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Results for “Translational repression”

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PubMed2006

Human let-7a miRNA blocks protein production on actively translating polyribosomes.

MicroRNAs (miRNAs) regulate gene expression at a post-transcriptional level through base-pairing to 3' untranslated regions (UTRs) of messenger RNAs. The mechanism by which human let-7a miRNA regulates mRNA translation was examined in HeLa cells expressing reporter mRNAs containing the Caenorhabditis elegans lin-41 3' UTR. let-7a miRNA strongly repressed translation, yet the majority of control and lin-41-bearing RNAs sedimented with polyribosomes in sucrose gradients; these polyribosomes, together with let-7a miRNA and the miRISC protein AGO, were released from those structures by puromycin. RNA containing the lin-41 3' UTR and an iron response element in the 5' UTR sedimented with polysomes when cells were incubated with iron, but showed ribosome run-off when the iron was chelated. These data indicate that let-7a miRNA inhibits actively translating polyribosomes. Nascent polypeptide coimmunoprecipitation experiments further suggest that let-7a miRNA interferes with the accumulation of growing polypeptides.

Base Sequence↗

PubMed2000

Dynamic interplay between antagonistic pathways controlling the sigma 32 level in Escherichia coli.

The heat-shock response in Escherichia coli depends primarily on the transient increase in the cellular level of heat-shock sigma factor final sigma(32) encoded by the rpoH gene, which results from both enhanced synthesis and transient stabilization of normally unstable final sigma(32). Heat-induced synthesis of final sigma(32) was previously shown to occur at the translation level by melting the mRNA secondary structure formed within the 5' coding sequence of rpoH including the translation initiation region. The subsequent decrease in the final sigma(32) level during the adaptation phase has been thought to involve both shutoff of synthesis (translation) and destabilization of final sigma(32)-mediated by the DnaK-DnaJ chaperones, although direct evidence for translational repression was lacking. We now show that the heat-induced synthesis of final sigma(32) does not shut off at the translation level by using a reporter system involving translational coupling. Furthermore, the apparent shutoff was not observed when the synthesis rate was determined by a very short pulse labeling (15 s). Examination of final sigma(32) stability at 10 min after shift from 30 to 42 degrees C revealed more extreme instability (t(1/2)=20 s) than had previously been thought. Thus, the dynamic change in final sigma(32) stability during the heat-shock response largely accounts for the apparent shutoff of final sigma(32) synthesis observed with a longer pulse. These results suggest a mechanism for maintaining the intricate balance between the antagonistic pathways: the rpoH translation as determined primarily by ambient temperature and the turnover of final sigma(32) as modulated by the chaperone (and presumably protease)-mediated autogenous control.

Amino Acid Sequence↗

PubMed1998

Translational control: a general mechanism for gene regulation during T cell activation.

Distributional changes of individual mRNAs between free ribonucleoprotein particles (mRNP) and ribosome-bound transcripts are used to assess translational control. Simultaneous analysis of many mRNA species is required to estimate the overall contribution of translation to the regulation of gene expression. To this purpose, total cytoplasmic RNA was fractionated in sucrose step gradients and poly(A)+ RNA was prepared from mRNP and ribosome-bound fractions. Since direct, simultaneous analysis of a profusion of mRNAs is not feasible, distribution of their in vitro translation products was examined after separation in 2-dimensional gels, followed by computer-based analysis of autoradiographs. When this analysis was applied to antigenically stimulated T cells, 36% of in vitro translation products showed a greater than 10-fold increase in intensity, suggesting transcriptional activation of the corresponding mRNAs. In comparison, 7.9% of individual mRNAs (54 of 685 species) were translationally activated. They were redistributed from free mRNP to ribosome-associated fractions; 4.7% (32 species) were translationally repressed, as indicated by the opposite pattern. The differential recruitment of 12.6% of mRNA species demonstrates specificity and the general significance of translational control during T cell activation, which implies that translation may play a similar role in regulating gene expression in a variety of physiological processes.

Animals↗

PubMed2006

Post-transcriptional small RNA pathways in plants: mechanisms and regulations.

Small RNAs are riboregulators that have critical roles in most eukaryotes. They repress gene expression by acting either on DNA to guide sequence elimination and chromatin remodeling, or on RNA to guide cleavage and translation repression. This review focuses on the various types of post-transcriptional small RNA-directed pathways in plants, describing their roles and their regulations.

DNA Methylation↗

PubMed1979

Translational regulation of polysome formation during dormancy of Physarum polycephalum.

The translational activity of actively growing microplasmodia and dormant microsclerotia of Physarum polycephalum was investigated by analyzing the distribution of ribosomes in polysomes. Microplasmodial post-mitochondrial fractions contained substantial amounts of polysomes and ribosomal subunits but very few native monosomes. During the starvation period which preceded microsclerotium formation, polysome levels remained constant, whereas the subunit titer began to increase. During encystment ribosomal subunits continued to accumulate as the level of polysomes gradually decreased. Dormant microsclerotia contained a large surplus of stored ribosomal subunits but no detectable polysomes. However, incubation of microsclerotia with concentrations of cycloheximide sufficient to slow polypeptide elongation without affecting initiation caused the gradual reappearance of polysomes at the expense of the subunits. Under these conditions the percentage of subunits driven into polysomes reached values similar to those of actively growing microplasmodia. Microsclerotia returned to nutrient medium contained very low levels of polysomes during the lag period which preceded germination. These were formed with preexisting, stored messenger ribonucleic acid. During the germination period, polysome levels were markedly increased. This elevation was dependent on new ribonucleic acid transcription. It is concluded that dormant microsclerotia contain functional messenger ribonucleic acid and ribosomes which are subject to translational repression at the level of initiation.

Cycloheximide↗

PubMed2005

Contrasting mechanisms of regulating translation of specific Drosophila germline mRNAs at the level of 5'-cap structure binding.

Translational control is a key genetic regulatory mechanism underlying the initial establishment of the major spatial axes of the Drosophila embryo. Many translational control mechanisms target eIF4E (eukaryotic initiation factor 4E), an initiation factor that recognizes the 5'-cap structure of the mRNA. Cap recognition by eIF4E, in complex with eIF4G, is essential for recruitment of the mRNA to the small ribosomal subunit. One established mechanism for repressing translation involves eIF4E-binding proteins, which competitively inhibit the eIF4E-eIF4G interaction. Our group has uncovered a novel mechanism for repression in which an eIF4E cognate protein called d4EHP, which cannot bind eIF4G, binds to the 5'-cap structure of cad mRNA thus rendering it translationally inactive. These two related, but distinct, mechanisms are discussed and contrasted in this review.

Animals↗

PubMed1999

Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies.

We addressed the impact of deleting TNF AU-rich elements (ARE) from the mouse genome on the regulation of TNF biosynthesis and the physiology of the host. Absence of the ARE affected mechanisms responsible for TNF mRNA destabilization and translational repression in hemopoietic and stromal cells. In stimulated conditions, TNF ARE were required both for the alleviation and reinforcement of message destabilization and translational silencing. Moreover, the mutant mRNA was no longer responsive to translational modulation by the p38 and JNK kinases, demonstrating that TNF ARE are targets for these signals. Development of two specific pathologies in mutant mice, i.e., chronic inflammatory arthritis and Crohn's-like inflammatory bowel disease, suggests that defective function of ARE may be etiopathogenic for the development of analogous human pathologies.

3' Untranslated Regions↗

PubMed2006

Distinct roles of the Pumilio and FBF translational repressors during C. elegans vulval development.

The C. elegans PUF and FBF proteins regulate various aspects of germline development by selectively binding to the 3' untranslated region of their target mRNAs and repressing translation. Here, we show that puf-8, fbf-1 and fbf-2 also act in the soma where they negatively regulate vulvaI development. Loss-of-function mutations in puf-8 cause ectopic vulval differentiation when combined with mutations in negative regulators of the EGFR/RAS/MAPK pathway and suppress the vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signalling. PUF-8 acts cell-autonomously in the vulval cells to limit their temporal competence to respond to the extrinsic patterning signals. fbf-1 and fbf-2, however, redundantly inhibit primary vulval cell fate specification in two distinct pathways acting in the soma and in the germline. The FBFs thereby ensure that the inductive signal selects only one vulval precursor cell for the primary cell fate. Thus, translational repressors regulate various aspects of vulval cell fate specification, and they may play a conserved role in modulating signal transduction during animal development.

Animals↗

PubMed2001

Repetitive elements in the 5' untranslated region of a human zinc-finger gene modulate transcription and translation efficiency.

A substantial proportion of the human genome consists of repetitive sequences. Although most of these sequences are nonessential for the organism, retroelements, such as Alu sequences, L1s, and HERVs (human endogenous retroviruses), have recently been implicated in the regulation of various genes. Our laboratory previously identified a novel, alternatively spliced zinc-finger gene, ZNF177, which incorporates Alu L1, and HERV segments into the 5' untranslated region (UTR) of transcripts. In this study, we investigated the genomic structure and functional significance of the repetitive sequences in the 5' UTR of ZNF177 mRNAs. Using luciferase and GFP reporter constructs, we assessed the effect of the HERV, Alu, and L1 sequences on gene expression levels. Our results indicate that the presence of the retroelement sequences, particularly the Alu and L1 segments which form one 5' UTR exon, modifies the expression level of both reporter genes. We present evidence that the Alu and L1 sequences alter both the RNA and protein levels of reporter genes by increasing transcription efficiency while decreasing translation efficiency. Our findings indicate that the Alu and L1 repeats in the 5' UTR of ZNF177 exert a positive transcriptional enhancer effect, but repress translation of the zinc finger gene. In addition, our analysis of a 5' UTR database suggests that 4% of human 5' UTRs harbor Alu sequences, indicating that the expression of many genes might be influenced by Alu repeats. These results illustrate the complex regulatory effects that retroelements can have on human gene expression.

5' Untranslated Regions↗

PubMed1992

5' end-dependent translation initiation of hepatitis C viral RNA and the presence of putative positive and negative translational control elements within the 5' untranslated region.

Hepatitis C virus (HCV) is a distant relative of pestiviruses and flaviviruses, but it has a 5' untranslated region (UTR) with some features structurally similar to that of picornaviruses. In order to test the role of the 5' UTR in controlling the expression of the HCV polyprotein, we fused full-length or deleted versions of the 5' UTR of HCV-1 RNA to chloramphenicol acetyl transferase (CAT) mRNA to monitor CAT activity in vivo. We found: (1) the full-length 5' UTR of HCV-1 RNA is translationally inactive while 5' deletions which mimic a 5' subgenomic RNA detected in vivo are active, (2) an efficient cis-acting element which represses translation is found at the 5' terminus, (3) a putative element which enhances translation is found near the 3' terminus of the 5' UTR, (4) additional cis-acting elements including small open reading frames (ORFs) upstream from the putative enhancer element downregulate translation. We did not find evidence supporting the existence of an internal ribosome entry site in the 5' UTR of HCV-1 RNA. These data suggest that HCV may employ a distinctive translation control strategy such as the generation of subgenomic viral mRNA in infected cells. Translational control of HCV might be responsible for some of the characteristic pathobiology seen in viral infection.

Base Sequence↗

PubMed1999

Ligand-mediated induction of thymidylate synthase occurs by enzyme stabilization. Implications for autoregulation of translation.

Thymidylate synthase (TS) is indispensable in the de novo synthesis of dTMP. As such, it has been an important target at which anti-neoplastic drugs are directed. The fluoropyrimidines 5-fluorouracil and 5-fluoro-2'-deoxyuridine are cytotoxic as a consequence of inhibition of TS by the metabolite 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). This inhibition occurs through formation of a stable ternary complex among the enzyme, the nucleotide analog, and the co-substrate N5, N10-methylenetetrahydrofolate. Numerous studies have shown that cellular concentrations of TS undergo about a 2-4-fold induction following treatment with TS inhibitors. An extensive body of in vitro studies has led to the proposal that this induction occurs because of relief of the translational repression brought on by the binding of TS to its own mRNA. In the current study, we have tested several predictions of this autoregulatory translation model. In contrast to expectations, we find that fluoropyrimidines do not cause a change in the extent of ribosome binding to TS mRNA. Furthermore, mutations within the mRNA that abolish its ability to bind TS have no effect on the induction. Finally, enzyme turnover measurements show that the induction is associated with an increase in the stability of the TS polypeptide. Our results, in total, indicate that enzyme stabilization, rather than translational derepression, is the primary mechanism of TS induction by fluoropyrimidines and call into question the general applicability of the autoregulatory translation model.

Base Sequence↗

PubMed2003

Cup-ling oskar RNA localization and translational control.

RNA localization and spatially restricted translational control can serve to deploy specific proteins to particular places within a cell. oskar (osk) RNA is a key initiatior of posterior patterning and germ cell specification in Drosophila, and its localization and translation are under elaborate control. In this issue, Wilhelm et al. (2003) show that the protein Cup both promotes osk localization and participates in repressing translation of unlocalized osk.

Animals↗

PubMed2000

Controlled translation initiation on insulin-like growth factor 2-leader 1 during Xenopus laevis embryogenesis.

A number of growth factors, whose spatio-temporal expression is essential for embryonic development, are encoded by mRNAs with a complex 5'UTR. Human insulin-like growth factor 2 mRNA contains a long (592 nucleotides) 5'UTR (IGFl1) with one upstream open reading frame and stable stem-loop structures, elements which might be important for controlled translation. To investigate whether these unusual features of IGFl1 can control translation initiation during embryogenesis, we examined the initiation efficiency on this 5'UTR during development of Xenopus laevis. The results demonstrate that IGFl1 strongly represses translation of a reporter in early embryos, compared with the Xenopus beta-globin 5'UTR. The inhibition is alleviated soon after the midblastula transition, suggesting a stimulatory effect of the transcription start. However, a similar stimulation of IGFl1-driven translation is seen in embryos in which de novo transcription was inhibited by actinomycin D. Furthermore, it is shown that up-regulation of IGFl1 activity is independent of eIF4E levels, and activity of IGFl1 is observed in all tissues of transgenic Xenopus embryos. These results indicate that post-translational modulation of a trans-acting factor enables efficient initiation on this complex 5'UTR after the midblastula transition.

5' Untranslated Regions↗

PubMed2006

Structural basis of RNA binding discrimination between bacteriophages Qbeta and MS2.

Sequence-specific interactions between RNA stem-loops and coat protein (CP) subunits play vital roles in the life cycles of the RNA bacteriophages, e.g., by allowing translational repression of their replicase cistrons and tagging their own RNA genomes for encapsidation. The CPs of bacteriophages Qbeta and MS2 each discriminate in favor of their cognate translational operators, even in the presence of closely related operators from other phages in vivo. Discrete mutations within the MS2 CP have been shown to relax this discrimination in vitro. We have determined the structures of eight complexes between such mutants and both MS2 and Qbeta stem-loops with X-ray crystallography. In conjunction with previously determined in vivo repression data, the structures enable us to propose the molecular basis for the discrimination mechanism.

Bacteriophages↗

PubMed1988

DNA polymerase of bacteriophage T4 is an autogenous translational repressor.

In bacteriophage T4 the protein product of gene 43 (gp43) is a multifunctional DNA polymerase that is essential for replication of the phage genome. The protein harbors DNA-binding, deoxyribonucleotide-binding, DNA-synthesizing (polymerase) and 3'-exonucleolytic (editing) activities as well as a capacity to interact with several other T4-induced replication enzymes. In addition, the T4 gp43 is a repressor of its own synthesis in vivo. We show here that this protein is an autogenous repressor of translation, and we have localized its RNA-binding sequence (translational operator) to the translation initiation domain of gene 43 mRNA. This mechanism for regulation of T4 DNA polymerase expression underscores the ubiquity of translational repression in the control of T4 DNA replication. Many T4 DNA polymerase accessory proteins and nucleotide biosynthesis enzymes are regulated by the phage-induced translational repressor regA, while the T4 single-stranded DNA-binding protein (T4 gp32) is, like gp43, autogenously regulated at the translational level.

Base Sequence↗

PubMed1998

Nuclear history of a pre-mRNA determines the translational activity of cytoplasmic mRNA.

The pathways of synthesis and maturation of pre-messenger RNA in the nucleus have a direct effect on the translational efficiency of mRNA in the cytoplasm. The transcription of intron-less mRNA in vivo directs this mRNA towards translational silencing. The presence of an intron at the 5' end of the transcript relieves this silencing, whereas an intron at the 3' end further represses translation. These regulatory events are strongly dependent on the transcription of pre-mRNA in the nucleus. The impact of nuclear history on regulatory events in the cytoplasm provides a novel mechanism for the control of gene expression.

Animals↗

PubMed1996

Cloning and characterization of the gene for the somatic form of DNA topoisomerase I from Xenopus laevis.

Two distinct tissue-specific forms of DNA topoisomerase I with M(r) of 165 and 110 kDa have been purified from oocytes and somatic cells respectively of the African frog Xenopus laevis. In this paper, cDNAs encoding a Xenopus topoisomerase I were cloned using PCR primers derived from sequences of yeast and human topoisomerase I. A polypeptide expressed from a portion of the coding sequence was recognized by an antiserum directed against the somatic topoisomerase I that had previously been shown to be unable to cross-react with the oocyte enzyme. Thus, the clone encodes the somatic cell topoisomerase I. An antiserum raised against a synthetic peptide containing the sequence surrounding the active site tyrosine of the somatic topoisomerase I reacts with the enzymes purified from both oocytes and somatic cells, indicating that the two enzymes share some limited sequence homology. RNA blot hybridization showed that oocytes contain an abundant store of somatic topoisomerase I mRNA that is not efficiently polyadenylated in oocytes. This stored RNA contains a consensus cytoplasmic polyadenylation element that is found in a variety of mRNAs that are translationally repressed in oocytes. Microinjection into oocytes of in vitro transcribed mRNA prepared from a Myc-tagged construct of the somatic topoisomerase I sequence is translated to yield a 110 kDa product. This suggests that the oocyte-specific 165 kDa topoisomerase I is not produced by tissue-specific post-translational modification of the somatic topoisomerase I. The oocyte enzyme appears to be produced from a minor mRNA species in oocytes that has not yet been identified.

Amino Acid Sequence↗

PubMed2005

Translational regulation of MOS messenger RNA in pig oocytes.

The temporal and spatial translation control of stored mRNA in oocytes is regulated by elements in their 3'-untranslated region (3'-UTR). The MOS 3'-UTR in pig oocytes is both heterogeneous (180, 480, or 530 nucleotides), and it contains multiple U-rich elements and extensive A-rich sequences (CA13CA5CA5CA6). We have examined the role of these potential regulatory elements by fusing wild-type or mutant MOS 3'-UTRs to luciferase mRNA and then injecting these chimeric transcripts into oocytes. We draw six main conclusions. First, the length of the MOS 3'-UTR tightly controls the level of translation of luciferase during oocyte maturation. Second, two U-rich (U5A) elements and the hexanucleotide signal (AAUAAA) are required for translation. Third, mutations, duplications, or relocations of the A-rich sequence reduce or block translation. Fourth, the relative importance of the A-rich and U-rich elements in controlling the level of translation differs. Fifth, none of our MOS 3'-UTR manipulations relieved translational repression before germinal vesicle breakdown. Sixth, all the MOS mRNA variants underwent polyadenylation during maturation. Whereas mutations to the hexanucleotide signal block both polyadenylation and translation, mutations to either the A-rich sequence or the U-rich elements block translation without fully blocking polyadenylation. We conclude that MOS mRNA translation in pig oocytes is subject to a more extensive series of controls than that in lower vertebrates.

3' Untranslated Regions↗