modes of action of imatinib mesylate and rapamycin in treatment of CML. They also suggest that aberrant cap-dependent mRNA translation may be a therapeutic target in Bcr-Abl-driven malignancies.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Translation ability of mitochondrial tRNAsSer with unusual secondary structures in an in vitro translation system of bovine mitochondria.

BACKGROUND: Metazoan mitochondrial (mt) tRNAs are structurally quite different from the canonical cloverleaf secondary structure. The mammalian mt tRNASerGCU for AGY codons (Y = C or U) lacks the entire D arm, whereas tRNASerUGA for UCN codons (N = A, G, C or U) has an extended anti-codon stem. It has been a long-standing problem to prove experimentally how these tRNAsSer work in the mt translation system. RESULTS: To solve the above-mentioned problem, we examined their translational abilities in an in vitro bovine mitochondrial translation system using transcripts of altered tRNASer analogues derived from bovine mitochondria. Both tRNASer analogues had almost the same ability to form ternary complexes with mt EF-Tu and GTP. The D-arm-lacking tRNASer GCU analogue had considerably lower translational activity than the tRNASerUGA analogue and produced mostly short oligopeptides, up to a tetramer. In addition, tRNASerGCU analogue was disfavoured by the ribosome when other tRNAs capable of decoding the cognate codon were available. CONCLUSION: Both mt tRNASerGCU and tRNASerUGA analogues with unusual secondary structure were found to be capable of translation on the ribosome. However, the tRNASerGCU analogue has some molecular disadvantage on the ribosome, which probably derives from the lack of a D arm.

Animals↗

PubMed2005

Cap-binding protein 1-mediated and eukaryotic translation initiation factor 4E-mediated pioneer rounds of translation in yeast.

Nonsense-mediated mRNA decay (NMD) in mammalian cells is restricted to newly synthesized mRNA that is bound at the 5' cap by the major nuclear cap-binding complex and at splicing-generated exon-exon junctions by exon junction complexes. This messenger ribonucleoprotein has been called the pioneer translation initiation complex and, accordingly, NMD occurs as a consequence of nonsense codon recognition during a pioneer round of translation. Here, we characterize the nature of messenger ribonucleoprotein that is targeted for NMD in Saccharomyces cerevisiae. Data indicate that NMD targets both cap-binding complex (Cbc)1p- and eukaryotic translation initiation factor (eIF)4E-bound mRNAs, unlike in mammalian cells, where NMD does not detectably target eIF4E-bound mRNA. First, intron-containing pre-mRNAs in yeast are detectably bound by either Cbc1p, or, unlike in mammalian cells, eIF4E, indicating that mRNAs can be derived from either Cbc1p- or eIF4E-bound pre-mRNAs. Second, the ratio of nonsense-containing Cbc1p-bound mRNA to nonsense-free Cbc1p-bound mRNA, which was < 0.4 for those mRNAs tested here, is essentially identical to the ratio of the corresponding nonsense-containing eIF4E-bound mRNA to nonsense-free eIF4E-bound mRNA, and both ratios increase in cells treated with the translational inhibitor cycloheximide (CHX). These data, together with data presented here and elsewhere showing that Cbc1p-bound transcripts are precursors to eIF4E-bound transcripts, demonstrate that Cbc1p-bound mRNA is targeted for NMD. In support of the idea that eIF4E-bound mRNA is also targeted for NMD, eIF4E-bound mRNA is targeted for NMD in strains that lack Cbc1p. These results suggest that both Cbc1p- and eIF4E-mediated pioneer rounds of translation occur in yeast.

Base Sequence↗

PubMed1993

A protein complex of translational regulators of GCN4 mRNA is the guanine nucleotide-exchange factor for translation initiation factor 2 in yeast.

In Saccharomyces cerevisiae, phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2) by protein kinase GCN2 stimulates translation of GCN4 mRNA. In mammalian cells, phosphorylation of eIF-2 alpha inhibits the activity of eIF-2B, the GDP-GTP exchange factor for eIF-2. We present biochemical evidence that five translational regulators of GCN4 encoded by GCD1, GCD2, GCD6, GCD7, and GCN3 are components of a protein complex that stably interacts with eIF-2 and represents the yeast equivalent of eIF-2B. In vitro, this complex catalyzes guanine nucleotide exchange on eIF-2 and overcomes the inhibitory effect of GDP on formation of eIF-2.GTP.Met-initiator tRNA(Met) ternary complexes. This finding suggests that mutations in GCD-encoded subunits of the complex derepress GCN4 translation because they mimic eIF-2 alpha phosphorylation in decreasing eIF-2B activity. Our results indicate that translational control of GCN4 involves a reduction in eIF-2B function, a mechanism used in mammalian cells to regulate total protein synthesis in response to stress.

Amino Acid Sequence↗

PubMed2000

Co-translational folding of an eukaryotic multidomain protein in a prokaryotic translation system.

Continuous monitoring of the enzymatic activity of newly synthesized firefly luciferase in Escherichia coli cell-free translation system was performed to record folding kinetics of this multidomain eukaryotic protein in the prokaryotic cytosol. Whereas in vitro refolding of denatured luciferase in prokaryotic cytosol occurred with a low yield of active enzyme and took about an hour, the enzyme acquired its native structure immediately upon release from the ribosome, as seen from the immediate halt of active luciferase accumulation upon blocking of translation with inhibitors. The nascent luciferase was also capable of acquiring the active conformation prior to release from the ribosome, when its C terminus was extended with a polypeptide segment. Specific enzymatic activity of the firefly luciferase was found to be equally high irrespective of whether this protein was synthesized in eukaryotic or prokaryotic translation systems. The data presented demonstrate the fundamental ability of prokaryotic cytosol to support effective co-translational protein folding in general and co-translational folding of multidomain proteins in particular.

Base Sequence↗

PubMed2005

Translation of eukaryotic translation initiation factor 4GI (eIF4GI) proceeds from multiple mRNAs containing a novel cap-dependent internal ribosome entry site (IRES) that is active during poliovirus infection.

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential scaffolding protein required to recruit the 43 S complex to the 5'-end of mRNA during translation initiation. We have previously demonstrated that eIF4GI protein expression is translationally regulated. This regulation is mediated by cis-acting RNA elements, including an upstream open reading frame and an IRES that directs synthesis of five eIF4GI protein isoforms via alternative AUG initiation codon selection. Here, we further characterize eIF4GI IRES function and show that eIF4GI is expressed from several distinct mRNAs that vary via alternate promoter use and alternate splicing. Several mRNA variants contain the IRES element. We found that IRES activity mapped to multiple regions within the eIF4GI RNA sequence, but not within the 5'-UTR per se. However, the 5'-UTR enhanced IRES activity in vivo and played a role in initiation codon selection. The eIF4GI IRES was active when transfected into cells in an RNA form, and thus, does not require nuclear processing events for its function. However, IRES activity was found to be dependent upon the presence, in cis, of a 5' m7guanosine-cap. Despite this requirement, the eIF4GI IRES was activated by 2A protease cleavage of eIF4GI, in vitro, and retained the ability to promote translation during poliovirus-mediated inhibition of cap-dependent translation. These data indicate that intact eIF4GI protein is not required for the de novo synthesis of eIF4GI, suggesting its expression can continue under stress or infection conditions where eIF4GI is cleaved.

5' Untranslated Regions↗

PubMed2004

Iron regulates hepatitis C virus translation via stimulation of expression of translation initiation factor 3.

BACKGROUND: Although the response to treatment with interferon- alpha in individuals with chronic hepatitis C virus (HCV) infection is negatively associated with increased liver iron stores, the underlying mechanisms at work have remained elusive to date. The translation initiation factor 3 (eIF3) is essential for HCV translation, and thus the effects that iron perturbations have on eIF3 expression and HCV translation were studied here. METHODS: eIF3 expression was analyzed by TaqMan polymerase chain reaction, Northern and Western blot analysis of HepG2 cells, and liver biopsies. Functional effects of iron on HCV mRNA translation were estimated by use of transient transfection experiments with bicistronic vectors. RESULTS: Iron treatment of HepG2 cells increased eIF3 mRNA and protein expression, whereas iron chelation reduced it. Accordingly, iron-dependent stimulation of eIF3 specifically induced the expression of reporter genes under the control of regulatory HCV mRNA stem-loop structures. Moreover, a positive association between liver iron levels, eIF3 expression, and HCV expression was found when liver-biopsy samples from HCV-infected patients were analyzed. CONCLUSION: Iron promotes the translation of HCV by stimulating the expression of eIF3, which may be one reason for the negative association between liver iron overload and HCV infection. Modulation of the affinity of eIF3 to bind to HCV mRNA may be a promising target for the treatment of chronic HCV infection.

Biopsy↗

PubMed1995

The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging.

Rous sarcoma virus (RSV) RNA leader contains three short upstream open reading frames. We have shown recently that both uORFs 1 and 3 influence in vivo translation of the downstream gag gene and are involved in the virus RNA packaging process. In this report, we have studied the translational events occurring at the upstream AUGs in vivo. We show that (i) the first and third AUGs are efficient translational initiation sites; (ii) ribosomes reinitiate efficiently at AUG3; and (iii) deletions in the intercistronic distance between uORF1 and 3 (which is well conserved among avian strains) prevent ribosome initiation at AUG3, thus increasing translation efficiency at the downstream AUGgag. The roles of the uORFs in translation and packaging are discussed.

Animals↗

PubMed1996

Translational stop codons in the precore sequence of hepatitis B virus pre-C RNA allow translation reinitiation at downstream AUGs.

Hepatitis B virus (HBV) wild-type pre-C RNA directs the synthesis of the HBeAg precursor but does not serve as mRNA for translation of the adjacent downstream C gene which encodes the core protein. Using bicistronic mRNA constructs that mimick pre-C RNA, we have demonstrated that this RNA likewise does not serve as messenger for translation of the P gene, which is located downstream of the C gene. However, when the pre-C RNA contains a translational stop codon at position 2 or 28 of the pre-C sequence (as in certain HBV mutants), it no longer directs synthesis of the HBeAg precursor but instead translation is initiated at downstream C and P gene AUGs. We propose that this occurs by a translation reinitiation mechanism.

Base Sequence↗

PubMed1996

The translational cis-regulatory element of mammalian ribosomal protein mRNAs is recognized by the plant translational apparatus.

The translational efficiency of mammalian ribosomal protein mRNAs correlates with the growth status of the cells and its control is mediated through a 5' terminal oligopyrimidine tract (5' TOP) common to all these mRNAs. In the present study, we demonstrate that the plant translational apparatus, as represented by wheat-germ extract, discriminates against mammalian mRNAs containing this motif to the same extent as do quiescent mammalian cells. Moreover, mutations in the 5' TOP, which abolish the growth-dependent translational control of the respective mRNAs in mammalian cells, render these mRNAs refractory to discrimination in the plant cell-free system. This selective discrimination reflects neither the specific instability of 5' TOP-containing mRNAs during the incubation in vitro nor a lower competitive potential for the cap-binding protein. The lower in vitro translational efficiency of these mRNAs is an inherent feature which is independent of whether they were derived from polysomes or messenger ribonucleoprotein particles of the transfected mammalian cells. The conservation of the discriminatory property of the translational apparatus between the animal and plant kingdoms is discussed from mechanistic and evolutionary points of view.

3T3 Cells↗

PubMed2001

Eukaryotic translation initiation factor 4E-dependent translation is not essential for survival of starved yeast cells.

The eukaryotic translation initiation factor 4E (eIF4E) interacts with the mRNA 5' cap structure (m(7)GpppX) and is essential for the appropriate translation of the vast majority of eukaryotic mRNAs. Most studies of the yeast Saccharomyces cerevisiae CDC33 gene product, eIF4E, have been carried out with logarithmically growing cells, and little is known about its role in starved, nonproliferating cells that enter the stationary phase (SP). It has previously been found that the rate of translation in SP cells is more than 2 orders of magnitude lower than it is in dividing yeast cells. Here we show that this low rate of translation is essential for maintaining the viability of starved yeast cells that enter SP. Specifically, starved cells whose eIF4A is inactive or treated with cycloheximide rapidly lose viability. Moreover, after heat inactivation of the cdc33 temperature-sensitive product, the synthesis of most proteins is abolished and only a small group of proteins is still produced. Unexpectedly, starved cdc33 mutant cells whose eIF4E is inactive and which therefore fail to synthesize the bulk of their proteins remain viable for long periods of time, indistinguishable from their isogenic wild-type counterparts. Taken together, our results indicate that eIF4E-independent translation is necessary and sufficient for survival of yeast cells during long periods of starvation.

Eukaryotic Cells↗

PubMed1993

GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells. We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCN1. Inactivation of GCN1 did not affect the level of eIF-2 alpha phosphorylation when mammalian eIF-2 alpha kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2 alpha. In addition, while GCN1 is required in vivo for phosphorylation of eIF-2 alpha by GCN2, cell extracts from gcn1 delta strains contained wild-type levels of GCN2 eIF-2 alpha-kinase activity. On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA. GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species. This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.

Amino Acid Sequence↗

PubMed1998

Translation of vascular endothelial growth factor mRNA by internal ribosome entry: implications for translation under hypoxia.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic growth factor that promotes compensatory angiogenesis in circumstances of oxygen shortage. The requirement for translational regulation of VEGF is imposed by the cumbersome structure of the 5' untranslated region (5'UTR), which is incompatible with efficient translation by ribosomal scanning, and by the physiologic requirement for maximal VEGF production under conditions of hypoxia, where overall protein synthesis is compromised. Using bicistronic reporter gene constructs, we show that the 1,014-bp 5'UTR of VEGF contains a functional internal ribosome entry site (IRES). Efficient cap-independent translation is maintained under hypoxia, thereby securing efficient production of VEGF even under unfavorable stress conditions. To identify sequences within the 5'UTR required for maximal IRES activity, deletion mutants were analyzed. Elimination of the majority (851 nucleotides) of internal 5'UTR sequences not only maintained full IRES activity but also generated a significantly more potent IRES. Activity of the 163-bp long "improved" IRES element was abrogated, however, following substitution of a few bases near the 5' terminus as well as substitutions close to the translation start codon. Both the full-length 5'UTR and its truncated version function as translational enhancers in the context of a monocistronic mRNA.

3T3 Cells↗