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PubMed2003

Tissue expression and translational control of rat kynurenine aminotransferase/glutamine transaminase K mRNAs.

Kynurenic acid (KA) is an endogenous glutamate receptor antagonist at the level of the different ionotropic glutamate receptors. One of the enzymes responsible for the production of KA, kynurenine aminotransferase I (KATI), also catalyses the reversible transamination of glutamine to oxoglutaramic acid (GTK, EC 2.6.1.15). The enzyme exists in a cytosolic and in a mitochondrial form because of the presence of two different KATI mRNAs coding for a protein respectively with and without leader sequence targeting the protein into mitochondria. We have cloned from a phage library of rat kidney cDNA four new KATI cDNAs containing different 5' untranslated regions (UTRs). One of the transcripts (+14KATI cDNA) contains an alternative site of initiation of translation. The tissue distribution of the different transcripts was studied by RT-PCR. The study demonstrated that several KATI mRNAs are constitutively expressed in ubiquitous manner, while +14KATI mRNA is present only in kidney. The translational efficiency of the different transcripts was studied in vitro and enzymatic activities were measured in transiently transfected Cos-1 cells. Each KATI mRNA exhibits a different in vitro translational efficiency, which corresponds to different levels of KAT enzymatic activity in transfected cells. Both findings correlate with the predicted accessibility of the ribosomal binding sites of the different mRNAs. The structure of the rat KATI/GTK gene was also studied. The expression of several KATI mRNAs with different 5'UTRs represents an interesting example of transcriptional/translational control on the expression of pyridoxal phosphate (PLP)-dependent aminotransferases.

5' Untranslated Regions↗
PubMed2006

Translational control of gene expression during hypoxia.

Poor oxygenation is a unique and prevalent feature of solid tumors associated with poor patient prognosis. In part, this is caused by a series of adaptive cellular responses that together have a large impact on gene expression and cell phenotype. HIF plays a key role in this response by activating a transcriptional program that stimulates genes involved in angiogenesis, cell metabolism, cell survival and cell invasion. Recently, hypoxia has also been shown to suppress protein synthesis through the regulation of the initiation step of mRNA translation. This appears to be a common feature of the cell in response to hypoxia and is mediated by two distinct pathways. The first occurs rapidly, is transient, and is associated with activation of the unfolded protein response (UPR) that occurs in response to endoplasmic reticulum (ER) stress. Translation inhibition during this initial phase is due to phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) in a PERK dependent manner. Although this effect is transient, overall levels of translation remain low during hypoxia due to inhibition of a second eukaryotic initiation complex, eIF4F. This second mechanism is multi-factorial, but due at least in part to inhibition of the mTOR kinase. Although each of these pathways leads to a general inhibition in translation, the consequence at the individual gene level is highly variable. This is due to sequences in the 5' and 3' untranslated regions (UTRs) of mRNA that confer their ability to maintain, or even increase, translation efficiency in spite of the overall inhibition. Consequently, regulation of mRNA translation appears to be an important mediator of gene expression during hypoxia.

Cell Hypoxia↗
PubMed2004

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: II. Analysis of activating signal transduction pathways, transcriptional, post-transcriptional, and post-translational control.

CCAAT/enhancer binding protein delta (C/EBPdelta) plays a key role in mammary epithelial cell G0 growth arrest. C/EBPdelta gene expression is down-regulated in rodent mammary tumorigenesis and in human breast cancer, suggesting that "loss of function" alterations in C/EBPdelta gene expression are common in mammary gland malignancies. The goal of this study was to systematically investigate the mechanisms controlling C/EBPdelta gene expression in MCF-10A and MCF-12A human mammary epithelial cell lines. The results demonstrate that G0 growth arrest conditions (i.e., serum and growth factor withdrawal or Oncostatin M (OSM) treatment) result in the activation of JAK1, JAK2, and Tyk 2, members of the Janus kinase family of non-receptor tyrosine kinases, in MCF-10A and MCF-12A cells. Growth arrest or OSM treatment also specifically increases activated (phosphorylated) signal transduction and activators of transcription 3 (STAT3) levels, demonstrating that STAT3, not STAT1 or STAT5, is the downstream target of the activated Janus kinases in MCF-10A and MCF-12A cells. Whole cell lysates from G0 growth arrested (GA) and OSM-treated MCF-12A cells exhibit increased acute phase response element (APRE) binding compared to lysates from growing (GR) MCF-12A cells. Transient transfection using C/EBPdelta promoter-luciferase constructs demonstrated that the APRE (STAT3) consensus binding site is essential for growth arrest or OSM induction of the C/EBPdelta promoter. Mutation of the C/EBPdelta promoter STAT3 site or expression of a dominant negative STAT3 construct (STAT3delta) reduces C/EBPdelta promoter activity in response to growth arrest conditions. The human C/EBPdelta promoter also contains an Sp1 site at -61 bp (relative to the transcriptional start site) which is required for basal transcriptional activation. Mutation or deletion of the Sp1 site decreases promoter activity in response to growth arrest conditions. Treatment with the transcriptional inhibitor actinomycin D demonstrated that the C/EBPdelta mRNA exhibits a relatively short half-life (approximately 40 min). Similarly, treatment with the translational inhibitor anisomysin demonstrated that the C/EBPdelta protein half-life was also relatively short (approximately 160 min). These results indicate that the human C/EBPdelta gene is controlled at multiple levels, consistent with a role for C/EBPdelta in cell cycle control and/or cell fate determination.

Animals↗
PubMed1994

Translational control in production of transposase and in transposition of insertion sequence IS3.

IS3 (1258 bp in length) contains two open reading frames, orfA and orfB, which are out of phase and overlap each other. We show here that three proteins of 10, 32 and 42 kDa in size are encoded by IS3. The 10 kDa protein is the product of orfA and is here called OrfA. The ATG codon of orfB which overlaps the termination codon of orfA is utilized to produce the 32 kDa protein (here called OrfB), in a manner depending on translation of orfA. The 42 kDa protein is a transframe protein (here called OrfB), which is synthesized from orfA and orfB by -1 translational frameshifting at the A4G motif present in the overlapping region. Both the frameshifting event to produce OrfB and the coupled translation event to produce OrfB are greatly stimulated by a pseudo knot structure located in the overlapping region between orfA and orfB. A mutant IS3 with a single base insertion in the A4G motif efficiently produces the OrfB transframe protein without frameshifting. This mutant was found not to mediate co-integration but to mediate adjacent deletion to produce various miniplasmids and minicircles in large amounts. The OrfB transframe protein is necessary and sufficient for formation of these deletion products, implying that it is the transposase. Most of the minicircles consisted solely of the entire IS3 sequence and a three base-pair sequence between the IS3 ends. The significance of minicircle formation is discussed.

Amino Acid Sequence↗
PubMed2006

Post-translational control of Nur77.

Nur77 is a nuclear orphan receptor that has been implicated in both cell survival and apoptosis. With the exception of T-cells, translocation of Nur77 to the cytoplasm promotes cell death, while its retention in the nucleus promotes survival and proliferation. Nur77 appears to be a true orphan receptor, indicating that its activity must be controlled by ligand-independent mechanisms. Here, we discuss the role of phosphorylation in the regulation of Nur77.

DNA-Binding Proteins↗
PubMed2001

Multigene lentiviral vectors based on differential splicing and translational control.

Lentiviral vectors, so far, have been optimized for the expression of a single open reading frame. Certain practical applications of gene therapy will, however, require expression of multiple genes. The goal of this study was to explore the feasibility of directing expression of two marker genes from a lentiviral vector. We designed two types of multigene lentiviral vectors. First, we used a strategy based on the natural splicing signals of HIV-1, by which multiple mRNAs are generated from a single transcriptional unit. A second strategy was construction of a polycistronic mRNA using a translational cis-acting element, the encephalomyocarditis virus internal ribosome entry site (IRES). Our studies show that the inclusion of multiple genes in lentiviral vectors does not result in reduction in virus titers or in the loss of ability to infect nondividing cells. We introduced mutations in tat and/or rev to test whether splicing modulates the relative levels of expression of reporter genes. We also developed a truncated version of tat, which is devoid of the apoptosis-associated domain. Inclusion of this tat mutant in a lentiviral vector resulted in the generation of virus with titers similar to those of lentivirus vectors expressing wild-type tat.

Alternative Splicing↗
PubMed1980

Transcriptional and translational control of glucose-stimulated (pro)insulin biosynthesis.

Islets of Langerhans were isolated from the pancreata of fed or 48-h-fasted Wistar rats. The islets were incubated with either [3H]leucine of [3H]uridine. Inhibition of RNA synthesis by actinomycin D or by alpha-amanitin for 4 h had no influence on the (pro)insulin biosynthesis of isolated islets of fed rats. The (pro)insulin biosynthesis was not inhibited after two days incubation of islets of fed rats with alpha-amanitin either. Incorporation of labelled uridine into total RNA for 3 h was stimulated by glucose in islets of fasted, but not of fed rats. Therefore, it was concluded that transcriptional control does not participate, even for longer periods than believed previously, in acute regulation of (pro)insulin biosynthesis of islets isolated fed rats. Despite the strong and preferential stimulation of (pro)insulin biosynthesis of islets of fed rats by glucose the radioactivity of the [3H]uridine-labelled polysomes active in proinsulin synthesis remained unchanged. To interprete these experimental data we suggest that glucose triggers the transformation of a translationally inactive form of pre-proinsulin mRNA to a translationally active form.

Amanitins↗
PubMed1984

Post-translational control by carrier availability of amino acid transport in fetal human fibroblasts.

The action of serum on the expression of the starvation-enhanced amino acid transport by System A (as a part of the adaptive regulation mechanism) has been studied in cultured fetal human fibroblasts. Serum enhanced L-proline uptake of cells starved in serum-free medium. This effect was rapid, proportional to the amount of pre-existing transporters, insensitive to cycloheximide and kinetically characterized by an increase of transport Vmax. These results can be interpreted to indicate that serum is essential for a vectorial post-translational event leading to insertion of transport proteins into the cell membrane.

Amino Acids↗
PubMed1995

Translational control of oskar generates short OSK, the isoform that induces pole plasma assembly.

At the posterior pole of the Drosophila oocyte, oskar induces a tightly localized assembly of pole plasm. This spatial restriction of oskar activity has been thought to be achieved by the localization of oskar mRNA, since mislocalization of the RNA to the anterior induces anterior pole plasm. However, ectopic pole plasm does not form in mutant ovaries where oskar mRNA is not localized, suggesting that the unlocalized mRNA is inactive. As a first step towards understanding how oskar activity is restricted to the posterior pole, we analyzed oskar translation in wild type and mutants. We show that the targeting of oskar activity to the posterior pole involves two steps of spatial restriction, cytoskeleton-dependent localization of the mRNA and localization-dependent translation. Furthermore, our experiments demonstrate that two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full oskar activity. Finally, we show that when oskar RNA is localized, accumulation of Oskar protein requires the functions of vasa and tudor, as well as oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by oskar.

Animals↗
PubMed2004

Defective translational control facilitates vesicular stomatitis virus oncolysis.

Vesicular stomatitis virus (VSV) exerts potent antitumor activity, although the molecular mechanisms underlying its oncolytic properties remain to be fully clarified. Here, we demonstrate that normally resistant murine embryonic fibroblasts are rendered highly permissive to VSV replication following cellular transformation, a progression that appears to compromise the antiviral effects of interferon (IFN). Subsequent studies revealed normal dsRNA-dependent protein kinase (PKR) activation and phosphorylation of eukaryotic initiation factor 2 (eIF2) alpha. Nevertheless, eIF2B-mediated guanine nucleotide exchange activity downstream of eIF2 was frequently aberrant in transformed cells, neutralizing eIF2alpha phosphorylation and permitting VSV mRNA translation. Thus, defects in translational regulation can cooperate with impaired IFN signaling to facilitate VSV replication, and may represent a common hallmark of tumorigenesis.

Animals↗
PubMed2006

Transcriptional and translational control of the Salmonella fliC gene.

The flagellin gene fliC encodes the major component of the flagellum in Salmonella enterica serovar Typhimurium. This study reports the identification of a signal within the 5' untranslated region (5'UTR) of the fliC transcript required for the efficient expression and assembly of FliC into the growing flagellar structure. Primer extension mapping determined the transcription start site of the fliC flagellin gene to be 62 bases upstream of the AUG start codon. Using tetA-fliC operon fusions, we show that the entire 62-base 5'UTR region of fliC was required for sufficient fliC mRNA translation to allow normal FliC flagellin assembly, suggesting that translation might be coupled to assembly. To identify sequence that might couple fliC mRNA translation to FliC secretion, the 5' end of the chromosomal fliC gene was mutagenized by PCR-directed mutagenesis. Single base sequences important for fliC-dependent transcription, translation, and motility were identified by using fliC-lacZ transcriptional and translational reporter constructs. Transcription-specific mutants identified the -10 and -35 regions of the consensus flagellar class 3 gene promoter. Single base changes defective in translation were located in three regions: the AUG start codon, the presumed ribosomal binding site region, and a region near the very 5' end of the fliC mRNA that corresponded to a potential stem-loop structure in the 5'UTR. Motility-specific mutants resulted from base substitutions only in the fliC-coding region. The results suggest that fliC mRNA translation is not coupled to FliC secretion by the flagellar type III secretion system.

5' Untranslated Regions↗
PubMed1988

Translational control in heat-shocked Drosophila embryos. Evidence for the inactivation of initiation factor(s) involved in the recognition of mRNA cap structure.

Lysates from normally growing (25 degrees C) or heat shocked (37 degrees C, 45 min) Drosophila melanogaster embryos were obtained and the effect of analogues of the mRNA 5'-terminal cap, m7G(5')ppp(5')N structure and of potassium ions on their endogenous protein synthesis activity was studied. At optimal concentration of KCH3COO (75-80 mM), protein synthesis in normal lysates is strongly inhibited by cap analogues (m7GpppG, m7GDP, and m7GMP). At the same ionic conditions, heat shock lysates translate preferentially the heat shock messengers, and this translation is almost unaffected by the cap analogues. In contrast, residual synthesis of normal proteins in heat shock lysates was reduced by these compounds. By lowering the concentration of potassium ions it was possible to gradually reverse the inhibitory effect of the cap analogues in normal lysates and also to increase specifically the translation of normal mRNAs in heat shock lysates. Translation of normal mRNAs is also partial but specifically rescued by supplementing heat shock lysates with polypeptide chain initiation factors partially purified from rabbit reticulocytes. These data are consistent with the notion that the failure of normal mRNAs to be translated under heat shock conditions might be due, at least to some extent, to the inactivation of polypeptide chain initiation factor(s) involved in the recognition of the mRNA 5'-terminal cap structure.

Animals↗
PubMed2004

Change in protein phenotype without a nucleus: translational control in platelets.

For most cells the nucleus takes center stage. Not only is it the largest organelle in eukaryotic cells, it carries most of the genome and transcription of DNA to RNA largely takes place in the nucleus. Because transcription is a major step in gene regulation, the absence of a nucleus is limiting from a biosynthetic standpoint. Consequently, the anucleate status of platelets has stereotyped it as a cell without synthetic potential. It is now clear, however, that this viewpoint is far too simplistic. In response to physiologic stimuli, platelets synthesize biologically relevant proteins that are regulated via gene expression programs at the translational level. This process does not require a nucleus; instead, it uses mRNAs and other translational factors that appear to be retained in specialized fashion as megakaryocytes generate platelets during thrombopoiesis. We highlight the molecular machinery and pathways used by platelets to translate mRNA into protein and offer insight into how these synthesized products may regulate thrombotic and inflammatory events.

Animals↗
PubMed1986

Compensatory increase in levels of beta minor globin in murine beta-thalassemia is under translational control.

A 3.7-kilobase pair deletion including the entire beta major globin gene results in beta-thalassemia in a murine model of the disease (Skow, L. C., Burkhart, B. A., Johnson, F. M., Popp, R. A., Popp, D. M., Goldberg, S. Z., Anderson, W. F., Barnett, L. B., and Lewis, S. E. (1983) Cell 34, 1043-1052). There is a compensatory increase in synthesis of beta minor globin, resulting in a beta minor/alpha globin ratio of 0.75 in the homozygous thalassemic mouse, as compared to 0.2 in the normal homozygous diffuse mouse. The results presented here demonstrate that the increase in beta minor globin synthesis occurs at translation rather than at transcription. RNase T1 analysis of reticulocyte mRNA reveals that the beta/alpha ratio of globin mRNA is 0.3, significantly lower than the globin synthetic ratio of 0.7. However, the beta/alpha ratio of mRNA on polysomes is higher than unassociated mRNA, demonstrating that beta minor mRNA is preferentially translated. Elevated synthesis of beta minor globin is maintained during in vitro translation in thalassemic reticulocyte lysate. In this system, partial inhibition of translational elongation by cycloheximide decreases the beta minor/alpha globin synthetic ratio, whereas partial inhibition of initiation by hemin deficiency increases the beta minor/alpha synthetic ratio. This suggests that beta minor mRNA competes with alpha-mRNA for a limiting mRNA binding factor at initiation of translation.

Animals↗
PubMed2003

Transcriptional and translational control of Mcl-1 during apoptosis.

Mcl-1 is an antiapoptotic member of the Bcl-2 family whose protein and mRNA have a short half-life. In this report, we studied the changes in Mcl-1 protein and mRNA expression induced by staurosporine and aspirin. Both drugs induced apoptosis in Jurkat cells and reduced the levels of Mcl-1 protein. The caspase inhibitor Z-VAD.fmk and the proteasome inhibitor MG132 partially protected Mcl-1 from decay, indicating that both caspase-dependent and proteasome pathways are involved during apoptosis. Staurosporine also reduced Mcl-1 mRNA levels and this reduction was mostly caspase-dependent. In addition, staurosporine reduced the transcriptional activity of the Mcl-1 promoter fused to a luciferase gene reporter more than actinomycin D, a general inhibitor of transcription. Thus, we conclude that staurosporine down-regulates Mcl-1 mRNA levels by inhibiting transcription in a caspase-dependent manner and reduces Mcl-1 protein levels by a caspase-independent post-transcriptional mechanism. In contrast aspirin, at doses and times that induced loss of viability and decay of Mcl-1 protein, had no effect on Mcl-1 mRNA levels. Aspirin rapidly inhibited de novo protein synthesis before caspase activation. Moreover, the translational factor eIF2alpha was transiently phosphorylated and therefore inhibited very soon after aspirin treatment. Aspirin also inhibited the luciferase reporter activity of several attached promoter constructs, but it did not affect the luciferase activity of a construct containing an internal ribosome entry site (IRES) in its mRNA 5(')UTR. We conclude that staurosporine inhibits transcription and translation, whereas aspirin only inhibits cap-dependent translation. Treatment with cycloheximide, at doses that inhibit protein synthesis without affecting cell viability, also induced Mcl-1 protein decay. Mcl-1 disappearance might be necessary but not sufficient for the induction of apoptosis by staurosporine and aspirin. A model for the control of Mcl-1 during drug-induced apoptosis is presented.

Apoptosis↗
PubMed1987

Translational control of alpha-amylase gene expression in Aspergillus awamori.

Regulation of alpha-amylase gene expression in Aspergillus awamori was studied by analyzing the enzyme activity levels, rate of protein synthesis, and alpha-amylase-specific mRNA levels under various conditions of growth. alpha-Amylase synthesis was sensitive to catabolite repression as glucose repressed its synthesis by about fourfold. The stimulation of alpha-amylase synthesis in the presence of its substrate starch was shown to be due to derepression rather than induction as the enzyme was synthesized at similar rates in both starch and starvation media. Repression and derepression of enzyme synthesis was found to be mediated at the translational level. The cellular levels of alpha-amylase-specific mRNA as measured by an in vitro translation assay system, were almost identical under all conditions of enzyme synthesis. Relative in vivo and in vitro alpha-amylase mRNA template activities suggest that alpha-amylase mRNA is translated much more efficiently during the derepression than under the conditions of repressed synthesis.

Aspergillus↗
PubMed2003

Transcriptional silencing and translational control: key features of early germline development.

The germ lineage has been studied for a long time because of its crucial role in the propagation and survival of a species. While this lineage, in contrast to the soma, is clearly unique in its totipotent ability to produce a new organism, it has now been found also to have specific features at the cellular level. One feature, a period of transcriptional quiescence in the early germ cell precursors, has been observed in both Drosophila and C. elegans, where it is essential for the formation and the survival of the germline. In addition, there are numerous instances where these early germ cells are reliant on translational regulation, especially in Drosophila. The genes that are important for these two functions, the mechanisms of their action, and studies in vertebrate organisms that reveal similarities as well as some potential differences in early germ cell development are discussed.

Animals↗