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Results for “Translational repression”

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PubMed1993

Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae.

Starvation of the yeast Saccharomyces cerevisiae for an amino acid signals increased translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. We have isolated and characterized the GCD6 and GCD7 genes and shown that their products are required to repress GCN4 translation under nonstarvation conditions. We find that both GCD6 and GCD7 show sequence similarities to components of a high-molecular-weight complex (the GCD complex) that appears to be the yeast equivalent of translation initiation factor 2B (eIF-2B), which catalyzes GDP-GTP exchange on eIF-2. Furthermore, we show that GCD6 is 30% identical to the largest subunit of eIF-2B isolated from rabbit reticulocytes. Deletion of either GCD6 or GCD7 is lethal, and nonlethal mutations in these genes increase GCN4 translation in the same fashion described for defects in known subunits of eIF-2 or the GCD complex; derepression of GCN4 is dependent on short open reading frames in the GCN4 mRNA leader and occurs independently of eIF-2 alpha phosphorylation by protein kinase GCN2, which is normally required to stimulate GCN4 translation. Together, our results provide evidence that GCD6 and GCD7 are subunits of eIF-2B in S. cerevisiae and further implicate this GDP-GTP exchange factor in gene-specific translational control.

Amino Acid Sequence↗

PubMed1990

A morphogenetic gradient of hunchback protein organizes the expression of the gap genes Krüppel and knirps in the early Drosophila embryo.

Segmentation of the Drosophila embryo depends on a hierarchy of interactions among the maternal and zygotic genes in the early embryo. The anterior region is organized maternally by the bicoid (bcd) gene product, which forms a concentration gradient in the anterior half of the embryo. The gap genes are also involved in establishing the body plan, with hunchback (hb) being expressed both maternally and zygotically. Zygotic expression of hb is directly activated by the bcd gene product, leading to a subdivision of the embryo into an anterior half expressing zygotically provided hb protein and a posterior half that does not. A similar effect on maternally provided hb protein is caused by the gene nanos, which represses the translation of maternally provided transcripts in the posterior half. This regulation of hb protein is a prerequisite for abdomen development, because the presence of hb protein in the posterior half represses posterior segmentation. This repression mechanism suggests that posterior segmentation might not directly depend on maternal positional cues, but be solely organized at the zygotic level. Here we report further evidence to support this hypothesis and show that the hb protein itself is crucially involved in organizing abdominal segmentation. Differential concentrations of hb protein determine the anterior and posterior borders of expression of the gap gene Krüppel (Kr) and the anterior border of the gap gene knirps (kni), thus defining three positional values. These regulatory pathways are controlled in a redundant way, in part by bcd and in part by the maternal hb gene product.

Animals↗

PubMed1995

Crystal structure of the T4 regA translational regulator protein at 1.9 A resolution.

The translational regulator protein regA is encoded by the T4 bacteriophage and binds to a region of messenger RNA (mRNA) that includes the initiator codon. RegA is unusual in that it represses the translation of about 35 early T4 mRNAs but does not affect nearly 200 other mRNAs. The crystal structure of regA was determined at 1.9 A resolution; the protein was shown to have an alpha-helical core and two regions with antiparallel beta sheets. One of these beta sheets has four antiparallel strands and has some sequence homology to RNP-1 and RNP-2, which are believed to be RNA-binding motifs and are found in a number of known RNA-binding proteins. Structurally guided mutants may help to uncover the basis for this variety of RNA interaction.

Amino Acid Sequence↗

PubMed2003

Bacterial translational control at atomic resolution.

Translational regulation allows rapid adaptation of protein synthesis to environmental conditions. In prokaryotes, the synthesis of many RNA-binding proteins is regulated by a translational feedback mechanism involving a competition between their natural substrate and their binding site on mRNA, which are often thought to resemble each other. This article describes the case of threonyl-tRNA synthetase, which represses the translation of its own mRNA. Recent data provide the first opportunity to describe at the atomic level both the extent and the limit of mimicry between the way this enzyme recognizes tRNA(Thr) and its regulatory site in mRNA. The data also give some clues about how the binding of the synthetase to its mRNA inhibits translation.

Base Sequence↗

PubMed2000

Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.).

Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

5' Untranslated Regions↗

PubMedDate not supplied

CRD-BP: a c-Myc mRNA stabilizing protein with an oncofetal pattern of expression.

The Coding Region Determinant-Binding Protein (CRD-BP) is an RRM and KH-domain-containing protein that recognizes specifically at least three RNAs. It binds to one of the two c-myc mRNA instability elements, to the 5'Un Translated Region (UTR) of the leader 3 IGF-II mRNA and to the oncofetal H19 RNA. CRD-BP has been assigned a role in stabilizing c-myc mRNA by preventing its endonucleolytic cleavage and in repressing the translation of the leader 3 IGF-II mRNA, the major embryonic species of this message. CRD-BP is normally expressed only in fetal tissues. However, its expression is detected in primary tumors and transformed cell lines of different origins. The vast majority of colon (80%) and breast (60%) tumors and sarcomas (73%) express CRD-BP whereas in other tumor types, for example prostate carcinomas, its expression is rare. CRD-BP expression has also been detected in benign tumors such as breast fibroadenomas, meningiomas and other benign mesenchymal tumors, implying a role for this gene in abnormal cell proliferation. In breast carcinomas, CRD-BP expression and or gene copy number gains in the region encompassing the c-myc locus were detected in approximately 75% of tumors, implying that the deregulated expression of c-myc may be more widespread than previously believed. Infiltrated lymph nodes, corresponding to CRD-BP-positive primary tumors, were also found positive indicating that monitoring for CRD-BP could prove useful for the detection and monitoring of disseminated disease.

Animals↗

PubMed1995

Testis-brain RNA-binding protein, a testicular translational regulatory RNA-binding protein, is present in the brain and binds to the 3' untranslated regions of transported brain mRNAs.

Previous studies have demonstrated that a phosphoprotein in testis binds to transcript c, a sequence containing two highly conserved elements, Y and H, in the 3' untranslated region (UTR) of mouse protamine 2 mRNA (mP2) and represses its translation in vitro. When gel-retardation assays were performed with cytoplasmic extracts prepared from seven different mouse tissues, we found that brain in addition to testis contains a protein that binds to transcript c. Both the testis and brain proteins are found exclusively in the nonpolysomal fractions of their postmitochondrial extracts. The testis and brain proteins appear to be identical according to numerous criteria: the complexes they form with transcript c have identical mobility in native gels, identical optimal pH, identical lability to increased salt concentrations, identical chromatographic properties, identical molecular sizes as judged from UV crosslinking, and identical peptide mapping as revealed by V8 digestion of the UV crosslinked protein-RNA complexes. In addition to binding to the same conserved sequence in the 3'UTR of mP2, the phosphoprotein from testis and brain, hereafter called testis-brain RNA-binding protein (TB-RBP), also specifically binds to a similar sequence in the 3'UTR of brain Tau mRNA. Since TB-RBP binds to the 3'UTRs of several translationally regulated mRNAs in testis and since numerous transported brain mRNAs also contain the same conserved binding elements, we propose that TB-RBP plays a role in mRNA storage, translocation, and/or localization in brain and testis.

Ammonium Sulfate↗

PubMed1996

Evidence for posttranscriptional regulation of C/EBPalpha and C/EBPbeta isoform expression during the lipopolysaccharide-mediated acute-phase response.

The mRNAs of the CCAAT/enhancer-binding trans-activator proteins (C/EBPalpha and C/EBPbeta) serve as templates for the differential translation of several isoforms which have specific transcriptional regulatory functions. By using an oligonucleotide corresponding to the C/EBP binding site of the mouse alpha1-acid glycoprotein promoter, we detected multiple forms of C/EBPalpha and C/EBP++ beta proteins in the mouse liver that have DNA-binding activity. By using specific antisera, we detected C/EBPalphas with molecular masses of 42, 38, 30, and 20 kDa that have DNA-binding activity. The pool levels of the 42- and 30-kDa isoforms were high in control nuclear extracts and decreased significantly after lipopolysaccharide (LPS) treatment. The binding activity and protein levels of the 20-kDa isoform are low in controls and increase dramatically after LPS treatment. C/EBPbeta isoforms with molecular masses of 35, 20, and 16 kDa were also detected. The 35-kDa pool level did not change whereas the 20-kDa isoform was strongly induced in response to LPS. Western (immunoblot) and Southwestern (DNA-protein) analyses show that p42 C/EBPalpha forms specific complexes with the alpha1-acid glycoprotein oligonucleotide in control nuclear extract and that p20 C/EBP beta forms complexes in LPS-treated liver. Our studies suggest that synthesis of specific C/EBPalpha and C/EBPbeta isoforms occurred in the normal liver in vivo and that LPS mediated a differential initiation and inhibition of translation at specific AUG sites within each mRNA. The qualitative and quantitative changes in C/EBPalpha and C/EBPbeta isoform pool levels suggest that LPS or an LPS-stimulated factor can regulate the selection of AUG start sites for both activation and repression of translation. This regulation appears to involve an LPS-mediated down-regulation of initiation at the first AUG codon of the 42-kDa C/EBPalpha and dramatic translational up-regulation at the fifth AUG codon of the 20-kDa C/EBPalpha and the third AUG codon of the 20-kDa C/EBPbeta. These regulatory events suggest the existence of proteins that may act as translational trans-acting factors.

Amino Acid Sequence↗

PubMed1999

Overexpression of poly(A)-binding protein down-regulates the translation or the abundance of its own mRNA.

Poly(A)-binding protein (PABP) mRNA is subject to autoregulation through a 61 nucleotides long A-rich sequence in its 5' untranslated region (UTR). Here, we show that this mode of regulation is exerted in a cell type-specific manner. Thus, overexpression of PABP in mouse NIH 3T3 fibroblasts represses the translation of the respective endogenous mRNA or that of a chimeric mRNA containing just the 5' UTR of PABP mRNA. In contrast, ectopic expression of PABP in human embryonic kidney 293 cells down-regulates the abundance of the endogenous PABP mRNA, rather than affecting its translational efficiency. Transfection experiments with chimeric constructs suggest that the lack of translational autoregulation of endogenous PABP mRNA in these cells appears to reflect the presence of an overriding regulatory element outside the A-rich region.

3T3 Cells↗

PubMed2000

Essential role of NAT1/p97/DAP5 in embryonic differentiation and the retinoic acid pathway.

NAT1/p97/DAP5 is a newly identified protein that shares homology with the translation initiation factor eIF4G. Studies in vitro and in transfected cells indicated that NAT1 might suppress global translation, thereby repressing cellular proliferation. Here we studied the functions of NAT1 in vivo by disrupting its gene in mice. NAT1(-/-) embryos died during gastrulation, indicating a crucial role for NAT1 in embryogenesis. Undifferentiated NAT1(-/-) embryonic stem cells were normal in morphology, proliferation, global translation and gene expression profile. However, NAT1(-/-) cells exhibited an impaired ability to differentiate: they were resistant to differentiation induced by retinoic acid, and teratomas derived from them consisted of undifferentiated and poorly differentiated tissues. The expression of retinoic acid-responsive genes, such as the cell-cycle inhibitor p21(WAF1), was selectively impaired in NAT1(-/-) cells. Transcription from synthetic retinoic acid-responsive elements was also impaired. These data demonstrated that this translation initiation factor homolog controls specific gene expression pathways required for cellular differentiation.

Animals↗

PubMed1989

tRNA-like structures and gene regulation at the translational level: a case of molecular mimicry in Escherichia coli.

Escherichia coli threonyl-tRNA synthetase regulates the translation of its own mRNA by binding to it in a region, called the operator, located in front of the ribosomal binding site. The primary and secondary structures of the operator resemble those of the anticodon arm of several tRNA(Thr) isoacceptor species. We reasoned that if the interaction between the synthetase and its two partially analogous ligands, the tRNA and the mRNA, had some common features, single mutations in the enzyme should affect both interactions in a very similar way. We thus isolated synthetase mutants (called super-repressors) that repress the translation of their mRNA in trans to an extreme level, and other mutants that are completely unable to perform any repression. The super-repressors, which are suspected to bind their mRNA with high affinity, are shown to bind the tRNA with an increased affinity. The non-repressing mutants, which are suspected to have lost their capacity to bind the mRNA, are shown to bind their tRNA with less affinity. The binding properties of the mutant enzymes for the other substrates, ATP and threonine, are unchanged. The observed correlation between regulatory and aminoacylation defects strongly suggests that the synthetase recognizes the similar parts of its two RNA ligands--the anticodon-like arm of the mRNA and the true anticodon arm of the tRNA--in an analogous way.

Amino Acid Sequence↗

PubMed1991

Characteristics of the interaction of the ferritin repressor protein with the iron-responsive element.

The iron-responsive regulation of ferritin mRNA translation is mediated by the specific interaction of the ferritin repressor protein (FRP) with the iron-responsive element (IRE), a highly conserved 28-nucleotide sequence located in the 5' untranslated region of ferritin mRNAs. The IRE alone is necessary and sufficient to confer repression of translation by FRP upon a heterologous message, chloramphenicol acetyltransferase, in an in vitro translation system. The activity of FRP is sensitive to iron in vivo. Cytoplasmic extracts of rabbit kidney cells show reduction of FRP activity when grown in the presence of iron, as detected by RNA band shift assay. Using a nitrocellulose filter binding assay to examine the interaction of FRP with the IRE in more detail, we find that purified FRP has a single high-affinity binding site for the IRE with a Kd of 20-50 pM. Hemin pretreatment decreases the total amount of FRP which can bind to the IRE. This effect is dependent on hemin concentration. Interestingly, the FRP which remains active at a given hemin concentration binds to the IRE with the same high affinity as untreated FRP. A variety of hemin concentrations were examined for their effect on preformed FRP/IRE complexes. All hemin concentrations tested resulted in rapid complex breakdown. The final amount of complex breakdown corresponds to the concentration of hemin present in the reaction. The effect of hemin on FRP activity suggests that a specific hemin binding site exists on FRP.

Animals↗

PubMed2002

Lack of a consistent relationship between demethylation of the CD44 promoter and CD44 expression.

Methylation of the CD44 promoter at one or more CpG dinucleotides has been proposed as being important in the control of CD44 expression. In vitro methylation of all CpGs occurring between bases -1 to -1374 upstream of the position of translation initiation repressed the promoter activity of mouse CD44 4.5- to 12-fold in transient transfection experiments. Assaying the methylation of these 29 CpGs by genomic sequencing using differential base modification by sodium bisulfite indicated that a cluster of three CpGs immediately upstream of the position of translation initiation was heavily methylated in the mouse CD44-negative T-cell lymphoma AKR1. All 19 CpGs between bases -4 and -447, including the cluster heavily methylated in AKR1, were demethylated in the CD44-positive T-cell lymphoma BW5147, while CpGs further upstream showed no change in methylation pattern. The cluster of heavily methylated CpGs remained methylated, however, when CD44 was activated by transient transfection of c-jun into an AKR1 subline expressing polyoma large-T or by treatment of this subline with sodium butyrate, and no significant demethylation of other CpGs was observed. It is concluded, therefore, that no consistent demethylation event in the promoter-containing region encompassing the 1374 base pairs upstream of the position of CD44 translation initiation is required for CD44 expression.

Animals↗

PubMed2004

Induction, production, repression, and de-repression of exoglucanase synthesis in Aspergillus niger.

The influence of carbon and nitrogen sources on the production of cellulases was investigated. The enzyme production was variable according to the carbon source. Levels of beta-cellobiohydrolase (CBH) were minimal in the presence of even low concentrations of glucose. Enzyme production was stimulated by other carbohydrates. The enzyme is subject to carbon source control by easily metabolizable sugars. Wheat bran and cellulose were the most effective promoters of beta-cellobiohydrolase and filter paperase (FPase) activities respectively, followed by rice bran. Exogenously supplied glucose inhibited the synthesis of the enzyme in cultures of A. niger growing on wheat bran. In defined medium with cellobiose, the cellobiohydrolase titres were 2- to 110-fold higher with cells growing on monomeric sugars and 1.5 times higher than cells growing on other disaccharides. It appeared that synthesis of beta-cellobiohydrolase varied under an induction mechanism, and a repression mechanism which changed the rate of synthesis of beta-cellobiohydrolase and FPase in induced over non-induced cultures. In this organism, substantial synthesis of beta-cellobiohydrolase can be induced by cellobiose, cellodextrin, cellulose or cellulose and hemi-cellulose containing substrates which showed low volumetric substrate uptake rate. The organism required limiting concentration of carbon, nitrogen or phosphorous for production of beta-cellobiohydrolase and FPase. During growth of A. niger on wheat bran, maximum volumetric productivities (Qp) of beta-cellobiohydrolase and FPase were 39.6 and 32.5 IU/lh and were significantly higher than the values reported for some other potent fungi and bacteria. The addition of actinomycin D (a repressor of transcription) and cycloheximide, (a repressor of translation) completely repressed CBH/FPase biosynthesis, suggested that the regulation of CBH synthesis in this organism occurs at both transcriptional and translational level. Thermodynamic studies revealed that the culture exerted protection against thermal inactivation when exposed to different fermentation temperatures.

Aspergillus niger↗

PubMed2005

PERK and GCN2 contribute to eIF2alpha phosphorylation and cell cycle arrest after activation of the unfolded protein response pathway.

Exposure of cells to endoplasmic reticulum (ER) stress leads to activation of PKR-like ER kinase (PERK), eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation, repression of cyclin D1 translation, and subsequent cell cycle arrest in G1 phase. However, whether PERK is solely responsible for regulating cyclin D1 accumulation after unfolded protein response pathway (UPR) activation has not been assessed. Herein, we demonstrate that repression of cyclin D1 translation after UPR activation occurs independently of PERK, but it remains dependent on eIF2alpha phosphorylation. Although phosphorylation of eIF2alpha in PERK-/- fibroblasts is attenuated in comparison with wild-type fibroblasts, it is not eliminated. The residual eIF2alpha phosphorylation correlates with the kinetics of cyclin D1 loss, suggesting that another eIF2alpha kinase functions in the absence of PERK. In cells harboring targeted deletion of both PERK and GCN2, cyclin D1 loss is attenuated, suggesting GCN2 functions as the redundant kinase. Consistent with these results, cyclin D1 translation is also stabilized in cells expressing a nonphosphorylatable allele of eIF2alpha; in contrast, repression of global protein translation still occurs in these cells, highlighting a high degree of specificity in transcripts targeted for translation inhibition by phosphorylated eIF2alpha. Our results demonstrate that PERK and GCN2 function to cooperatively regulate eIF2alpha phosphorylation and cyclin D1 translation after UPR activation.

Animals↗

PubMed2004

Nanos maintains germline stem cell self-renewal by preventing differentiation.

Despite much progress in understanding how extrinsic signaling regulates stem cell self-renewal, little is known about how cell-autonomous gene regulation controls this process. In Drosophila ovaries, germline stem cells (GSCs) divide asymmetrically to produce daughter GSCs and cystoblasts, the latter of which develop into germline cysts. Here, we show that removing the translational repressor Nanos from either GSCs or their precursors, the primordial germ cells (PGCs), causes both cell types to differentiate into germline cysts. Thus, Nanos is essential for both establishing and maintaining GSCs by preventing their precocious entry into oogenesis. These functions are likely achieved by repressing the translation of differentiation factors in PGCs and GSCs.

Animals↗

PubMed1990

Escherichia coli threonyl-tRNA synthetase and tRNA(Thr) modulate the binding of the ribosome to the translational initiation site of the thrS mRNA.

Escherichia coli threonyl-tRNA synthetase binds to the leader region of its own mRNA at two major sites: the first shares some analogy with the anticodon arm of several tRNA(Thr) isoacceptors and the second corresponds to a stable stem-loop structure upstream from the first one. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. The enzyme is still able to bind to derepressed mRNA mutants resulting from single substitutions in the anticodon-like arm. This binding is restricted to the stem-loop structure of the second site. However, the interaction of the enzyme with this site fails to occlude ribosome binding. tRNA(Thr) is able to displace the wild-type mRNA from the enzyme at both sites and suppresses the inhibitory effect of the synthetase on the formation of the translational initiation complex. Our results show that tRNA(Thr) acts as an antirepressor on the synthesis of its cognate aminoacyl-tRNA synthetase. This repression/derepression double control allows precise adjustment of the rate of synthesis of threonyl-tRNA synthetase to the tRNA level in the cell.

Anticodon↗

PubMed2006

Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control.

Hypoxia has recently been shown to activate the endoplasmic reticulum kinase PERK, leading to phosphorylation of eIF2alpha and inhibition of mRNA translation initiation. Using a quantitative assay, we show that this inhibition exhibits a biphasic response mediated through two distinct pathways. The first occurs rapidly, reaching a maximum at 1-2 h and is due to phosphorylation of eIF2alpha. Continued hypoxic exposure activates a second, eIF2alpha-independent pathway that maintains repression of translation. This phase is characterized by disruption of eIF4F and sequestration of eIF4E by its inhibitor 4E-BP1 and transporter 4E-T. Quantitative RT-PCR analysis of polysomal RNA indicates that the translation efficiency of individual genes varies widely during hypoxia. Furthermore, the translation efficiency of individual genes is dynamic, changing dramatically during hypoxic exposure due to the initial phosphorylation and subsequent dephosphorylation of eIF2alpha. Together, our data indicate that acute and prolonged hypoxia regulates mRNA translation through distinct mechanisms, each with important contributions to hypoxic gene expression.

Adaptor Proteins, Signal Transducing↗