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Results for “Translational repression”

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PubMed1991

Translational control of globin chain ontogeny in hamster yolk sac erythroid cells.

Prior research has demonstrated that globin ontogeny of hamster proceeds nearly to completion during the several days that yolk sac erythroid cells (YSEC) circulate in the embryo; synthesis of embryonic globin chains gives way to synthesis of adult globin chains in these primitive cells. In the present study, we translated total cell RNA extracted from YSEC on days 9-13 of gestation in wheat germ cell-free extract, expecting to observe the same progressive rise that occurs in vivo in rates of translation of alpha- and beta-globin mRNA during ontogeny. The opposite occurred; translation rates of both globins decreased sharply. This disparity between synthesis of alpha- and beta-globins in vivo and in vitro suggested an element of control of translation attributable to the YSEC cytoplasm. We therefore assayed the effect of RNA-free clarified YSEC cytoplasm on cell-free translation of YSEC RNA. A repression of translation was detected of alpha- and beta-globin mRNA (not of embryonic globin mRNA), exercised strongly by cytoplasm from YSEC early in ontogeny (gestational day 9), and weakening as ontogeny progressed. The same effect was noted on alpha- and beta-globin mRNA of adult hamster and of rabbit. Heat treatment of cytoplasm abolished the greater part of the translation regulation, suggesting that the active agent is protein. Further characterization of this translational regulator included: (a) it binds to globin poly(A) mRNA but not to poly(A), (b) it was not detected in cell lysate of adult hamster brain, lung, or erythrocytes, and (c) it did not inhibit translation of adult hamster brain and liver RNA. We conclude that hamster globin ontogeny is substantially modulated by this translational regulation of alpha- and beta-globin expression.

Animals↗

PubMed2000

fhlA repression by OxyS RNA: kissing complex formation at two sites results in a stable antisense-target RNA complex.

OxyS is a small untranslated RNA that is induced in response to oxidative stress in Escherichia coli. This small RNA acts as a global regulator affecting the expression of multiple genes. OxyS represses the translation of fhlA, a transcriptional activator for formate metabolism. Previously, we have shown that fhlA repression by OxyS is mediated through base-pairing with a short sequence overlapping the ribosome binding site. Here we show that the OxyS-fhlA interaction involves a second site residing further downstream, within the coding region of fhlA. Mutations that disrupt pairing at this site affect the ability of OxyS to prevent 30 S ribosomes from binding to fhlA mRNA. Structure probing of fhlA mRNA demonstrates that both sites reside in the loops of two stem-loop structures. OxyS-fhlA pairing analysis shows that OxyS binds wild-type fhlA with an apparent dissociation constant of 25 nM, indicating that kissing complex formation between OxyS and fhlA results in a stable antisense-target complex. Mutations at either site, which disrupt pairing of OxyS to fhlA, decrease the stability of this complex. Our results indicate that kissing complex formation is sufficient to repress fhlA translation by OxyS.

Base Sequence↗

PubMed2002

Activation of GCN2 in UV-irradiated cells inhibits translation.

BACKGROUND: Mammalian cells subjected to ultraviolet (UV) irradiation actively repress DNA replication, transcription, and mRNA translation. While the effects of UV irradiation on DNA replication and transcription have been extensively studied, the mechanism(s) responsible for translational repression are poorly understood. RESULTS: Here, we demonstrate that UV irradiation elicits phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) by activating the kinase GCN2 in a manner that does not require SAPK/JNK or p38 MAP kinase. GCN2-/- cells, and cells expressing nonphosphorylatable eIF2alpha as their only source of eIF2alpha protein, fail to repress translation in response to UV irradiation. CONCLUSIONS: These results provide a mechanism for translation inhibition by UV irradiation and identify a hitherto unrecognized role for mammalian GCN2 as a mediator of the cellular response to UV stress.

Animals↗

PubMed2002

The 5' untranslated region of protein kinase Cdelta directs translation by an internal ribosome entry segment that is most active in densely growing cells and during apoptosis.

Protein kinase Cdelta (PKCdelta) is a member of the PKC family of phospholipid-dependent serine/threonine kinases and is involved in cell proliferation, apoptosis, and differentiation. Previous studies have suggested that different PKC isoforms might be translationally regulated. We report here that the 395-nt-long 5' untranslated region (5' UTR) of PKCdelta is predicted to form very stable secondary structures with free energies (deltaG values) of around -170 kcal/mol. The 5' UTR of PKCdelta can significantly repress luciferase translation in rabbit reticulocyte lysate but does not repress luciferase translation in a number of transiently transfected cell lines. By using a bicistronic luciferase reporter, we show that the 5' UTR of PKCdelta contains a functional internal ribosome entry segment (IRES). The activity of the PKCdelta IRES is greatest in densely growing cells and during apoptosis, when total protein synthesis and levels of full-length eukaryotic initiation factor 4G are reduced. However, the IRES activity of the 5' UTR of PKCdelta is not enhanced during serum starvation, another condition shown to inhibit cap-dependent translation, suggesting that its potency is dependent on specific cellular conditions. Accumulating data suggest that PKCdelta has a function as proliferating cells reach high density and in early and later events of apoptosis. Our studies suggest a mechanism whereby PKCdelta synthesis can be maintained under these conditions when cap-dependent translation is inhibited.

3T3 Cells↗

PubMed1990

Evidence for allosteric coupling between the ribosome and repressor binding sites of a translationally regulated mRNA.

Escherichia coli ribosomal protein S4 is a translational repressor regulating the expression of four ribosomal genes in the alpha operon. In vitro studies have shown that the protein specifically recognizes an unusual mRNA pseudoknot secondary structure which links sequences upstream and downstream of the ribosome binding site for rpsM (S13) [Tang, C. K., & Draper, D. E. (1989) Cell 57, 531]. We have prepared fusions of the rpsM translational initiation site and lacZ that allows us to detect repression in cells in which overproduction of S4 repressor can be induced. Twenty-five mRNA sequence variants have been introduced into the S13-lacZ fusions and the levels of translational repression measured. Sets of compensating base changes confirm the importance of the pseudoknot secondary structure for translational repression. An A residue in a looped, single-stranded sequence is also required for S4 recognition and may contact S4 directly. Comparison of translational repression levels and S4 binding constants for the set of mRNA mutations show that nine mutants are repressed much more weakly than predicted from their affinity for S4; in extreme cases no repression can be detected for variants with unchanged S4 binding. We suggest that the mRNA contains functionally distinct ribosome and repressor binding sites that are allosterically coupled. Mutations can relieve translational repression by disrupting the linkage between the two sites without altering S4 binding. This proposal assigns to the mRNA a more active role in mediating translational repression than found in other translational repression systems.

Allosteric Regulation↗

PubMed1997

Translational activation in coliphage Qbeta: on a polycistronic messenger RNA, repression of one gene can activate translation of another.

We present evidence for translational activation of the Qbeta coliphage maturation cistron, mediated by the presence of Qbeta replicase. This activation does not require RNA replication, translation of a second gene, or any direct protein-RNA binding at the maturation gene initiation site. Our data support a model in which the Qbeta maturation gene remains translationally "off" by two means: (1) the thermodynamic stability of an RNA structure that greatly discourages, but does not eliminate, ribosome access at the maturation start site; and (2) the presence of the stronger, proximal coat gene ribosome binding site. Moreover, maturation gene expression is switched "on" when ribosome entry at the coat initiation site, present on the same polycistronic RNA molecule, is repressed by Qbeta replicase, thereby allowing ribosomes to compete for the weaker, upstream maturation start site.

Allolevivirus↗

PubMed1994

Binding specificity of T4 DNA polymerase to RNA.

Bacteriophage T4 DNA polymerase, product of phage gene 43 (gp43), is a multifunctional DNA-binding protein and a key component of the phage DNA replicase. It is also an RNA-binding protein that selectively recognizes a site on its mRNA (the translational operator) and represses its own translation. We examined the ability of the protein to discriminate between DNA and RNA by using a gel mobility shift assay with defined RNA and DNA substrates. A higher affinity to RNA as compared with DNA (about 100-fold) was observed in assays that utilized synthetic DNA and in vitro transcribed RNA substrates bearing the T4 gene 43 translational operator sequence. The replacement of thymine with uracil in the synthetic DNA did not improve binding. The results suggest that the protein's selectivity for RNA is based in structure (intramolecular interactions) specific to the ribonucleotide sequence of the operator. Competition studies suggest that the protein determinants for RNA and DNA recognition are only partially overlapping.

Base Sequence↗

PubMed1999

The STAR protein, GLD-1, is a translational regulator of sexual identity in Caenorhabditis elegans.

The Caenorhabditis elegans sex determination gene, tra-2, is translationally regulated by elements in the 3'-untranslated region called TGEs. TGEs govern the translation of mRNAs in both invertebrates and vertebrates, indicating that this is a highly conserved mechanism for controlling gene activity. A factor called DRF, found in worm extracts binds the TGEs and may be a repressor of translation. Using the yeast three-hybrid screen and RNA gel shift analysis, we have found that the protein GLD-1, a germline-specific protein and a member of the STAR family of RNA-binding proteins, specifically binds to the TGEs. GLD-1 is essential for oogenesis, and is also necessary for spermatogenesis and inhibition of germ cell proliferation. Several lines of evidence demonstrate that GLD-1 is a translational repressor acting through the TGEs to repress tra-2 translation. GLD-1 can repress the translation of reporter RNAs via the TGEs both in vitro and in vivo, and is required to maintain low TRA-2A protein levels in the germline. Genetic analysis indicates that GLD-1 acts upstream of the TGE control. Finally, we show that endogenous GLD-1 is a component of DRF. The conservation of the TGE control and the STAR family suggests that at least a subset of STAR proteins may work through the TGEs to control translation.

Animals↗

PubMed2005

Identification and functional outcome of mRNAs associated with RNA-binding protein TIA-1.

The RNA-binding protein TIA-1 (T-cell intracellular antigen 1) functions as a posttranscriptional regulator of gene expression and aggregates to form stress granules following cellular damage. TIA-1 was previously shown to bind mRNAs encoding tumor necrosis factor alpha (TNF-alpha) and cyclooxygenase 2 (COX-2), but TIA-1 target mRNAs have not been systematically identified. Here, immunoprecipitation (IP) of TIA-1-RNA complexes, followed by microarray-based identification and computational analysis of bound transcripts, was used to elucidate a common motif present among TIA-1 target mRNAs. The predicted TIA-1 motif was a U-rich, 30- to 37-nucleotide (nt)-long bipartite element forming loops of variable size and a bent stem. The TIA-1 motif was found in the TNF-alpha and COX-2 mRNAs and in 3,019 additional UniGene transcripts (approximately 3% of the UniGene database), localizing preferentially to the 3' untranslated region. The interactions between TIA-1 and target transcripts were validated by IP of endogenous mRNAs, followed by reverse transcription and PCR-mediated detection, and by pulldown of biotinylated RNAs, followed by Western blotting. Further studies using RNA interference revealed that TIA-1 repressed the translation of bound mRNAs. In summary, we report a signature motif present in mRNAs that associate with TIA-1 and provide support to the notion that TIA-1 represses the translation of target transcripts.

Base Sequence↗

PubMed2006

PML inhibits HIF-1alpha translation and neoangiogenesis through repression of mTOR.

Loss of the promyelocytic leukaemia (PML) tumour suppressor has been observed in several human cancers. The tumour-suppressive function of PML has been attributed to its ability to induce growth arrest, cellular senescence and apoptosis. Here we identify PML as a critical inhibitor of neoangiogenesis (the formation of new blood vessels) in vivo, in both ischaemic and neoplastic conditions, through the control of protein translation. We demonstrate that in hypoxic conditions PML acts as a negative regulator of the synthesis rate of hypoxia-inducible factor 1alpha (HIF-1alpha) by repressing mammalian target of rapamycin (mTOR). PML physically interacts with mTOR and negatively regulates its association with the small GTPase Rheb by favouring mTOR nuclear accumulation. Notably, Pml-/- cells and tumours display higher sensitivity both in vitro and in vivo to growth inhibition by rapamycin, and lack of PML inversely correlates with phosphorylation of ribosomal protein S6 and tumour angiogenesis in mouse and human tumours. Thus, our findings identify PML as a novel suppressor of mTOR and neoangiogenesis.

Animals↗

PubMed2001

Expression of MSY2 in mouse oocytes and preimplantation embryos.

Translational control plays a central role during oocyte maturation and early embryogenesis, as these processes occur in the absence of transcription. MSY2, a member of a multifunctional Y-box protein family, is implicated in repressing the translation of paternal mRNAs. Here, we characterize MSY2 expression in mouse oocytes and preimplantation embryos. Northern blot analysis indicates that MSY2 expression is highly restricted and essentially confined to the oocyte in the female mouse. MSY2 transcript and protein, as assessed by reverse transcription-polymerase chain reaction and immunoblotting, respectively, are expressed in growing oocytes, metaphase II-arrested eggs, and 1-cell embryos, but then are degraded by the late 2-cell stage; no expression is detectable in the blastocysts. During oocyte maturation, MSY2 is phosphorylated and following fertilization it is dephosphorylated. Quantification of the mass amount of MSY2 reveals that it represents 2% of the total protein in the fully grown oocyte, i.e., it is a very abundant protein. Both endogenous MSY2 and MSY2-enhanced green fluorescent protein (EGFP), which is synthesized following microinjection of an mRNA encoding MSY2-EGFP, are primarily localized in the cytoplasm, and about 75% of the MSY2 remains associated with oocyte cytoskeletal preparations. Results of these studies are consistent with the proposal that MSY2 functions by stabilizing and/or repressing the translation of maternal mRNAs.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Repression of GCN4 mRNA translation by nitrogen starvation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae activates a regulatory network called "general control" that provides the cell with sufficient amounts of protein precursors during amino acid starvation. We investigated how starvation for nitrogen affects the general control regulatory system, because amino acid biosynthesis is part of nitrogen metabolism. Amino acid limitation results in the synthesis of the central transcription factor Gcn4p, which binds to specific DNA-binding motif sequences called Gcn4-protein-responsive elements (GCREs) that are present in the promoter regions of its target genes. Nitrogen starvation increases GCN4 transcription but efficiently represses expression of both a synthetic GCRE6::lacZ reporter gene and the natural amino acid biosynthetic gene ARO4. Repression of Gcn4p-regulated transcription by nitrogen starvation is independent of the ammonium sensing systems that include Mep2p and Gpa2p or Ure2p and Gln3p but depends on the four upstream open reading frames in the GCN4 mRNA leader sequence. Efficient translation of GCN4 mRNA is completely blocked by nitrogen starvation, even when cells are simultaneously starved for amino acids and eukaryotic initiation factor-2 alpha is fully phosphorylated by Gcn2p. Our data suggest that nitrogen starvation regulates translation of GCN4 by a novel mechanism that involves the four upstream open reading frames but that still acts independently of eukaryotic initiation factor-2 alpha phosphorylation by Gcn2p.

DNA-Binding Proteins↗

PubMed1998

Repression of interleukin-2 mRNA translation in primary human breast carcinoma tumor-infiltrating lymphocytes.

Human breast carcinoma tumor-infiltrating lymphocytes (TIL) express activation antigens in situ indicative of ongoing immune response-CD28, CD45RO, CD69, CD71, and DR. However, interleukin 2 (IL-2) receptor was poorly expressed: CD25 was detected in only 1/24 samples and CD122 in only 2/24 samples. Furthermore, isolated breast cancer TIL were defective in proliferative response but recover when treated with recombinant IL-2. Nineteen of 24 tumor samples expressed B7-1, B7-2, and CD28 protein, showing that absence of costimulator proteins or counter ligand was not the basis for TIL proliferative deficit. Expression of IL-2 activity was not detected; however, mRNA encoding IL-2 was produced and translatable in vitro. These findings show that human breast cancer tumor-induced repression of IL-2 RNA translation is the basis of failure of TIL to express the IL-2 receptor and subsequent T cell hyporesponsiveness.

Breast Neoplasms↗

PubMed1971

Characterization of the initial peptide of Q-beta RNA polymerase and control of its synthesis.

Bacteriophage Qbeta RNA directs the cell-free synthesis of two fMet-containing dipeptides prior to the first round of ribosomal translocation. One of these, fMet-Ala, corresponds to the initial segment of the Qbeta coat protein. In the present work we take advantage of the fact that translation of the Qbeta RNA polymerase cistron is repressed by its coat protein to correlate the other peptide, fMet-Ser, with the Qbeta RNA polymerase gene. Our experiments show that repression affects translation of the first two codons of the polymerase cistron, thereby isolating the effect of Qbeta coat repressor. Further studies, using single rounds of translocation of the Qbeta mRNA and codon-specific tRNAs, allow us to predict the first three amino acids of the Qbeta RNA polymerase protein, fMet-Ser-Lys, and to suggest that the initiation region of the Qbeta RNA polymerase cistron has the sequence ...AUG.UC[unk].AA[unk]...

Bacteriophages↗

PubMed1994

Interaction of the Chlamydia trachomatis histone H1-like protein (Hc1) with DNA and RNA causes repression of transcription and translation in vitro.

The 18 kDa histone H1-like protein from Chlamydia trachomatis (Hc1) is a DNA-binding protein thought to be involved in condensation of the chlamydial chromosome during late stages in the chlamydial life cycle. Expression of Hc1 in Escherichia coli results in an overall relaxation of DNA and severely affects DNA, RNA and protein synthesis. We have analysed the interaction of Hc1 with single-stranded DNA and RNA by Southwestern and Northwestern blotting. Furthermore, we show that purified, recombinant Hc1 dramatically affects transcription and translation in vitro at physiologically relevant concentrations. These results were found to coincide with the formation of condensed Hc1-DNA and Hc1-RNA complexes as revealed by agarose gel electrophoresis and electron microscopy. The implications of these results for possible functions of Hc1 in vivo are discussed.

Chlamydia trachomatis↗

PubMed1989

Control of the Saccharomyces cerevisiae regulatory gene PET494: transcriptional repression by glucose and translational induction by oxygen.

The product of the Saccharomyces cerevisiae nuclear gene PET494 is required to promote the translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII). The level of cytochrome c oxidase activity is affected by several different environmental conditions, which also influence coxIII expression. We have studied the regulation of PET494 to test whether the level of its expression might modulate coxIII translation in response to these conditions. A pet494::lacZ fusion was constructed and used to monitor PET494 expression. PET494 was regulated by oxygen availability: expression in a respiration-competent diploid strain grown anaerobically was one-fifth the level of expression in aerobically grown cells. However, since PET494 mRNA levels did not vary in response to oxygen deprivation, regulation by oxygen appears to occur at the translational level. This oxygen regulation was not mediated by heme, and PET494 expression was independent of the heme activator protein HAP2. The regulation of PET494 expression by carbon source was also examined. In cells grown on glucose-containing medium, PET494 was expressed at levels four- to sixfold lower than in cells grown on ethanol and glycerol. However, addition of ethanol to glucose-containing medium induced PET494 expression approximately twofold. PET494 transcript levels varied over a fourfold range in response to different carbon sources. The effects on PET494 expression of mutations in the SNF1, SNF2, SSN6, and HXK2 genes were also determined and found to be minimal.

Cloning, Molecular↗

PubMed2006

Exercise-induced alterations in extracellular signal-regulated kinase 1/2 and mammalian target of rapamycin (mTOR) signalling to regulatory mechanisms of mRNA translation in mouse muscle.

The present study examined the effects of an acute bout of treadmill exercise on signalling through the extracellular signal-regulated kinase (ERK)1/2 and mammalian target of rapamycin (mTOR) pathways to regulatory mechanisms involved in mRNA translation in mouse gastrocnemius muscle. Briefly, C57BL/6 male mice were run at 26 m min(-1) on a treadmill for periods of 10, 20 or 30 min, then the gastrocnemius was rapidly removed and analysed for phosphorylation and/or association of protein components of signalling pathways and mRNA translation regulatory mechanisms. Repression of global mRNA translation was suggested by disaggregation of polysomes into free ribosomes, which occurred by 10 min and was sustained throughout the time course. Exercise repressed the mTOR signalling pathway, as shown by dephosphorylation of the eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1), enhanced association of the regulatory-associated protein of mTOR with mTOR, and increased assembly of the tuberin-hamartin complex. In contrast, exercise caused no change in phosphorylation of either Akt/PKB or tuberin. Upstream of mTOR, exercise was associated with an increase in cAMP, protein kinase A activity, and AMP-activated protein kinase phosphorylation. Simultaneously, exercise caused a rapid and sustained activation of the MEK1/2-ERK1/2-p90RSK pathway, resulting in increased phosphorylation of downstream targets including eIF4E and the ribosomal protein (rp)S6 on S235/S236. Overall, the data are consistent with exercise-induced repression of mTOR signalling and global rates of mRNA translation, accompanied perhaps by up-regulated translation of selected mRNAs through regulatory mechanisms such as eIF4E and rpS6 phosphorylation, mediated by activation of the ERK1/2 pathway.

Animals↗

PubMed1999

Cell type-dependent and -independent control of HER-2/neu translation.

Overexpression of the HER-2 oncogene occurs in a variety of human tumors, including 25-30% of breast carcinomas, and has been associated with an adverse prognosis. Amplification of the HER-2 gene is frequently detected in tumors, but by itself may not fully account for HER-2 overexpression since transcriptional and post-transcriptional mechanisms also regulate HER-2 protein synthesis. Our studies reveal that the efficiency of HER-2 translation differs between primary and transformed cells. In primary human fibroblasts and human mammary epithelial cells, the HER-2 mRNA is associated with monosome and small polysome fractions. In contrast, in BT474 and MCF-7 human breast cancer cell lines and in COS-7 cells the mRNA co-sedimented with larger polysomes, indicating that it is more efficiently translated in these transformed cells. Northern analysis revealed no detectable mRNA size difference, and nuclease S1 protection and sequence analyses showed no differences between the HER-2 transcript leader in primary cells compared to transformed human cells. The transcript leader in all cell types contains a short upstream open reading frame that is also conserved in other mammalian species. Transient transfection assays revealed that the HER-2 transcript leader repressed downstream translation approximately five-fold in both primary and transformed cells and mutation of the upstream initiation codon alleviated most of the inhibitory effect. These results indicate that HER2 expression is translationally controlled both by a short upstream open reading frame that represses HER-2 translation in a cell type-independent manner, and by a distinct cell type-dependent mechanism that increases translational efficiency of HER-2 in transformed cells.

Animals↗