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Results for “Translational repression”

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PubMed1994

Mutations that increase the affinity of a translational repressor for RNA.

The coat protein of the RNA bacteriophage MS2 is a specific RNA binding protein that represses translation of the viral replicase gene during the infection cycle. As an approach to characterizing the RNA-binding site of coat protein we have isolated a series of coat mutants that suppress the effects of a mutation in the translational operator. Each of the mutants exhibits a super-repressor phenotype, more tightly repressing both the mutant and wild-type operators than does the wild-type protein. The variant coat proteins were purified and subjected to filter binding assays to determine their affinities for the mutant and wild-type operators. Each protein binds the operators from 3 to 7.5-fold more tightly than normal coat protein. The amino acid substitutions seem to extend the normal binding site by introducing new interactions with RNA.

Base Sequence↗

PubMed1990

Sequence and developmental expression of the mRNA encoding the seleno-protein of the sperm mitochondrial capsule in the mouse.

We have characterized cDNA clones encoding the selenium-containing polypeptide of the keratinous mitochondrial capsule in mouse sperm. The longest open reading frame encodes a polypeptide 143 amino acids long which contains 21% cysteine and 27% proline and closely resembles the size and amino acid composition of bull mitochondrial capsule seleno-protein (V. Pallini, B. Baccetti, and A. G. Burrini, 1979, in "The Spermatozoon," D. W. Fawcett and J. M. Bedford, Eds., pp. 141-151, Urban & Schwartzenberg, Baltimore/Munich). The reading frame encoding the mitochondrial capsule seleno-protein ends with an amber stop codon suggesting that selenium is not incorporated cotranslationally into the protein by an opal suppressor selenocysteyl-tRNA as has been found for several eukaryotic and bacterial proteins. Northern blots using RNA extracted from purified spermatogenic cells and staged prepuberal mice suggest that the mitochondrial capsule seleno-protein mRNA is first transcribed in late meiotic cells and that the levels of the mRNA increase after meiosis in early haploid cells. Southern blots demonstrate that there is one copy of the gene in the mouse genome. The identification of this cDNA clone, in combination with previous work (K. C. Kleene, 1989, Development 106, 367-373) demonstrates that the mRNA for the mitochondrial capsule seleno-protein is translationally repressed with long homogenous poly(A) tracts in round spermatids and translationally active with shortened heterogenous poly(A) tracts in elongating spermatids.

Amino Acid Sequence↗

PubMed1988

Control of the tRNA-tufB operon in Escherichia coli. 2. Mechanisms of the feedback inhibition of tufB expression studied in vivo and in vitro.

The mechanism underlying feedback inhibition of tufB expression has been studied in vivo by gene-dosage experiments and by gene and operon fusions involving lacZ. Raising the cellular EF-Tu content, by introducing a multicopy plasmid encoding EF-TuA into the cell, repressed the level of EF-TuB but left the content of tRNA(Thr)3, encoded by the tRNA-tufB operon, unaffected. This indicates that autoregulation of chromosomal tufB expression does not occur by modulating transcription initiation at the promoter of the tRNA-tufB operon. This conclusion is further substantiated by experiments with a tRNA':lacZ operon fusion. The molecular ratio of chromosome-borne tufA and tufB transcripts also remained unaltered under conditions of excess EF-Tu, though experiments with a tRNA-tufB':lacZ operon fusion showed a decrease of tufB transcripts. Our data further exclude drastic effects of the autogenous repressor on processing of the contranscript of the operon into monocistronic tufB RNA and on alteration of EF-TuB turnover. Two possible mechanisms remain, which cannot yet be decided between. One is modulation of EF-Tu by transcription termination either directly or indirectly by affecting antitermination. The second is translational repression. In vitro translation of transcripts derived from SP6 clones did not reveal any feedback inhibition of EF-TuB synthesis. Surprisingly, addition of EF-Tu to a coupled transcription/translation systems was found to block transcription initiation at the primary promoter of the tRNA-tufB operon by over 90%. Although this in vitro effect of EF-Tu could not be demonstrated in vivo, possibly because of a difference in higher-order structure between plasmid-borne and chromosome-borne DNA, it indicates that under certain conditions EF-Tu binds very specifically to the tRNA-tufB operon promoter or its upstream region.

Cloning, Molecular↗

PubMed2006

Recapitulation of short RNA-directed translational gene silencing in vitro.

microRNAs (miRNAs) are a large class of endogenous short RNAs that repress gene expression. Many miRNAs are conserved throughout evolution, and dysregulation of miRNA pathways has been correlated with an increasing number of human diseases. In animals, miRNAs typically bind to the 3' untranslated region (3'UTR) of target mRNAs with imperfect sequence complementarity and repress translation. Despite their importance in regulating biological processes in numerous organisms, the mechanisms of miRNA function are largely unknown. Here, we report in vitro reactions for miRNA-directed translational gene silencing. These reactions faithfully recapitulate known in vivo hallmarks of mammalian miRNA function, including a requirement for a 5' phosphate and perfect complementarity to the mRNA target in the 5' seed region. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a polyA tail, whereas increasing polyA tail length alone can increase miRNA silencing activity.

Animals↗

PubMed2002

Crystallization and characterization of Smaug: a novel RNA-binding motif.

During Drosophila embryogenesis, Smaug protein represses translation of Nanos through an interaction with a specific element in its 3(')UTR. The repression occurs in the bulk cytoplasm of the embryo; Nanos is, however, successfully translated in the specialized cytoplasm of the posterior pole. This generates a gradient of Nanos emanating from the posterior pole that is essential for organizing proper abdominal segmentation. To understand the structural basis of RNA binding and translational control, we have crystallized a domain of Drosophila Smaug that binds RNA. The crystals belong to the space group R3 with unit cell dimensions of a=b=129.3A, c=33.1A, alpha=beta=90 degrees, gamma=120 degrees and diffract to 1.80A with synchrotron radiation. Initial characterization of this domain suggests that it encodes a novel RNA-binding motif.

3' Untranslated Regions↗

PubMed1998

Rapamycin induces the G0 program of transcriptional repression in yeast by interfering with the TOR signaling pathway.

The macrolide antibiotic rapamycin inhibits cellular proliferation by interfering with the highly conserved TOR (for target of rapamycin) signaling pathway. Growth arrest of budding yeast cells treated with rapamycin is followed by the program of molecular events that characterizes entry into G0 (stationary phase), including the induction of polymerase (Pol) II genes typically expressed only in G0. Normally, progression into G0 is characterized by transcriptional repression of the Pol I and III genes. Here, we show that rapamycin treatment also causes the transcriptional repression of Pol I and III genes. The down-regulation of Pol III transcription is TOR dependent. While it coincides with translational repression by rapamycin, transcriptional repression is due in part to a translation-independent effect that is evident in extracts from a conditional tor2 mutant. Biochemical experiments reveal that RNA Pol III and probably transcription initiation factor TFIIIB are targets of repression by rapamycin. In view of previous evidence that TFIIIB and Pol III are inhibited when protein phosphatase 2A (PP2A) function is impaired, and that PP2A is a component of the TOR pathway, our results suggest that TOR signaling regulates Pol I and Pol III transcription in response to nutrient growth signals.

Antifungal Agents↗

PubMed1997

Substitution of just five nucleotides at and around the transcription start site of rat beta-actin promoter is sufficient to render the resulting transcript a subject for translational control.

Vertebrate mRNAs with a 5' terminal oligopyrimidine tract (5' TOP), including those encoding ribosomal proteins and elongation factors, are candidates for translational control in a growth-dependent fashion. The present study was designed to determine the minimal cis-regulatory element involved in this mode of regulation. We selected rat beta-actin mRNA, a typical translationally uncontrolled transcript, as a subject for gain-of-function analysis. Mutations at and around its cap site leading to the formation of a 7 pyrimidines long 5' TOP render the resulting transcript translationally repressed upon growth arrest of lymphosarcoma cells. In contrast, growth-dependent translational control of this mRNA in fibroblasts requires, in addition, a GC motif downstream of the 5' TOP. A similar motif is present in all ribosomal prtein mRNAs shown to be translationally controlled.

3T3 Cells↗

PubMed2001

Human testis expresses a specific poly(A)-binding protein.

In testis mRNA stability and translation initiation are extensively under the control of poly(A)-binding proteins (PABP). Here we have cloned a new human testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical residues to the ubiquitous PABP1. A northern blot of multiple human tissues hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2 kb) was present in all tested tissues. In human adult testis, PABP3 mRNA expression was restricted to round spermatids, whereas PABP1 was expressed in these cells as well as in pachytene spermatocytes. PABP3-specific antibodies identified a protein of 70 kDa in human testis extracts. This protein binds poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3 gene is intronless with a transcription start site 61 nt upstream from the initiation codon. A sequence of 256 bp upstream from the transcription start site drives the promoter activity of PABP3 and its tissue-specific expression. The expression of PABP3 might be a way to bypass PABP1 translational repression and to produce the amount of PABP needed for active mRNA translation in spermatids.

Amino Acid Sequence↗

PubMed2003

Inhibitors of p38 mitogen-activated protein kinase: potential as anti-inflammatory agents in asthma?

Asthma is an inflammatory disease of the airways, which in patients with mild to moderate symptoms is adequately controlled by either beta(2)-adrenoceptor agonists or corticosteroids, or a combination of both. Despite this, there are classes of patients that fail to respond to these treatments. In addition, there is a general trend towards increasing morbidity and mortality due to asthma, which suggests that there is a need for new and improved treatments. The p38 mitogen-activated protein kinases (MAPKs) represent a point of convergence for multiple signalling processes that are activated in inflammation and that impact on a diverse range of events that are important in inflammation. Small molecule pyridinyl imidazole inhibitors of p38 MAPK have proved to be highly effective in reducing various parameters of inflammation, in particular cytokine expression. Like corticosteroids, inhibitors of p38 MAPK appear to be able to repress gene expression at multiple levels, for example, by transcriptional, posttranscriptional and translational repression, and this raises the possibility of a similarly broad spectrum of anti-inflammatory activities. Indeed these molecules have proved to be effective in numerous in vitro and in vivo models of inflammation and septicaemia, which suggests that such compounds may be effective as therapeutic agents against inflammatory disorders. Despite these very promising indications of the possible therapeutic use of p38 MAPK inhibitors, a number of events that are p38-dependent are in fact also beneficial to the resolution or modulation of diseases such as asthma. We conclude that the overall effect of p38 MAPK inhibition would be beneficial in inflammatory diseases such as rheumatoid arthritis and asthma. However, these drugs may result in a complex phenotype that will require careful evaluation. Currently, a number of second or third generation inhibitors of p38 MAPK are being tested in phase I and phase II clinical trials.

Anti-Inflammatory Agents↗

PubMed1987

Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs.

Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.

Animals↗

PubMed2001

Translational control in vertebrate development.

Translational control plays a large role in vertebrate oocyte maturation and contributes to the induction of the germ layers. Translational regulation is also observed in the regulation of cell proliferation and differentiation. The features of an mRNA that mediate translational control are found both in the 5' and in the 3' untranslated regions (UTRs). In the 5' UTR, secondary structure, the binding of proteins, and the presence of upstream open reading frames can interfere with the association of initiation factors with the cap, or with scanning of the initiation complex. The 3' UTR can mediate translational activation by directing cytoplasmic polyadenylation and can confer translational repression by interference with the assembly of initiation complexes. Besides mRNA-specific translational control elements, the nonspecific RNA-binding proteins contribute to the modulation of translation in development. This review discusses examples of translational control and their relevance for developmental regulation.

Animals↗

PubMed2001

Dual interactions of the translational repressor Paip2 with poly(A) binding protein.

The cap structure and the poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This effect is mediated by a direct interaction of eukaryotic initiation factor 4G and poly(A) binding protein (PABP), which brings about circularization of the mRNA. Of the two recently identified PABP-interacting proteins, one, Paip1, stimulates translation, and the other, Paip2, which competes with Paip1 for binding to PABP, represses translation. Here we studied the Paip2-PABP interaction. Biacore data and far-Western analysis revealed that Paip2 contains two binding sites for PABP, one encompassing a 16-amino-acid stretch located in the C terminus and a second encompassing a larger central region. PABP also contains two binding regions for Paip2, one located in the RNA recognition motif (RRM) region and the other in the carboxy-terminal region. A two-to-one stoichiometry for binding of Paip2 to PABP with two independent K(d)s of 0.66 and 74 nM was determined. Thus, our data demonstrate that PABP and Paip2 could form a trimeric complex containing one PABP molecule and two Paip2 molecules. Significantly, only the central Paip2 fragment, which binds with high affinity to the PABP RRM region, inhibits PABP binding to poly(A) RNA and translation.

Amino Acid Sequence↗

PubMed2006

Effects of C/EBPalpha and C/EBPbeta in BCR/ABL-expressing cells: differences and similarities.

C/EBPalpha and C/EBPbeta, two transcription factors of the C/EBP family play important roles in the proliferation and differentiation of various cell types including myeloid progenitors. Expression of C/EBPalpha and C/EBPbeta is repressed in myeloid blast crisis of Chronic Myelogenous Leukemia by mechanisms that involve translation repression which depends on the interaction of RNA-binding proteins with conserved binding sites in the 5'UTR of c/ebpalpha and c/ebpbeta mRNA. Ectopic expression of C/EBPalpha and C/EBPbeta in myeloid progenitors expressing the BCR/ABL oncogene inhibits proliferation, induces differentiation and suppresses leukemogenesis in mice, but C/EBPalpha is markedly more effective than C/EBPbeta. The more potent effects of C/EBPalpha probably depends on protein-protein interaction with cell-cycle regulatory proteins, but the pattern of genes modulated by C/EBPalpha and C/EBPbeta is not completely overlapping. This suggests that transcription-dependent and -independent effects are both involved and support the therapeutic potential of reactivating C/EBPalpha and C/EBPbeta expression in leukemic cells.

Animals↗

PubMed2000

Isolation of translationally controlled mRNAs by differential screening.

Translationalregulation plays an important role in the control of gene expression. Changes in translation initiation rates are the most common translation-regulating mechanisms, resulting in alterations in mRNA loading of ribosomes. This differential mobilization of mRNAs onto polyribosomes was used in differential screening to directly identify cDNAs whose transcripts are translationally controlled during antigenic stimulation of primary human T lymphocytes. Ribosome-free and polysome-bound mRNAs were prepared from quiescent and activated T cells and used as templates to synthesize four cDNA pools. These in turn were used as probes to hybridize four identical replicas of a T cell library or, alternatively, four cDNA arrays. Translational activation was indicated by redistribution of the hybridization signals from the ribosome-free fraction in resting T cells to the polysome-associated fraction in activated T cells. Translational repression corresponded to the opposite hybridization pattern. Fifty-two cDNAs were identified as translationally controlled by screening 472 genes in a cDNA array; 12 additional ones were obtained by screening a cDNA library. Several of the transcripts corresponded to mRNAs previously reported to be translationally controlled, thus validating the method. For the majority, however, such regulation had not yet been described. Translational control was verified for representative examples by demonstrating the redistribution of the corresponding mRNAs on polysome gradients in response to T cell activation. Our strategy therefore provides an efficient tool to directly isolate or identify translationally controlled mRNAs in a variety of physiological situations. Moreover, differential screening using arrays enables simultaneous analysis of both transcriptional and translational regulation, further enhancing the power of gene expression analysis.

Cells, Cultured↗

PubMed2004

MicroRNA-directed cleavage of HOXB8 mRNA.

MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs, some of which are known to play important regulatory roles in animals by targeting the messages of protein-coding genes for translational repression. We find that miR-196, a miRNA encoded at three paralogous locations in the A, B, and C mammalian HOX clusters, has extensive, evolutionarily conserved complementarity to messages of HOXB8, HOXC8, and HOXD8. RNA fragments diagnostic of miR-196-directed cleavage of HOXB8 were detected in mouse embryos. Cell culture experiments demonstrated down-regulation of HOXB8, HOXC8, HOXD8, and HOXA7 and supported the cleavage mechanism for miR-196-directed repression of HOXB8. These results point to a miRNA-mediated mechanism for the posttranscriptional restriction of HOX gene expression during vertebrate development and demonstrate that metazoan miRNAs can repress expression of their natural targets through mRNA cleavage in addition to inhibiting productive translation.

3' Untranslated Regions↗

PubMed2000

The control of cyclin B1 mRNA translation during mouse oocyte maturation.

In maturing mouse oocytes, protein synthesis is required for meiotic maturation subsequent to germinal vesicle breakdown (GVBD). While the number of different proteins that must be synthesized for this progression to occur is unknown, at least one of them appears to be cyclin B1, the regulatory subunit of M-phase-promoting factor. Here, we investigate the mechanism of cyclin B1 mRNA translational control during mouse oocyte maturation. We show that the U-rich cytoplasmic polyadenylation element (CPE), a cis element in the 3' UTR of cyclin B1 mRNA, mediates translational repression in GV-stage oocytes. The CPE is also necessary for cytoplasmic polyadenylation, which stimulates translation during oocyte maturation. The injection of oocytes with a cyclin B1 antisense RNA, which probably precludes the binding of a factor to the CPE, delays cytoplasmic polyadenylation as well as the transition from GVBD to metaphase II. CPEB, which interacts with the cyclin B1 CPE and is present throughout meiotic maturation, becomes phosphorylated at metaphase I. These data indicate that CPEB is involved in both the repression and the stimulation of cyclin B1 mRNA and suggest that the phosphorylation of this protein could be involved in regulating its activity.

3' Untranslated Regions↗

PubMed2005

A novel RNA-binding protein in neuronal RNA granules: regulatory machinery for local translation.

Local translation in neuronal dendrites is an important basis for long-term synaptic plasticity, and RNA granules in the dendrites are involved in the local translation. Here, we identify RNG105 (RNA granule protein 105), a novel RNA-binding protein, as a component of the RNA granules in dendrites of hippocampal neurons. The RNG105-localizing RNA granules contain mRNAs, the translational products of which play key roles in synaptic plasticity. RNG105 has an ability to repress translation both in vitro and in vivo, consistent with the finding that the RNA granule is translationally arrested in the basal conditions. Dissociation of RNG105 from the RNA granules is induced by BDNF, a growth factor responsible for synaptic plasticity. The RNG105 dissociation is coincident with the induction of local translation near the granules. These findings suggest that RNG105 is a translational repressor in the RNA granules and provide insight into the link between RNG105 dynamics and local translational regulation.

Amino Acid Sequence↗

PubMed2003

Transcriptional and post-transcriptional mechanisms regulating the rat pituitary vasopressin V1b receptor gene.

The number of V1b vasopressin receptors (V1bR) in the anterior pituitary plays an important role during adaptation of the hypothalamic-pituitary-adrenal axis to stress in rats. Regulation of V1bR expression involves transcriptional and translational mechanisms. One of the elements mediating transcriptional activation of the rat V1bR gene is a long stretch of GAGA repeats (GAGA box) in the promoter located near the transcription start point capable of binding a protein complex of 127 kDa present in pituitary nuclear extracts. There is a lack of correlation between changes in V1bR mRNA and the number of VP binding sites, suggesting that V1bR expression depends on the efficiency of V1b R mRNA translation into protein. Two mechanisms by which the 5' untranslated region (5'-UTR) of the rat V1bR mRNA can mediate either inhibition or activation of V1bR mRNA translation have been identified. First, upstream open reading frames (ORF) present in the 5'-UTR repress translation of the major ORF encoding the V1b receptor, and secondly, an internal ribosome entry site (IRES) activates V1bR translation. Stimulation of IRES activity through protein kinase C-mediated pathways results in V1bR mRNA translation increasing V1bR protein levels. The existence of multiple loci of regulation for the V1bR at transcriptional and translational levels provides a mechanism to facilitate plasticity of regulation of the number of pituitary vasopressin receptors according to physiological demand.

5' Untranslated Regions↗