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Results for “Translational repression”

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PubMed1998

Translational induction of the c-myc oncogene via activation of the FRAP/TOR signalling pathway.

Previous studies on the regulation of c-myc have focused on the transcriptional control of this proto-oncogene. We have investigated the signalling pathways involved under circumstances in which there is a translational upregulation in the levels of c-myc protein. We have demonstrated an up to tenfold serum-dependent increase of c-myc protein levels in Epstein-Barr virus immortalized B-cell lines 2-4 h after disruption of cellular aggregates, which is not accompanied by an equivalent increase in mRNA. Overall protein synthesis rates only increased threefold suggesting that the c-myc message was being selectively translated. We observed increases in the phosphorylation of p70 and p85 S6 kinases and of initiation factor eIF-4E binding protein 1 (4E-BP1) 1-2 h after stimulation, suggesting activation of the FRAP/TOR signalling pathway. The increased phosphorylation of 4E-BP1 led to a decrease in its association with eIF-4E and an increase in its association with the eIF-4G component of the eIF-4F initiation complex. The signalling inhibitors rapamycin and wortmannin blocked the phosphorylation of 4E-BP1 and abolished the translational component of the c-myc response. Our data suggest that dissociation of eIF-4E from 4E-BP1, leading to an increase in the formation of the eIF-4F initiation complex, relieves the translation repression imposed on the c-myc mRNA by its structured 5'UTR.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Characterization of a cis-acting regulatory element in the protein-coding region of human dihydrofolate reductase mRNA.

Previous studies have shown that human DHFR (dihydrofolate reductase), in addition to its critical role in DNA biosynthesis, functions as an RNA-binding protein. The interaction between DHFR and its own mRNA results in translational repression. In this study, we characterized the cis-acting elements on human DHFR mRNA that are required for the DHFR mRNA-DHFR protein interaction. Using a series of gel-shift and nitrocellulose filter-binding assays, a 164 nt RNA sequence, corresponding to nt 401-564, was identified within the coding region that binds to DHFR protein with an affinity similar to that of full-length DHFR mRNA. To document in vivo biological activity, various DHFR sequences contained within the coding region were cloned on to the 5' end of a luciferase reporter plasmid, and transient transfection experiments were performed using human colon cancer RKO cells. In cells transfected with p644/DHFR:401-564, luciferase activity was decreased by 50% when compared with cells transfected with the p644 plasmid alone. Luciferase mRNA levels were identical under each of these conditions, as determined by Northern-blot analysis. In cells transfected with p644/DHFR:401-564, luciferase activity was restored to almost 100% of control when cells were treated with the antifolate analogue methotrexate or with a short-interfering RNA targeting DHFR mRNA. These findings provide evidence that the DHFR 401-564 sequence is a DHFR-response element. In vitro and in vivo studies further localized this cis-element to an 82 nt sequence corresponding to nt 401-482. This work provides new insights into critical elements that mediate RNA-protein interactions.

Binding Sites↗

PubMed1999

Striking multiplicity of eIF4E-BP1 phosphorylated isoforms identified by 2D gel electrophoresis regulation by heat shock.

Eukaryotic initiation factor eIF4E-binding protein 1 (eIF4E-BP1), or PHAS-I, is multiply phosphorylated by insulin-stimulated protein kinase(s). Estimates for the number of phosphorylation sites range from two to greater than eight. IEF/SDS/PAGE can precisely differentiate protein isoforms based on their differences in charge (phosphorylation) and molecular mass. In this study, the diversity of eIF4E-BP1 isoforms was determined using IEF/SDS/PAGE/immunoblotting of unfractionated cell lysates. To investigate the molecular regulation of phosphorylation, alterations in eIF4E-BP1 in response to heat shock in HeLa cells were determined. In exponentially growing cells, 8-10 prominent eIF4E-BP1 isoforms were detected. Following heat shock, a rapid, temperature-dependent dephosphorylation of eIF4E-BP1 occurs roughly concurrent with protein synthesis inhibition; during recovery from heat shock rephosphorylation of eIF4E-BP1 parallels restoration of protein synthesis. However, eIF4E-BP1 and eIF4E kinases remain highly active during heat shock, as okadaic acid treatment restores phosphorylation of both factors in heat shocked cells. eIF4E-BP1 dephosphorylation is associated with eIF4E dissociation from large molecular mass complexes and increased binding to eIF4E-BP1. The amount of eIF4E-BP1 converted to the dephosphorylated state is sufficient to titrate all the eIF4E present. eIF4E-BP1 phosphorylation changes regulated by heat shock also occur in Drosophila. Of the 10 isoforms of eIF4E-BP1 resolved by IEF/SDS/PAGE, at least seven are labelled with [32P] and all 10 are recognized by (eIF4E-BP1)-specific antibodies. These results identify a complex set of eIF4E-BP1 phosphorylation isoforms; changes in the expression of these isoforms in response to stresses such as heat shock may contribute to translation repression.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Redundant control of the Caenorhabditis elegans sperm/oocyte switch by PUF-8 and FBF-1, two distinct PUF RNA-binding proteins.

PUF proteins control both growth and differentiation in the C. elegans germ line. These conserved RNA-binding proteins inhibit expression of target mRNAs, either by repressing translation or promoting degradation. Previous studies showed that PUF-8, a PUF protein with striking similarity to human Pumilio, prevents return of primary spermatocytes to the mitotic cell cycle [Subramaniam, K. & Seydoux, G. (2003) Curr. Biol. 13, 134-139]. We now report that PUF-8 is also critical for the hermaphrodite sperm/oocyte switch. Most puf-8 mutant hermaphrodites make both sperm and oocytes and are self-fertile, but some make a vast excess of sperm and fail to switch into oogenesis. This puf-8 defect is dramatically enhanced by removal of another puf gene called fbf-1: all fbf-1 puf-8 double mutants fail in the hermaphrodite sperm/oocyte switch. Therefore, puf-8 and fbf-1 act redundantly to control this decision. Epistasis analyses place puf-8 and fbf-1 upstream of fog-2, a gene near the top of the germ-line sex determination pathway. Furthermore, the abundance of FOG-2 increases dramatically in the distal region of fbf-1 puf-8 double mutants. We suggest that PUF-8 and FBF-1 may control fog-2 expression, and that the sperm/oocyte decision occurs in the distal germ line.

Animals↗

PubMed2005

Three small RNAs jointly ensure secondary metabolism and biocontrol in Pseudomonas fluorescens CHA0.

In many Gram-negative bacteria, the GacS/GacA two-component system positively controls the expression of extracellular products or storage compounds. In the plant-beneficial rhizosphere bacterium Pseudomonas fluorescens CHA0, the GacS/GacA system is essential for the production of antibiotic compounds and hence for biological control of root-pathogenic fungi. The small (119-nt) RNA RsmX discovered in this study, together with RsmY and RsmZ, forms a triad of GacA-dependent small RNAs, which sequester the RNA-binding proteins RsmA and RsmE and thereby antagonize translational repression exerted by these proteins in strain CHA0. This small RNA triad was found to be both necessary and sufficient for posttranscriptional derepression of biocontrol factors and for protection of cucumber from Pythium ultimum. The same three small RNAs also positively regulated swarming motility and the synthesis of a quorum-sensing signal, which is unrelated to N-acyl-homoserine lactones, and which autoinduces the Gac/Rsm cascade. Expression of RsmX and RsmY increased in parallel throughout cell growth, whereas RsmZ was produced during the late growth phase. This differential expression is assumed to facilitate fine tuning of GacS/A-controlled cell population density-dependent regulation in P. fluorescens.

Bacterial Proteins↗

PubMed2005

MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation.

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression primarily through translational repression. In erythropoietic (E) culture of cord blood CD34+ progenitor cells, the level of miR 221 and 222 is gradually and sharply down-modulated. Hypothetically, this decline could promote erythropoiesis by unblocking expression of key functional proteins. Indeed, (i) bioinformatic analysis suggested that miR 221 and 222 target the 3' UTR of kit mRNA; (ii) the luciferase assay confirmed that both miRs directly interact with the kit mRNA target site; and (iii) in E culture undergoing exponential cell growth, miR down-modulation is inversely related to increasing kit protein expression, whereas the kit mRNA level is relatively stable. Functional studies show that treatment of CD34+ progenitors with miR 221 and 222, via oligonucleotide transfection or lentiviral vector infection, causes impaired proliferation and accelerated differentiation of E cells, coupled with down-modulation of kit protein: this phenomenon, observed in E culture releasing endogenous kit ligand, is magnified in E culture supplemented with kit ligand. Furthermore, transplantation experiments in NOD-SCID mice reveal that miR 221 and 222 treatment of CD34+ cells impairs their engraftment capacity and stem cell activity. Finally, miR 221 and 222 gene transfer impairs proliferation of the kit+ TF-1 erythroleukemic cell line. Altogether, our studies indicate that the decline of miR 221 and 222 during exponential E growth unblocks kit protein production at mRNA level, thus leading to expansion of early erythroblasts. Furthermore, the results on kit+ erythroleukemic cells suggest a potential role of these miRs in cancer therapy.

Animals↗

PubMed2001

X-ray analysis of an RNA tetraplex (UGGGGU)(4) with divalent Sr(2+) ions at subatomic resolution (0.61 A).

Four-stranded guanine tetraplexes in RNA have been identified to be involved in crucial biological functions, such as dimerization of retroviral RNA, translational repression, and mRNA turnover. However, the structural basis for these biological processes is still largely unknown. Here we report the RNA tetraplex structure (UGGGGU)(4) at ultra-high resolution (0.61 A). The space group is P42(1)2, and cell constants are a = b = 36.16 A and c = 74.09 A. The structure was solved by the multiple-wavelength anomalous dispersion method using a set of three-wavelength data of the isomorphous bromo derivative (br)UGGGGU and refined to 0.61-A resolution. Each of the four strands in the asymmetric unit forms a parallel tetraplex with symmetry-related molecules. The tetraplex molecules stack on one another in opposite polarity (head-to-head or tail-to-tail) to form a pseudocontinuous column. All of the 5'-end uridines rotate around the backbone of G2, swing out, and form unique octaplexes with the neighboring G tetraplexes, whereas the 3'-end uridines are stacked-in and form uridine tetrads. All of the bases are anti, and the riboses are in the mixed C2'- and C3'-puckering mode. Strontium ions are observed in every other guanine tetrad plane, sitting on the fourfold axis and associated to the eight O6 atoms of neighboring guanine bases in a bipyramidal-antiprism geometry. The hydrogens are clearly observed in the structure.

Base Pairing↗

PubMed2002

The archetype STYX/dead-phosphatase complexes with a spermatid mRNA-binding protein and is essential for normal sperm production.

Differentiation of spermatids into spermatozoa is regulated via phosphorylated RNA-binding proteins that modulate the expression of stage-specific mRNAs. We demonstrate that the phosphoserine, -threonine or -tyrosine, interaction protein, Styx, complexes with a testicular RNA-binding protein and is essential for normal spermiogenesis. Ablation of Styx expression in mouse disrupts round and elongating spermatid development, resulting in a >1,000-fold decrease in spermatozoa production. Moreover, Styx(-/-) males are infertile because of structural head abnormalities in residual epididymal sperm. Immunoprecipitation of Styx with Crhsp-24, a phosphorylated RNA-binding protein implicated in translational repression of histone mRNAs, provides a strategy for regulating posttranscriptional gene expression.

Animals↗

PubMed1987

Bacteriophage T4 regA protein binds to mRNAs and prevents translation initiation.

The bacteriophage T4 regA protein is a translational repressor of a subset of phage mRNAs. We show here that purified regA protein binds specifically to target mRNAs near the initiating AUG and occludes binding of ribosomes. Translational repression by regA protein diminishes expression of many genes whose mRNA sequences around the initiating AUG codons are different. A comparison of nucleotide sequences from several regA-repressed mRNAs suggests that the initiating AUG is an important, but not sufficient, sequence for regA binding.

Base Sequence↗

PubMed1995

Independent signaling of grp78 gene transcription and phosphorylation of eukaryotic initiator factor 2 alpha by the stressed endoplasmic reticulum.

Perturbation of endoplasmic reticular (ER) function signals increased expression of the gene encoding the ER resident chaperone Grp78/BiP and rapid suppression of translational initiation accompanied by phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 alpha phosphorylation and grp78 mRNA induction were measured in GH3 pituitary cells subjected to varied degrees of ER stress to ascertain whether activation of an eIF-2 alpha kinase is involved in both events. grp78 mRNA was induced at low concentrations of ionomycin and dithiothreitol that did not provoke eIF-2 alpha phosphorylation or inhibition of amino acid incorporation. Mobilization of the bulk of cell-associated Ca2+ and the induction of grp78 mRNA occurred at comparable low concentrations of ionomycin, whereas phosphorylation of eIF-2 alpha and inhibition of protein synthesis required higher ionophore concentrations. Pretreatment for 1 h with cycloheximide suppressed grp78 mRNA induction and eIF-2 alpha phosphorylation in response to either stressor. Prolonged (17 h) cycloheximide blockade increased eIF-2 alpha phosphorylation without inducing grp78 mRNA. Upon release from the blockade, grp78 mRNA was induced and eIF-2 alpha was dephosphorylated. Translational tolerance to ionomycin or dithiothreitol, accompanied by dephosphorylation of eIF-2 alpha, was observed whenever grp78 mRNA was induced. Induction of grp78 mRNA preceded significant eIF-2 alpha phosphorylation during treatment with brefeldin A. It is concluded that signaling of grp78 gene transcription can occur independently of eIF-2 alpha phosphorylation or translational repression and that greater degrees of ER stress are required for eIF-2 alpha phosphorylation than for grp78 mRNA induction.

Brefeldin A↗

PubMed1997

Interleukin-4 and -13 inhibit tumor necrosis factor-alpha mRNA translational activation in lipopolysaccharide-induced mouse macrophages.

The production of tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide (LPS)-stimulated macrophages can be markedly inhibited by the two closely related cytokines, interleukin (IL)-4 and IL-13. To investigate the molecular mechanism of this inhibition, we analyzed the effect of the two cytokines on TNF-alpha production and TNF-alpha mRNA accumulation in the mouse macrophage cell lines RAW 264.7 and J774 stimulated by LPS. Whereas LPS-induced TNF-alpha production is strongly suppressed by both cytokines, TNF-alpha mRNA accumulation is not significantly affected, indicating that IL-4 and IL-13 induce a translational repression of TNF-alpha mRNA. Transfection of reporter gene constructs containing different regions of the TNF-alpha gene revealed that the inhibitory action of IL-4 and IL-13 is mediated by the UA-rich sequence present in the TNF-alpha mRNA 3'-untranslated region.

Animals↗

PubMed2000

GI domain-mediated association of the eukaryotic initiation factor 2alpha kinase GCN2 with its activator GCN1 is required for general amino acid control in budding yeast.

In response to the starvation of a single amino acid, the budding yeast Saccharomyces cerevisiae activates numerous genes involved in various amino acid biosynthetic pathways, all of which are under the control of transcription factor GCN4. This general amino acid control response is based on de-repressed translation of GCN4 mRNA, which is induced by the activation of the eIF2alpha kinase, GCN2. Although it is known that in vivo activation of GCN2 requires GCN1, the mode of GCN1 action remains to be elucidated at the molecular level. Here, we show that GCN2 interacts with GCN1 via the GI domain, a novel protein-binding module that occurs at the N terminus; mutations to conserved residues of this domain abolish its binding to GCN1. Furthermore, the yeast cells with GCN2 defective in interaction with GCN1 fail to display general control response. A similar phenotype is observed in cells overexpressing the GI domain of GCN2 or its target region on GCN1. Thus, GI domain-mediated association of GCN2 to GCN1 is required for general amino acid control. This finding provides the first insight into the molecular mechanism for the activation of GCN2 by GCN1.

Amino Acid Sequence↗

PubMed2003

RNase L mediates transient control of the interferon response through modulation of the double-stranded RNA-dependent protein kinase PKR.

The transient control of diverse biological responses that occurs in response to varied forms of stress is often a highly regulated process. During the interferon (IFN) response, translational repression due to phosphorylation of eukaryotic initiation factor 2alpha, eIF2alpha, by the double-stranded RNA-dependent protein kinase, PKR, constitutes a means of inhibiting viral replication. Here we show that the transient nature of the IFN response against acute viral infections is regulated, at least in part, by RNase L. During the IFN antiviral response in RNase L-null cells, PKR mRNA stability was enhanced, PKR induction was increased, and the phosphorylated form of eIF2alpha appeared with extended kinetics compared with similarly treated wild type cells. An enhanced IFN response in RNase L-null cells was also demonstrated by monitoring inhibition of viral protein synthesis. Furthermore, ectopic expression of RNase L from a plasmid vector prevented the IFN induction of PKR. These results suggest a role for RNase L in the transient control of the IFN response and possibly of other cytokine and stress responses.

Animals↗

PubMed2003

How bacterial ribosomal protein L20 assembles with 23 S ribosomal RNA and its own messenger RNA.

In bacteria, the expression of ribosomal proteins is often feedback-regulated at the translational level by the binding of the protein to its own mRNA. This is the case for L20, which binds to two distinct sites of its mRNA that both resemble its binding site on 23 S rRNA. In the present work, we report an NMR analysis of the interaction between the C-terminal domain of L20 (L20C) and both its rRNA- and mRNA-binding sites. Changes in the NMR chemical shifts of the L20C backbone nuclei were used to show that the same set of residues are modified upon addition of either the rRNA or the mRNA fragments, suggesting a mimicry at the atomic level. In addition, small angle x-ray scattering experiments, performed with the rRNA fragment, demonstrated the formation of a complex made of two RNAs and two L20C molecules. A low resolution model of this complex was then calculated using (i) the rRNA/L20C structure in the 50 S context and (ii) NMR and small angle x-ray scattering results. The formation of this complex is interesting in the context of gene regulation because it suggests that translational repression could be performed by a complex of two proteins, each interacting with the two distinct L20-binding sites within the operator.

Bacterial Proteins↗

PubMed2004

A Gcn4p homolog is essential for the induction of a ribosomal protein L41 variant responsible for cycloheximide resistance in the yeast Candida maltosa.

Cycloheximide (CYH) resistance in the yeast Candida maltosa is based on the inducible expression of genes encoding a variant of ribosomal protein L41-Q, with glutamine at position 56 instead of the proline found in normal L41. The promoter of L41-Q2a, one of the L41-Q gene alleles encoding L41-Q, has an element similar to the Gcn4p-responsive element of Saccharomyces cerevisiae. In a previous study, this element was shown to be essential for the induction of L41-Q by CYH. In the present study, a C. maltosa GCN4 homolog, C-GCN4, was cloned. It had a long 5'-leader region with three upstream open reading frames. Enhanced expression of the C-GCN4 reporter fusion gene upon the addition of 3-aminotriazole or by mutations in start codons of all three upstream open reading frames indicates that C-GCN4 expression is under translation repression as was seen with GCN4. The C-GCN4-depleted mutant was unable to grow in a nutrient medium containing CYH and did not express L41-Q genes. Recombinant C-Gcn4p bound to the consensus DNA element for Gcn4p, 5'-(G/A)TGACTCAT-3', located upstream of L41-Q2a. Thus, C-Gcn4p, which likely functions in the general control of amino acid biosynthesis, is essential for the expression of L41-Q genes.

Antifungal Agents↗

PubMed2004

Intron retention generates a novel Id3 isoform that inhibits vascular lesion formation.

The expression of intron-containing messages has been shown to occur in a variety of diseases including lactic acidosis, Cowden Syndrome, and several cancers. However, it is unknown whether these intron-containing messages result in protein production in vivo. Indeed, intron-containing RNAs are typically retained in the nucleus, targeted for degradation, or are repressed translationally. Here, we show that during vascular lesion formation in rats, an alternative isoform of the helix-loop-helix transcription factor Id3 (Id3a) generated by intron retention is abundantly expressed. We demonstrate that Id3 is expressed early in lesion formation when the proliferative index of the neointima is highest and that Id3 promotes smooth muscle cell (SMC) proliferation and S-phase entry and inhibits transcription of the cell-cycle inhibitor p21(Cip1). Using an Id3a-specific antibody developed by our laboratory, we show that Id3a protein is induced during vascular lesion formation and that Id3a expression peaks late when the proliferative index is low or declining and extensive apoptosis is observed. Furthermore, Id3a fails to promote SMC growth and S-phase entry or to inhibit p21(Cip1) promoter transactivation. In contrast, Id3a stimulates SMC apoptosis and inhibits endogenous Id3 production. Adenoviral delivery of Id3a inhibited lesion formation in balloon-injured rat carotid arteries in vivo. These data describe a novel feedback loop whereby intron retention generates an Id3 isoform that acts to limit SMC growth during vascular lesion formation, providing the first evidence that regulated intron retention can modulate a pathologic process in vivo.

Adenoviridae↗

PubMed2004

Targeted knockdown of the RNA-binding protein CRD-BP promotes cell proliferation via an insulin-like growth factor II-dependent pathway in human K562 leukemia cells.

The c-myc mRNA coding region determinant-binding protein (CRD-BP) was first identified as a masking protein that stabilizes c-myc mRNA in a cell-free mRNA degradation system. Thus, CRD-BP is thought to promote cell proliferation by maintaining c-Myc at critical levels. CRD-BP also appears to be an oncofetal protein, based upon its expression during mammalian development and in some tumors. By using K562 leukemia cells as a model, we show that CRD-BP gene silencing by RNA interference significantly promoted proliferation, indicating an inhibitory effect of CRD-BP on proliferation. Unexpectedly, CRD-BP knockdown had no discernible effect on c-myc mRNA levels. CRD-BP is also known as insulin-like growth factor II (IGF-II) mRNA-binding protein-1. It has been reported to repress translation of a luciferase reporter mRNA containing an IGF-II 5'-untranslated region known as leader 3 but not one containing IGF-II leader 4. CRD-BP knockdown markedly increased IGF-II mRNA and protein levels but did not alter translation of luciferase reporter mRNAs containing 5'-untranslated regions consisting of either IGF-II leader 3 or leader 4. Addition of antibody against IGF-II to cell cultures inhibited the proliferative effect of CRD-BP knockdown, suggesting that regulation of IGF-II gene expression, rather than c-myc mRNA levels, mediates the proliferative effect of CRD-BP knockdown. Thus, we have identified a dominant function for CRD-BP in cell proliferation of human K562 cells, involving a possible IGF-II-dependent mechanism that appears independent of its ability to serve as a c-myc mRNA masking protein.

5' Untranslated Regions↗

PubMed2006

Hydrodynamic studies on the quaternary structure of recombinant mouse Purbeta.

Purbeta is a gene regulatory factor belonging to a family of highly conserved nucleic acid-binding proteins related by their ability to preferentially bind single-stranded DNA or RNA sequences rich in purine nucleotides. In conjunction with Puralpha, Purbeta has been implicated in transcriptional and translational repression of genes encoding contractile proteins found in the heart and vasculature. Although several models of sequence-specific DNA recognition, strand separation, and activator inhibition by oligomeric Puralpha and Purbeta have been proposed, it is currently unclear whether protein-protein interaction is a prerequisite to, or a consequence of nucleic acid binding. In this study, a recombinant protein purification scheme was devised to yield homogenous mouse Purbeta devoid of nucleic acid. Recombinant Purbeta was then subjected to light scattering and analytical ultracentrifugation analyses to assess the size, shape, and oligomeric state of the purified protein in solution. Results of laser light scattering and sedimentation velocity experiments indicated that Purbeta reversibly self-associates in the absence of nucleic acid. Both approaches independently showed that the hydrodynamic shape of the Purbeta homodimer is markedly asymmetric and non-spherical. Sedimentation velocity analyses indicated that dimeric Purbeta has a sedimentation coefficient of 3.96 Svedberg, a frictional coefficient ratio (f/f(0)) of 1.60, and a hydrodynamic radius of 4.43 nm. These values were consistent with those determined by independent dynamic light scattering studies. Sedimentation equilibrium analyses confirmed that Purbeta self-associates in a reversible monomer-dimer equilibrium characterized by a K(d) = 1.13 +/- 0.27 microm.

Animals↗