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Results for “Translational repression”

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PubMed1995

RNA-protein interactions of the bacteriophage RB69 RegA translational repressor protein.

We report the RNA-protein interactions of phage RB69 RegA and RegA691 proteins. In RNase protection assays, RB69 RegA protects from 10 (44 mRNA) to >70 (rpbA) nucleotides; for rpbA in particular, the length of the protected region is RegA-concentration dependent. Quantitative fluorescence quenching measurements with RB69 wildtype RegA and RegA691 show that both proteins bind poly(U) similarly, but RegA691 binds with lower affinity to both 44 and rpbA RNA. The results indicate that both RB69 RegA RNA-protein and protein-protein interactions contribute to a translational repression hierarchy.

Bacteriophage T4↗

PubMed1995

p53-dependent repression of CDK4 translation in TGF-beta-induced G1 cell-cycle arrest.

Transforming growth factor beta 1 (TGF-beta 1) can cause a cell-cycle arrest in G1. Inhibition of cyclin-dependent kinase 4 (cdk4) synthesis plays a significant role in the mechanism by which this cytokine causes G1 growth arrest. Deregulated expression of cdk4 confers resistance to TGF-beta 1. Here, we show that TGF-beta 1 down-regulates cdk4 expression by inhibiting its translation. Moreover, mutant p53 confers resistance to TGF-beta 1 by interfering with the down-regulation of cdk4 in response to the cytokine. In contrast, we demonstrate that wild-type p53 represses the translation of CDK4. Regulation of cdk4 synthesis by both p53 and TGF-beta 1 is mediated by the 5'-untranslated region of the CDK4 message. Thus, regulation of CDK4 translation may be involved in control of G1 progression by p53.

Base Sequence↗

PubMed2003

Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA.

The 16S rRNA-binding ribosomal protein S15 is a key component in the assembly of the small ribosomal subunit in bacteria. We have shown that S15 from the extreme thermophile Thermus thermophilus represses the translation of its own mRNA in vitro, by interacting with the leader segment of its mRNA. The S15 mRNA-binding site was characterized by footprinting experiments, deletion analysis and site-directed mutagenesis. S15 binding triggers a conformational rearrangement of its mRNA into a fold that mimics the conserved three-way junction of the S15 rRNA-binding site. This conformational change masks the ribosome entry site, as demonstrated by direct competition between the ribosomal subunit and S15 for mRNA binding. A comparison of the T.thermophilus and Escherichia coli regulation systems reveals that the two regulatory mRNA targets do not share any similarity and that the mechanisms of translational inhibition are different. Our results highlight an astonishing plasticity of mRNA in its ability to adapt to evolutionary constraints, that contrasts with the extreme conservation of the rRNA-binding site.

Bacterial Proteins↗

PubMed2000

Translational control of cyclin B1 mRNA during meiotic maturation: coordinated repression and cytoplasmic polyadenylation.

Translational control is prominent during meiotic maturation and early development. In this report, we investigate a mode of translational repression in Xenopus laevis oocytes, focusing on the mRNA encoding cyclin B1. Translation of cyclin B1 mRNA is relatively inactive in the oocyte and increases dramatically during meiotic maturation. We show, by injection of synthetic mRNAs, that the cis-acting sequences responsible for repression of cyclin B1 mRNA reside within its 3'UTR. Repression can be saturated by increasing the concentration of reporter mRNA injected, suggesting that the cyclin B1 3'UTR sequences provide a binding site for a trans-acting repressor. The sequences that direct repression overlap and include cytoplasmic polyadenylation elements (CPEs), sequences known to promote cytoplasmic polyadenylation. However, the presence of a CPE per se appears insufficient to cause repression, as other mRNAs that contain CPEs are not translationally repressed. We demonstrate that relief of repression and cytoplasmic polyadenylation are intimately linked. Repressing elements do not override the stimulatory effect of a long poly(A) tail, and polyadenylation of cyclin B1 mRNA is required for its translational recruitment. Our results suggest that translational recruitment of endogenous cyclin B1 mRNA is a collaborative effect of derepression and poly(A) addition. We discuss several molecular mechanisms that might underlie this collaboration.

3' Untranslated Regions↗

PubMed1998

Inhibition of CCAAT/enhancer-binding protein alpha and beta translation by upstream open reading frames.

CCAAT/enhancer-binding protein (C/EBP) alpha is a bZIP transcription factor whose expression is restricted to specific cell types. Analysis of C/EBPalpha mRNA and protein levels in various mammalian cells indicates that expression of this gene is controlled both transcriptionally and post-transcriptionally. We report here that C/EBPalpha translation is repressed in several cell lines by an evolutionarily conserved upstream open reading frame (uORF), which acts in cis to inhibit C/EBPalpha translation. Mutations that disrupt the uORF completely abolished translational repression of C/EBPalpha. The related c/ebpbeta gene also contains an uORF that suppresses translation. The length of the spacer sequence between the uORF terminator and the ORF initiator codon (7 bases in all c/ebpalpha genes and 4 bases in c/ebpbeta homologs) is precisely conserved. The effects of insertions, deletions, and base substitutions in the C/EBPalpha spacer showed that both the length and nucleotide sequence of the spacer are important for efficient translational repression. Our data indicate that the uORFs regulate translation of full-length C/EBPalpha and C/EBPbeta and do not play a role in generating truncated forms of these proteins, as has been suggested by start site multiplicity models.

3T3 Cells↗

PubMed1989

SecA protein autogenously represses its own translation during normal protein secretion in Escherichia coli.

The Escherichia coli secA gene, whose expression is responsive to the protein secretion status of the cell, is the second gene in an operon. We found that both the basal and induced levels of SecA biosynthesis are dependent on prior translation of the upstream gene, gene X, and identified two large gene X-secA transcripts. The 10-fold derepression of secA expression by protein export defects was at the translational level since no further increases in gene X or secA mRNA levels were detected during this period, and a secA-lacZ protein fusion but not an operon fusion was appropriately derepressed. Furthermore, overexpression of the SecA protein severely reduced expression of only the secA-lacZ protein fusion, indicating that SecA autogenously represses its own translation.

Bacterial Proteins↗

PubMed1997

Purification and characterisation of cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP), a protein that represses cathepsin B mRNA translation.

We reported that the 3'-untranslated region (3'-UTR) of cathepsin B mRNA of Sarcophaga peregrina (flesh fly) is necessary for the repression of its translation, and detected the 3'-UTR-binding protein in lysates of larval hemocytes in which cathepsin B mRNA translation was repressed [Yano, T., Kurata, S. & Natori, S. (1995) Eur. J. Biochem. 234, 39-43]. In this study, we purified the 3'-UTR-binding protein from an embryonic cell line of Sarcophaga. The purified protein (CBBP) was found to repress cathepsin B mRNA translation in a rabbit reticulocyte lysate. We found that the CBBP contents of the hemocytes did not change during metamorphosis, although the cathepsin B mRNA became translatable only at the pupal stage. Moreover, we found that pupal, but not larval hemocytes, contained a factor that inhibited the binding of CBBP to the 3'-UTR. A regulatory mechanism of cathepsin B expression in Sarcophaga hemocytes is discussed.

Animals↗

PubMed1998

Repression of human thymidylate synthase mRNA translation by antisense 2'-O-methyl oligoribonucleotides.

Previous studies have shown that translation of thymidylate synthase (TS) mRNA is controlled by its own protein end product TS in a negative autoregulatory manner. Disruption of this process results in increased synthesis of TS and may be associated with the development of cellular drug resistance to TS-directed anticancer agents. As one strategy to inhibit TS expression, we have designed antisense RNA oligoribonucleotides (ORNs) that directly target the 5'-upstream binding site (nt 80-109) of TS mRNA, a critical cis-acting regulatory element. ORNs were analyzed for their ability to specifically inhibit translation of human TS mRNA in an in vitro rabbit reticulocyte lysate translation system. Native 2'-hydroxyl(OH) ORNs inhibited TS mRNA translation in a dose-dependent manner but did not repress translation of control mRNAs, including p53 or Escherichia coli TS. A control sense 2'-OH ORN was unable to repress translation of either human TS mRNA or control mRNAs. Modified antisense ORNs with 2'-O-methyl phosphodiester or 2'-O-methyl phosphorothioate backbones (or both) repressed human TS mRNA translation in a dose-dependent manner, and they were both more effective than the respective 2'-OH ORN. However, nonspecific effects on mRNA translation were observed with the 2'-O-methyl phosphorothioate ORN. In vitro translation experiments revealed that in the presence of antisense ORNs, the target TS mRNA remained intact. These findings demonstrate that antisense ORNs targeted at the 5'-upstream cis-acting element represent effective inhibitors of TS mRNA translation.

Animals↗

PubMedDate not supplied

[The levels and nature of translation in mouse preimplantation embryos with repressed cytokinesis].

The translational activity of embryos cultured up to the 8-cell stage in cytochalasin D-supplemented medium was studied. These embryos remained unicellular during the entire preimplantation period. Blastocoel formation and hatching began in the cytochalasin-treated embryos at the same time as in the control. We have studied the overall translation rates of "one-cell embryos" and estimated the relative translational differences for individual polypeptides compared to the control embryos. Up to the early blastocyst stage, the translation levels in these embryos, measured by 35S-methionine incorporation, were about two times lower than in the control. At later stages, the differences were fivefold. The ppm values of approximately one-third of all individual spots on two-dimensional electrophoregrams differed three times or more between the control and cytochalasin-treated embryos. These results suggest that the first morphogenetic events in mammalian ontogenesis may be controlled autonomously and the timing of morphogenetic transitions is controlled by acquisition of a definite set of stage-specific factors which serves as the signal for subsequent development.

Animals↗

PubMed2003

Small non-coding RNAs, co-ordinators of adaptation processes in Escherichia coli: the RpoS paradigm.

Adaptation to the changing environment requires both the integration of external signals and the co-ordination of internal responses. Around 50 non-coding small RNAs (sRNAs) have been described in Escherichia coli; the levels of many of these vary with changing environmental conditions. This suggests that they play a role in cell adaptation. In this review, we use the regulation of RpoS (sigma38) translation as a paradigm of sRNA-mediated response to environmental conditions; rpoS is currently the only known gene regulated post-transcriptionally by at least three sRNAs. DsrA and RprA stimulate RpoS translation in response to low temperature and cell surface stress, respectively, whereas OxyS represses RpoS translation in response to oxidative shock. However, in addition to regulating RpoS translation, DsrA represses the translation of HNS (a global regulator of gene expression), whereas OxyS represses the translation of FhlA (a transcriptional activator), allowing the cell to co-ordinate different pathways involved in cell adaptation. Environmental cues affect the synthesis and stability of specific sRNAs, resulting in specific sRNA-dependent translational control.

Adaptation, Biological↗

PubMed2001

Translational activation and repression by distinct elements within the 5'-UTR of ENaC alpha-subunit mRNA.

The rat amiloride-sensitive epithelial Na(+) channel (rENaC), the rate-limiting step in epithelial Na(+) transport, consists of three subunits, alpha, beta, and gamma. We hypothesized that alpha-rENaC translation is regulated via its 5'-untranslated region (UTR). Transient transfections of alpha-rENaC promoter-reporter constructs in representative epithelial cell lines demonstrated up to fivefold differences in activity among constructs containing different amounts of the alpha-rENaC 5'-UTR sequence. Differences in reporter protein activity did not parallel differences in reporter mRNA, demonstrating that 5'-UTR regulation must be at the level of translation. Specifically, translation was enhanced by a region extending from +53 to +211 bp downstream from the transcription start site and repressed by the region between +367 and +499 bp. Examination of the 5'-UTR sequence revealed an out-of-frame initiation codon within the repressive region, 43 bp upstream from the start of the alpha-rENaC open reading frame. Mutational analysis of this upstream start codon indicated that it plays, at most, a minor role in impeding translation both in vitro and in vivo, suggesting that additional mechanisms of translational regulation are operative.

5' Untranslated Regions↗

PubMed1996

The translational inhibitor cycloheximide represses growth factor depletion-induced apoptosis in an alb-SV40T transgenic rat liver cell line.

A transgenic rat line carrying the alb-SV40A transgene has been described by this laboratory. Several cell lines have been established from the livers of two of these rats. One of these cell lines, L37, exhibits a large nuclear/cytoplasmic ratio and a well-differentiated cytoplasm containing numerous organelles. When L37 cells are placed into culture medium lacking necessary growth factors, cellular proliferation continues for 48 hours after medium change. Subsequent to the initial 48 hours, cells begin to shrink and lose contact with adjacent cells, eventually sloughing off the culture plate surface, with most cell deaths occurring between 48 and 96 hours after medium change. Microscopic examination of sloughing cells indicates they possess highly convoluted and blebbed plasma membranes, a morphological characteristic of apoptosis. Ultrastructural studies demonstrate the ubiquitous presence of apoptotic bodies. When DNA isolated from growth factor-depleted cells is resolved on agarose gels, DNA fragmentation ladders are observed at times of maximum apoptotic change. Quantitative analysis of L37 cells between 48 and 96 hours after the removal of the culture medium shows that 59% +/- 2% of the cells undergo apoptosis. When cycloheximide, puromycin, or actinomycin D is added to the L37 cultures, only cycloheximide is able to repress apoptosis, indicating that the mechanism of apoptosis in the L37 liver-derived cell line requires a cycloheximide-sensitive translational event. The extremely high rate of apoptosis, together with the maintenance of hepatocellular characteristics, indicates the usefulness of this cell line as a model in which to study the mechanisms of hepatocellular apoptosis.

Albumins↗

PubMed1999

Glycerol-3-phosphate-mediated repression of malT in Escherichia coli does not require metabolism, depends on enzyme IIAGlc and is mediated by cAMP levels.

malT encodes the central activator of the maltose system in Escherichia coli, a gene that is typically under positive control of the cAMP/CAP catabolite repression system. When cells were grown in tryptone broth, the addition of glycerol reduced malT expression two- to threefold. Phosphorylation of glycerol to glycerol-3-phosphate (G3P) was necessary for this repression, but further metabolism to dihydroxyacetone phosphate was not. Mutants lacking adenylate cyclase and harbouring a crp* mutation (synthesizing a cAMP receptor protein that is independent of cAMP) no longer repressed a transcriptional malT-lacZ fusion but still repressed a translational malT-lacZ fusion. Similar results were obtained with a mutant lacking enzyme IIAGlc. For the translational fusion (in a cya crp* genetic background) to be repressed by glycerol, a drop to pH 5 of the growth medium was necessary. Thus, while transcriptional repression by glycerol requires enzyme IIAGlc, cAMP and CAP, pH-mediated translational repression is cAMP independent. Other sugars that are not transported by the phosphotransferase system, most notably D-xylose, showed the same effect as glycerol.

Artificial Gene Fusion↗

PubMed1998

Growth-dependent and PKC-mediated translational regulation of the upstream stimulating factor-2 (USF2) mRNA in hematopoietic cells.

Upstream stimulating factor (USF2) is a basic helix-loop-helix leucine zipper transcription factor, which is found in most tissues. A critical role for USF2 in cellular proliferation has been proposed based on its importance in the regulation of various cyclins and P53 and its capability to antagonize c-myc. In this paper we report that IL-3, which is a major growth factor for mast cells, induces USF2 protein synthesis in murine mast cells (MC-9). Surprisingly, it does not significantly affect the level of USF2 mRNA in these cells at any of the time points tested. Using polysomal fractionation and RNA analysis we then demonstrated that this translational regulation is mostly the result of increased USF2 translational efficiency. Moreover, protein kinase C (PKC) inhibitors prevented both the induction of USF2 protein synthesis and the increase in USF2 translational efficiency in IL-3-activated mast cells. Two other hematopoietic cell lines were used to determine whether the translational regulation of USF2 is of a more general nature: mouse lymphosarcoma cells whose proliferation is inhibited by dexamethasone; and mouse erythroleukemia cells that differentiate upon exposure to hexamethylen bisacetamide. In both cell types, USF2 translation was repressed in the non-dividing cells. This strongly implies that USF2 is translationally repressed in quiescent hematopoietic cells. Considering the proposed role of USF in proliferation it seems that translational regulation of USF2 might have an important role in cellular growth.

Animals↗

PubMed2000

Kinetic proofreading scanning models for eukaryotic translational initiation: the cap and poly(A) tail dependency of translation.

Two simplified kinetic proofreading scanning (KPS) models were proposed to describe the 5' cap and 3' poly(A) tail dependency of eukaryotic translation initiation. In Model I, the initiation factor complex starts scanning and unwinding the secondary structure of the 5' untranslated region (UTR) from the 5' terminus of mRNA. In Model II, the initiation factor complex starts scanning from any binding site in the 5' UTR. In both models, following ATP hydrolysis, the initiation factor complex either dissociates from mRNA or continues to scan and unwind RNA secondary structure in the 5' UTR. This step repeats n times until the AUG codon is reached. These two models show very different cap and/or poly(A) tail dependency of translation initiation. The models predict that both cap and poly(A) tail dependencies of translation, and translatability of mRNAs are coupled with the structure of 5' UTR: the translation of mRNA with structured 5' UTR is strongly cap- and poly(A) tail-dependent; while translation of mRNA with unstructured 5' UTR is less cap- and poly(A) tail-dependent. We use these two models to explain: (1) the cap and poly(A) tail dependence of translation; (2) the effect of exogenous poly(A) on translation; (3) repression of host mRNA and translation of late adenovirus mRNA in the late phase of adenovirus infection; (4) repression of host mRNA and translation of Vaccinia virus mRNA in virus-infected cell; (5) heat shock repression of translation of normal mRNA and stimulation of translation of hsp mRNA; and (6) the synergistic effect of cap and poly(A) tail on stimulating translation. The kinetic proofreading scanning models provide a coherent interpretation of those phenomena.

5' Untranslated Regions↗

PubMed1999

Translational effects of mutations and polymorphisms in a repressive upstream open reading frame of the human cytomegalovirus UL4 gene.

The human cytomegalovirus (HCMV) gpUL4 mRNA contains a 22-codon upstream open reading frame (uORF2), the peptide product of which represses downstream translation by blocking translation termination at its own stop codon and by causing ribosomes to stall on the mRNA. A distinctive feature of this unusual mechanism is its strict dependence on the uORF2 peptide sequence. To delineate sequence elements that function in the inhibitory mechanism, deletions and missense mutations affecting the previously uncharacterized amino-terminal region of uORF2 were analyzed in transient-transfection and infection assays. These experiments identified multiple codons in this region that are necessary for inhibition of downstream translation by uORF2 and, in conjunction with previous results, demonstrated that amino acids dispersed throughout the uORF2 peptide participate in the repressive mechanism. In contrast to the highly conserved carboxy terminus, the amino-terminal portion of the uORF2 peptide is polymorphic. A survey of uORF2 sequences in HCMV clinical isolates revealed that although most have uORF2 sequences that are predicted to retain the uORF2 inhibitory activity, approximately 15% contain polymorphisms at codons that are essential for full inhibition by uORF2. Consistent with predictions based on analyses of engineered mutations, two viral isolates with uORF2 sequences that do not inhibit downstream translation in transfection assays expressed much more gpUL4 protein but similar levels of UL4 mRNA compared to the levels produced by the prototypic laboratory strain HCMV (Towne) and another clinical isolate with an inhibitory variant uORF2. These results demonstrate that uORF2 is polymorphic in sequence and repressive activity and suggest that the uORF2 regulatory mechanism, although prevalent among natural HCMV isolates, is not absolutely essential for viral replication.

Amino Acid Sequence↗

PubMed2005

RNA transport and local control of translation.

In eukaryotes, the entwined pathways of RNA transport and local translational regulation are key determinants in the spatio-temporal articulation of gene expression. One of the main advantages of this mechanism over transcriptional control in the nucleus lies in the fact that it endows local sites with independent decision-making authority, a consideration that is of particular relevance in cells with complex cellular architecture such as neurons. Localized RNAs typically contain codes, expressed within cis-acting elements, that specify subcellular targeting. Such codes are recognized by trans-acting factors, adaptors that mediate translocation along cytoskeletal elements by molecular motors. Most transported mRNAs are assumed translationally dormant while en route. In some cell types, especially in neurons, it is considered crucial that translation remains repressed after arrival at the destination site (e.g., a postsynaptic microdomain) until an appropriate activation signal is received. Several candidate mechanisms have been suggested to participate in the local implementation of translational repression and activation, and such mechanisms may target translation at the level of initiation and/or elongation. Recent data indicate that untranslated RNAs may play important roles in the local control of translation.

Animals↗

PubMed1997

Effects of cyclin A2 noncoding regions on reporter gene translation during early development of Xenopus laevis.

The repression of translation of Xenopus cyclin A2 transcripts during early development was examined by analyzing the effects of cyclin A2 noncoding regions using a CAT reporter system. On their own, the 5' and 3' UTRs (untranslated regions) were unable to inhibit reporter translation until approximately the time of the midblastula transition. Transcripts containing the 3' UTR were polyadenylated after fertilization and the midblastula transition. When both noncoding regions flanked a CAT reporter gene, translation was repressed at all stages of development examined in spite of their polyadenylation after fertilization. From these data, we conclude that the 5' and 3' UTRs interact synergically to prevent translation during early development and that the poly(A) tail is insufficient to promote their translation.

Animals↗