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At least 433 records · Page 24Linked to original sources
PubMed2004

Calcineurin is required for translational control of protein synthesis in rat pancreatic acini.

CCK increases the rate of net protein synthesis in rat pancreatic acini by activating initiation and elongation factors required for translation. The immunosuppressant FK506 inhibits the Ca(2+)-calmodulin-dependent phosphatase calcineurin in pancreatic acinar cells and blocks pancreatic growth induced by chronic CCK treatment. To test a requirement for calcineurin in the activation of the translational machinery stimulated by CCK, we evaluated the effects of FK506 on protein synthesis and on regulatory initiation and elongation factors in rat pancreatic acini in vitro. CCK acutely increased protein synthesis in acini from normal rats with a maximum increase at 100 pM CCK to 170 +/- 11% of control. The immunosuppressant FK506 dose-dependently inhibited CCK-stimulated protein synthesis over the same concentration range that blocked calcineurin activity, as assessed by dephosphorylation of the calcineurin substrate calcium-regulated heat-stable protein of 24 kDa. Another immunosuppressant, cyclosporin A, inhibited protein synthesis, but its effects appeared more complex. FK506 also inhibited protein synthesis stimulated by bombesin and carbachol. FK506 did not significantly affect the activity of the initiation factor-2B, or the phosphorylation of the initiation factor-2alpha, ribosomal protein protein S6, or the mRNA cap binding protein eukaryotic initiation factor (eIF) 4E. Instead, blockade of calcineurin with FK506 reduced the phosphorylation of the eIF4E binding protein, reduced the formation of the eIF4F complex, and increased the phosphorylation of eukaryotic elongation factor 2. From these results, we conclude that calcineurin activity is required for protein synthesis, and this action may be related to an effect on the formation of the mRNA cap binding complex and the elongation processes.

Animals↗
PubMed2006

Cancer-specific targeting of an adenovirus-delivered herpes simplex virus thymidine kinase suicide gene using translational control.

Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV-1 thymidine kinase (HSV-Tk), was modified with a complex 5' upstream-untranslated region (5'-UTR) that limits efficient translation to cells expressing high levels of the translation initiation factor, eIF4E. Since previous studies have shown that most tumor cells express elevated levels of eIF4E, tumor-specific gene delivery was optimized by incorporation of the 5'-UTR-modified suicide gene (HSV-UTk) into an adenovirus vector (Ad-CMV-UTk). The efficacy of this novel approach of targeting suicide gene expression and limiting cytotoxicity by means of translational restriction was tested in vitro with the use of the human breast cancer cell lines (MCF-7, MDA-MB435, and ZR-75-1). As controls, normal MCF10A, HMEC, and HMSC cell lines that express relatively low levels of eIF4E were used. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantify HSV-Tk mRNA for cells infected with Ad-CMV-UTk as well as with Ad-CMV-Tk (a control adenovirus in which HSV-Tk is not regulated at the level of translation). Translation of HSV-Tk in the Ad-infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined following treatment with the pro-drug ganciclovir (GCV) using an MTT viability assay. Finally, microPET imaging was used to assess cancer cell-specific expression of HSV-Tk and expression in normal tissues in vivo after intraperitoneal injection of Ad-CMV-Tk or Ad-CMV-UTk. These data collectively showed enhanced cancer cell-specific gene expression and reduced normal tissue gene expression for the Ad-HSV-UTk compared to the Ad-CMV-Tk, leading to increased cancer cell-enhanced GCV cytotoxicity. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro and in vivo is effective and may provide a platform for enhanced cancer gene therapy specificity.

Adenoviruses, Human↗
PubMed1988

Translational control in early sea urchin embryogenesis: initiation factor eIF4F stimulates protein synthesis in lysates from unfertilized eggs of Strongylocentrotus purpuratus.

We have used cell-free translation systems from unfertilized eggs and embryos of the sea urchin Strongylocentrotus purpuratus to analyze the mechanisms limiting protein synthesis in early embryogenesis. Unfertilized egg lysates supplemented with nuclease-treated reticulocyte lysate were stimulated 2-4-fold in incorporation of radioactive amino acid into protein. Thirty-minute zygote lysates supplemented in this way were not stimulated. These results suggested that a component limiting translation in the unfertilized egg lysate was provided by the nuclease-treated lysate and that this component was no longer limiting protein synthesis following fertilization. In view of these results, partially fractionated lysates and individual purified translational components from mammalian cells were tested for stimulation of the unfertilized egg lysate. A 1000000g supernatant devoid of ribosomal subunits also stimulated the unfertilized egg lysate. Thus, the stimulation was not due to the addition of active ribosomal subunits but to soluble elements in the reticulocyte lysate. Of the soluble components tested, only the cap-binding protein complex eIF4F caused a dramatic stimulation of the unfertilized egg lysate (2-3.5-fold). The 30-min zygote lysate was not stimulated by eIF4F or by any of the other components tested, supporting the hypothesis that a block in the translational machinery is removed at fertilization. A rabbit reticulocyte shift assay was used to analyze whether mRNA is limiting in early development. When unfertilized egg lysate was added to the shift assay, there was no shift in radioactivity from 43S to 80S complexes, indicating the unfertilized egg mRNA is not available for translation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Translational control of hippocampal synaptic plasticity and memory by the eIF2alpha kinase GCN2.

Studies on various forms of synaptic plasticity have shown a link between messenger RNA translation, learning and memory. Like memory, synaptic plasticity includes an early phase that depends on modification of pre-existing proteins, and a late phase that requires transcription and synthesis of new proteins. Activation of postsynaptic targets seems to trigger the transcription of plasticity-related genes. The new mRNAs are either translated in the soma or transported to synapses before translation. GCN2, a key protein kinase, regulates the initiation of translation. Here we report a unique feature of hippocampal slices from GCN2(-/-) mice: in CA1, a single 100-Hz train induces a strong and sustained long-term potentiation (late LTP or L-LTP), which is dependent on transcription and translation. In contrast, stimulation that elicits L-LTP in wild-type slices, such as four 100-Hz trains or forskolin, fails to evoke L-LTP in GCN2(-/-) slices. This aberrant synaptic plasticity is mirrored in the behaviour of GCN2(-/-) mice in the Morris water maze: after weak training, their spatial memory is enhanced, but it is impaired after more intense training. Activated GCN2 stimulates mRNA translation of ATF4, an antagonist of cyclic-AMP-response-element-binding protein (CREB). Thus, in the hippocampus of GCN2(-/-) mice, the expression of ATF4 is reduced and CREB activity is increased. Our study provides genetic, physiological, behavioural and molecular evidence that GCN2 regulates synaptic plasticity, as well as learning and memory, through modulation of the ATF4/CREB pathway.

Animals↗
PubMed1996

The upstream open reading frame of the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive translational control element.

S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the pathway of polyamine biosynthesis. The cellular levels of the polyamines specifically regulate AdoMetDC translation through the 5'-leader of the mRNA, which contains a small upstream open reading frame (uORF) 14 nucleotides from the cap. Mutating the initiation codon of the uORF, which encodes a peptide product with the sequence MAGDIS, abolished regulation. In addition, the uORF is sufficient, by itself, to provide polyamine regulation when inserted into the 5'-leader of the human growth hormone mRNA. Changing the amino acid sequence at the carboxyl terminus of the peptide product of the uORF abolished polyamine regulation. In contrast, altering the nucleotide sequence of the uORF at degenerate positions, without changing the amino acid sequence of the peptide, did not affect regulation. Extending the distance between cap and uORF, thereby changing the rate of initiation at the initiator AUG of the uORF, did not alter polyamine regulation. When the uORF was extended so as to overlap, out of frame, the downstream major cistron, polyamine regulation was abolished. We propose that polyamines do not modulate the rate of recognition of the uORF but rather regulate interaction of the peptide product of the uORF with its target.

Adenosylmethionine Decarboxylase↗
PubMed1996

The "+70 pause": hypothesis of a translational control of membrane protein assembly.

Sequences of 66 genes encoding bacterial or yeast membrane proteins have been examined for the respective positioning of putative transmembrane domains and translational pauses. The latter were operationally defined as clusters of at least 17 non-preferred codons along the mRNA. The putative transmembrane domains were defined as stretches of at least 17 hydrophobic amino acids in the encoded protein. For yeast non-mitochondrial membrane proteins, it was observed that clusters of non-preferred codons occur more frequently about 56 to 75 codons after a hydrophobic stretch in the encoded protein. About 40 amino acid residues are required to span the large ribosomal subunit. Such clusters were thus predicted to cause a severe slow-down in peptide elongation, just when the hydrophobic stretch fully protrudes from the ribosome. This transient slow-down of the ribosome pace has consequently been named the "+70 pause". This pause was not observed for mitochondrial or bacterial membrane proteins, which are thought to insert post-translationally in their respective membranes. Because insertion of yeast proteins in the endoplasmic reticulum membrane is generally cotranslational instead, it is possible that the "+70 pause" reflects the coupling of translation, targeting, insertion and folding in this case. The pause may, for instance, give time for productive interaction of the newly synthesized hydrophobic domain with the proper targeting/insertion machineries. Thus, it would favor entrance of the stalled protein domain into the proper pathway.

Codon↗
PubMed1985

Gene expression in the polycistronic operons of Escherichia coli heat-labile toxin and cholera toxin: a new model of translational control.

A new model is proposed based on the suggestion that stable local secondary structures of mRNA interfere with ribosome movement on mRNA and consequently reduce the translation rate. This model accounts for a different level of translation for each cistron in the polycistronic mRNA of Escherichia coli heat-labile toxin (LT) and cholera toxin. We also conclude that the mRNA secondary structures have been conserved during the evolution of the toxin genes for its functional importance.

Bacterial Toxins↗
PubMed1974

Translational control of hemoglobin synthesis in thalassemic bone marrow.

Previous studies of beta-thalassemic reticulocytes have implied a decreased amount of functional beta-mRNA but unimpaired translation of the beta-mRNA present. However, the beta/alpha synthetic ratios in beta-thalassemic marrow are higher than those observed in reticulocytes of the same patients. This could imply that marrow cells contain an abnormally functioning beta-mRNA no longer active in reticulocytes. To test the function of mRNA found in marrow, intact cells were incubated with [(35)S]methionine and the relative amounts of nascent alpha- and beta-chains on polysomes of different sizes were measured by tryptic digestion and determination of the specific activities of the respective peptides. Results showed that in normal and beta-thalassemic marrow, as well as in reticulocytes, beta-chain production, though deficient, occurs predominantly on larger polysomes than the production of alpha-chains. In one patient with severe thalassemia and very little production of beta-chains in marrow or reticulocytes, delta-chain synthesis was found predominantly on larger polysomes than alpha-chain synthesis. These results indicate that in beta-thalassemic as well as in nonthalassemic marrow and reticulocytes, each beta- and delta-mRNA initiates protein synthesis at a rate faster than does each alpha-mRNA, and suggest that the beta-mRNA in contact with polyribosomes is normally functioning but quantitatively deficient in beta-thalassemic marrow as well as in reticulocytes. No translational defect was detected in a similar study performed in reticulocytes of a patient with hemoglobin H disease, suggesting a normally functioning mRNA in contact with polyribosomes in this condition as well. In both thalassemias, unbalanced synthesis of alpha- and beta-chains was more pronounced on polysomes than in completed chains. This difference possibly reflects a compensatory delay in translation of the nonthalassemic chain, which is present in excess.

Bone Marrow↗
PubMedDate not supplied

[Translational control by the poly(A) binding protein: a check for mRNA integrity].

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the capbinding protein eIF4E and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this "closed loop" mRNP among other effects enhance the affinity of eIF4E for the 5' cap by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picomavirus' internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to initiation complex formation. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP.

Animals↗
PubMed1995

Mechanism of post-segregational killing by hok-homologue pnd of plasmid R483: two translational control elements in the pnd mRNA.

The pnd system of plasmid R483 mediates plasmid stabilization by killing of plasmid-free cells. The pnd mRNA is very stable and can be translated into PndA protein, a cell toxin which kills the cells from within by damaging the cell membrane. Translation of the pnd mRNA is inhibited by the PndB antisense, a small labile RNA of 63 nt. The rapid decay of the PndB antidote leads to onset of PndA synthesis in plasmid-free segregants or after addition of rifampicin. Surprisingly however, the full-length pnd mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was found to be active. We have therefore suggested previously, that the 3'-end of the full-length pnd mRNA encodes a fold-back inhibitory sequence (fbi), which prevents its translation. Here we present an analysis of the metabolism of the pnd mRNAs. A mutational analysis shows that single point mutations in the fbi motif results in more rapid truncation. The fbi mutations could not be complemented by second-site mutations in either of the pndA or pndC Shine-Dalgarno (SD) elements. Surprisingly, mutations in the pndC SD element also lead to a more rapid truncation. The effect of these latter mutations was, however, complemented by mutations in a proposed anti-SD element upstream of the pndC SD. Mutations in the anti-SD element were lethal. These results show, that the pnd mRNA contains two negative control elements, one located in its very 3'-end (fbi), and one located just upstream of the pndC SD region (the anti-SD element). These observations add to the complexity of the induction scheme previously proposed to explain activation of pndA expression in plasmid-free cells: In addition to its negative effect of translation, the fbi structure also maintains a reduced processing rate in the 3'-end of the mRNA. This permits the accumulation of a reservoir of pnd mRNA, which can be activated by 3'-end processing in plasmid-free cells. The anti-SD may prevent translation of the pnd mRNA during transcription, thus preventing detrimental synthesis of toxin.

Bacterial Proteins↗
PubMed1982

Yeast use translational control to compensate for extra copies of a ribosomal protein gene.

The efficient assembly of ribosomes requires a balanced synthesis of ribosomal RNA and each ribosomal protein. In an attempt to establish the mechanisms responsible for such balanced synthesis we have altered the gene dosage for one of the components by introducing into yeast an autonomously replicating plasmid containing the gene tcm1, which codes for ribosomal protein L3. The plasmid is maintained at 5-10 copies per cell by selection for expression of its URA3 gene. The plasmid-containing cells transcribe 7.5 times as much L3 mRNA as control cells, maintain 3.5 times as much L3 mRNA as control cells and synthesize no more than 1.2 times as much L3 protein as control cells. We conclude that the balanced synthesis of ribosomal proteins is maintained by modulating both the efficiency of translation and the lifetime of their mRNAs.

DNA, Recombinant↗
PubMed2006

Signaling pathways and molecular mechanisms through which branched-chain amino acids mediate translational control of protein synthesis.

BCAAs stimulate protein synthesis in in vitro preparations of skeletal muscle. Likewise, the stimulation of protein synthesis in skeletal muscle produced by intake of a mixed meal is due largely to BCAAs. Of the three BCAAs, leucine is the one primarily responsible for the stimulation of protein synthesis under these circumstances. The stimulatory effect of leucine on protein synthesis is mediated through upregulation of the initiation of mRNA translation. A number of mechanisms, including phosphorylation of ribosomal protein S6 Kinase, eukaryotic initiation factor (eIF)4E binding protein-1, and eIF4G, contribute to the effect of leucine on translation initiation. These mechanisms not only promote global translation of mRNA but also contribute to processes that mediate discrimination in the selection of mRNA for translation. A key component in a signaling pathway controlling these phosphorylation-induced mechanisms is the protein kinase, termed the mammalian target of rapamycin (mTOR). The activity of mTOR toward downstream targets is controlled in part through its interaction with the regulatory-associated protein of mTOR (known as raptor) and the G protein beta-subunit-like protein. Signaling through mTOR is also controlled by upstream members of the pathway such as the Ras homolog enriched in brain (Rheb), a GTPase that activates mTOR, and tuberin (also known as TSC2), a GTPase-activating protein, which, with its binding partner hamartin (also known as TSC1), acts to repress mTOR. Candidates for mediating the action of leucine to stimulate signaling through the mTOR pathway include TSC2, Rheb, and raptor. The current state of our understanding of how leucine acts on these signaling pathways and molecular mechanisms to stimulate protein synthesis in skeletal muscle is summarized in this article.

Animals↗
PubMed2003

Conditional gene expression by controlling translation with tetracycline-binding aptamers.

We present a conditional gene expression system in Saccharomyces cerevisiae which exploits direct RNA-metabolite interactions as a mechanism of genetic control. We inserted preselected tetracycline (tc) binding aptamers into the 5'-UTR of a GFP encoding mRNA. While aptamer insertion generally reduces GFP expression, one group of aptamers displayed an additional, up to 6-fold, decrease in fluorescence upon tc addition. Regulation is observed for aptamers inserted cap-proximal or near the start codon, but is more pronounced from the latter position. Increasing the thermodynamic stability of the aptamer augments regulation but reduces expression of GFP. Decreasing the stability leads to the opposite effect. We defined nucleotides which influence the regulatory properties of the aptamer. Exchanging a nucleotide probably involved in tc binding only influences regulation, while mutations at another position alter expression in the absence of tc, without affecting regulation. Thus, we have developed and characterized a regulatory system which is easy to establish and controlled by a non-toxic, small ligand with good cell permeability.

5' Untranslated Regions↗
PubMed2004

Loss of translational control in yeast compromised for the major mRNA decay pathway.

The cytoplasmic fate of mRNAs is dictated by the relative activities of the intimately connected mRNA decay and translation initiation pathways. In this study, we have found that yeast strains compromised for stages downstream of deadenylation in the major mRNA decay pathway are incapable of inhibiting global translation initiation in response to stress. In the past, the paradigm of the eIF2alpha kinase-dependent amino acid starvation pathway in yeast has been used to evaluate this highly conserved stress response in all eukaryotic cells. Using a similar approach we have found that even though the mRNA decay mutants maintain high levels of general translation, they exhibit many of the hallmarks of amino acid starvation, including increased eIF2alpha phosphorylation and activated GCN4 mRNA translation. Therefore, these mutants appear translationally oblivious to decreased ternary complex abundance, and we propose that this is due to higher rates of mRNA recruitment to the 40S ribosomal subunit.

Amino Acids↗
PubMed2003

Mechanisms of translational control in dendrites.

Synaptogenesis and synaptic plasticity demonstrate the ability of neurons to mature and respond to various stimuli. Both of these events require protein synthesis, in particular, localized translation within dendrites. Dendrites localize specific mRNAs in proximity to dendritic spines and have the capacity to actively translate these mRNAs within various 'hotspots' in the dendritic space. Rates of dendritic translation are stimulated and inhibited by various agents, suggesting that several signaling pathways that modulate localized protein synthesis may exist within the dendrite. This review will cover several suggested pathways for regulation of dendritic translation and propose a correlation between deregulated dendritic translation and disease.

Animals↗
PubMed1989

Protamine 3'-untranslated sequences regulate temporal translational control and subcellular localization of growth hormone in spermatids of transgenic mice.

Although the mouse protamine 1 gene (mP1) is first transcribed in round spermatids, its mRNA is not translated until about 1 week later in elongating spermatids. To determine what mP1 sequences are important for its transcriptional and translational regulation, we have constructed fusions between mP1 and the human growth hormone (hGH) structural gene and analyzed their expression in transgenic mice. We show that mP1 sequences 5' to the start of transcription are sufficient to confer spermatid-specific expression on the hGH gene. We also show that 156 nucleotides of mP1 3'-untranslated sequence is sufficient to confer mP1-like translational regulation on the hGH mRNA. Interestingly, the subcellular localization of hGH was dependent on the time during spermiogenesis that it was made. Synthesis of hGH in early round spermatids resulted in localization in the acrosome, whereas synthesis in late elongating spermatids resulted in intracellular, but not acrosomal, localization.

Acrosome↗