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At least 919 records · Page 51Linked to original sources
PubMed2003

Cleavage of the A site mRNA codon during ribosome pausing provides a mechanism for translational quality control.

Cells employ many mechanisms to ensure quality control during protein biosynthesis. Here, we show that, during the pausing of a bacterial ribosome, the mRNA being translated is cleaved at a site within or immediately adjacent to the A site codon. The extent of this A site mRNA cleavage is correlated with the extent of ribosome pausing as assayed by tmRNA-mediated tagging of the nascent polypeptide. Cleavage does not require tmRNA, the ribosomal alarmone (p)ppGpp, or bacterial toxins such as RelE which have been shown to stimulate a similar activity. Translation is required for cleavage, suggesting that the ribosome participates in the reaction in some fashion. When normal protein synthesis is compromised, A site mRNA cleavage and the tmRNA system provide a mechanism for reducing translational errors and the production of aberrant and potentially harmful polypeptides.

Bacterial Toxins↗
PubMed2005

Transforming growth factor-beta induces airway smooth muscle hypertrophy.

Although smooth muscle hypertrophy is present in asthmatic airways, little is known about the biochemical pathways regulating airway smooth muscle protein synthesis, cell size, or accumulation of contractile apparatus proteins. We sought to develop a model of airway smooth muscle hypertrophy in primary cells using a physiologically relevant stimulus. We hypothesized that transforming growth factor (TGF)-beta induces hypertrophy in primary bronchial smooth muscle cells. Primary human bronchial smooth muscle cells isolated from unacceptable lung donor tissue were studied. Cells were seeded on uncoated plastic dishes at 50% confluence and TGF-beta was added. Experiments were performed in the absence of serum. TGF-beta increased cell size and total protein synthesis, expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain, formation of actomyosin filaments, and cell shortening to acetylcholine. Further, TGF-beta increased airway smooth muscle alpha-actin synthesis in the presence of the transcriptional inhibitor actinomycin D, evidence that translational control is a physiologically important element of the observed hypertrophy. TGF-beta induced the phosphorylation of eukaryotic translation initiation factor-4E-binding protein, a signaling event specifically involved in translational control. Finally, two inhibitors of 4E-binding protein phosphorylation, the phosphoinositol 3-kinase inhibitor LY294002 and a phosphorylation site mutant of 4E-binding protein-1 that dominantly inhibits eukaryotic initiation factor-4E, each blocked TGF-beta-induced alpha-actin expression and cell enlargement. We conclude that TGF-beta induces hypertrophy of primary bronchial smooth muscle cells. Further, phosphorylation of 4E-binding protein is required for the observed hypertrophy.

Acetylcholine↗
PubMed2000

A conserved mechanism for controlling the translation of beta-F1-ATPase mRNA between the fetal liver and cancer cells.

To characterize the mechanisms governing the biogenesis of mitochondria in cancer, we studied the mitochondrial phenotype and the mechanisms controlling the expression of the beta subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) gene in the rat FAO and AS30D hepatomas. When compared with normal adult rat liver, the relative cellular content of the mitochondrial beta-F1-ATPase and glutamate dehydrogenase, as well as of mitochondrial DNA, was severely reduced in both cell lines. A paradoxical increase in the cellular abundance of beta-F1-ATPase mRNA was observed in cancer cells. Run-on transcription assays and the estimation of mRNA half-lives revealed that the increased abundance of beta-F1-ATPase mRNA results from the stabilization of the transcript in cancer. In vitro translation assays revealed a specific inhibition of the synthesis of the beta-precursor when translation reactions were carried out in the presence of extracts derived from cancer cells. The inhibitory effect was recapitulated using an RNA chimera that contained the 3'-untranslated region of beta-F1-ATPase mRNA. Hepatoma extracts also contained an increased activity of the developmentally regulated translation-inhibitory proteins that bind the 3'-untranslated region of beta-F1-ATPase mRNA. The results indicate that the expression of this gene in hepatoma cells is controlled by the same mechanisms that regulate its expression in the liver during fetal development.

3' Untranslated Regions↗
PubMed1992

Inducible expression bovine papillomavirus shuttle vectors containing ferritin translational regulatory elements.

The combination of transcriptional and translational control elements in an inducible expression vector suitable for use in stably transformed cell lines was explored. To this end, ferritin translational control elements have been inserted downstream from a mouse metallothionein (mMT-I) transcriptional promoter (PmMT-I), and upstream from various reporter protein-encoding open reading frames (ORFs), all carried on a bovine papillomavirus shuttle vector. Protocols which stimulate transcription (with zinc) and translation (with iron) were developed to optimize the induction of reporter protein synthesis. It was found that insertion of an iron regulatory element between the PmMT-I and a reporter ORF bestowed a sixfold inducibility of reporter protein synthesis with iron and a 90-fold inducibility with iron plus zinc in a classical superinduction protocol. Surprisingly, inclusion of other rabbit ferritin light chain sequences (rFL), including the ORF, enhanced reporter inducibilities to over 15- and 500-fold, respectively. These additional rFL sequences not only increased inducibility but also (i) increased the half-life of the mRNA and (ii) strongly inhibited translation of an ORF located downstream from the 5' proximal ORF. The maximum levels of reporter proteins attained in transformed cells after prolonged induction represented from 1% to 7% of total cellular protein. These inducible expression vectors should prove useful for the production and study of cytotoxic proteins.

Animals↗
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↗
PubMed2002

Dendritic BC1 RNA: functional role in regulation of translation initiation.

In neurons, local protein synthesis in synaptodendritic microdomains has been implicated in the growth and plasticity of synapses. Prerequisites for local translation are the targeted transport of RNAs to distal sites of synthesis in dendrites and translational control mechanisms to limit synthesis to times of demand. Here we identify dendritic BC1 RNA as a specific repressor of translation. Experimental use of internal ribosome entry mechanisms and sucrose density gradient centrifugation showed that BC1-mediated repression targets translation at the level of initiation. Specifically, BC1 RNA inhibited formation of the 48S preinitiation complex, i.e., recruitment of the small ribosomal subunit to the messenger RNA (mRNA). However, 48S complex formation that is independent of the eukaryotic initiation factor 4 (eIF4) family of initiation factors was found to be refractory to inhibition by BC1 RNA, a result that implicates at least one of these factors in the BC1 repression pathway. Biochemical experiments indicated a specific interaction of BC1 RNA with eIF4A, an RNA unwinding factor, and with poly(A)-binding protein. Both proteins were found enriched in synaptodendritic microdomains. Significantly, BC1-mediated repression was shown to be effective not only in cap-dependent translation initiation but also in eIF4-dependent internal initiation. The results suggest a functional role of BC1 RNA as a mediator of translational control in local protein synthesis in nerve cells.

Animals↗
PubMed2006

The cap-dependent translation apparatus integrates and amplifies cancer pathways.

Deregulation of a plethora of cancer genes causes pathological changes in only a small set of pathways that confer a cell with malignant properties. This principle of convergence of oncogenic signaling-the ability of several hundred oncogenes to focus their effects on a few critical regulatory nodes that impart autonomy to the cell-motivates the search for putative focal points. Genomic, transcriptional and posttranslational mechanisms that regulate the function of cancer gene pathways are all well established. It has recently become evident that cancer is also subject to translational control. Here we discuss cancer-related regulatory events that are mediated by the cap-dependent mRNA binding stage of translation initiation. This information implicates the cap-dependent protein synthesis pathway as a pleotropic integrator and amplifier of many essential oncogenic signals, and the translational control network as a bona fide molecular target for anti-cancer therapeutic interventions.

Active Transport, Cell Nucleus↗
PubMed2006

Global alterations in mRNA polysomal recruitment in a cell model of colorectal cancer progression to metastasis.

Tumour onset and progression are due to the accumulation of genomic lesions, which alter gene expression and ultimately proteome activities. These lesions are thought to affect primarily the transcriptional control of gene expression. In the present study, we aimed at evaluating the genome-wide occurrence of alterations in the translational control exploiting an isogenic, phenotypically validated cellular model of colorectal cancer (CRC) transition from invasive carcinoma to metastasis. In this model, microarray profiling shows that changes in the level of messenger ribonucleic acid (mRNA) association with polysomes occur more than 2-fold than changes in the level of total cellular mRNA. When common to both the total and polysomal compartments, these changes are also homodirectional, being amplified in magnitude at the polysomal level. Comparison between the transcriptional and the translational fluctuations revealed distinct signatures of statistically over-represented gene functions, involving the program of cell proliferation for both levels of analysis, while the apoptosis and the translation programs were affected mainly at translation. Looking for an upstream determinant of translational deregulation, we found an increase in the hyperphosphorylated form of the 4E-BP1 protein in the metastatic cell line, possibly resulting in an increased activation of cap-dependent translation due to increased activity of the eIF4E protein. Analysis of the distribution profiles for the 5' untranslated region (5'-UTR) length of the changed genes showed an association between longer 5'-UTRs and the probability for the relevant gene to be altered translationally, consistent with enhanced eIF4E function. This genome-wide analysis is in favour of a model of profound alteration of translational control in late CRC progression. It also suggests polysomal mRNA profiles as a new, informative dimension for the study of transcriptome imbalance in cancer.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Endotoxin (LPS) stimulates 4E-BP1/PHAS-I phosphorylation in macrophages.

INTRODUCTION: Translational control of cytokine production in endotoxin (LPS)-stimulated macrophages is poorly characterized but likely important. An early step in protein translation is engagement of mRNA by eukaryotic initiation factor 4E (eIF-4E). Translation initiation can be prevented by small 4E-binding proteins (4E-BP1 or PHAS-I) which must be phosphorylated in order to disengage eIF-4E. We examined whether LPS alters 4E-BP1 phosphorylation in macrophages. MATERIALS AND METHODS: Elicited rat peritoneal macrophages and Raw 264.7 macrophages were treated with signal transduction inhibitors and then LPS. Cells were harvested and equal protein amounts were electrophoresed (SDS-PAGE). Western blots (WB) were developed with 4E-BP1 antibody. Alternatively cell lysates were exposed to 7-methyl GTP Sepharose beads in order to isolate the cap-binding protein eIF-4E. The relative amounts of 4E-BP1 associated with eIF-4E were then determined by WB. RESULTS: Macrophage 4E-BP1 is phosphorylated upon stimulation by LPS as evidenced by the appearance of a more slowly migrating gamma (hyperphosphorylated) band on gel electrophoresis. Inhibition of both the p42/p44 MAPK pathway (PD 98059) and the p38 MAPK pathway (SB 203580) failed to alter LPS-induced 4E-BP1 phosphorylation. Rapamycin (FRAP/mTOR inhibitor) blocked 4E-BP1 phosphorylation causing a predominance of the alpha (hypophosphorylated) band. This was confirmed further by 7-methyl-GTP Sepharose isolation of eIF-4E with which 4E-BP1 coprecipitates. CONCLUSION: LPS stimulates 4E-BP1 phosphorylation in macrophages through FRAP/mTOR signaling. This pathway may contribute to the translational control of cytokine gene expression in macrophages.

Animals↗
PubMed1985

Mechanism of activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid.

We have reported previously that calcium ions and phospholipid activate the heme-stabilized proinhibitor form (pro-HCI) of the heme-controlled translational inhibitor (HCI) in reticulocyte lysates and promote the first step of the reaction pro-HCI in equilibrium reversible HCI----irreversible HCI. This suggested the possible involvement of a Ca2+/phospholipid-dependent protein kinase (protein kinase C) in the activation. However, further investigation revealed, among other things, that polyunsaturated fatty acids (e.g., arachidonic acid) were as effective as Ca2+/phospholipid in promoting translational inhibition and phosphorylation of the alpha subunit of the chain-initiation factor eIF-2 and, moreover, HCI activation could be prevented or reversed in either case by NADPH-generating systems or by dithiols. Our results suggest that pro-HCI is activated by lipoperoxides produced in reticulocyte lysates from either phospholipid or polyunsaturated fatty acids; the presence of Ca2+ is required in the former but not in the latter case. The reversible activation of HCI by Ca2+ and phospholipid might suggest a possible modulatory role of Ca2+ in translational control.

Calcium↗
PubMed2005

Levels of mTOR and its downstream targets 4E-BP1, eEF2, and eEF2 kinase in relationships with tau in Alzheimer's disease brain.

The pathogenesis of formation of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) brains is unknown. One of the possibilities might be that translation of tau mRNA is aberrantly regulated in AD brains. In the current study, levels of various translation control elements including total and phosphorylated (p) forms of mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1), eukaryotic elongation factor 2 (eEF2), and eEF2 kinase were investigated in relationship with tau in homogenates of the medial temporal cortex from 20 AD and 10 control brains. We found that levels of p-mTOR (Ser2481), and p-4E-BP1 (Thr70 and Ser65) dramatically increase in AD, and are positively significantly correlated with total tau and p-tau. Levels of p-eEF2K were significantly increased, and total eEF2 significantly decreased in AD, when compared to controls. The changes of p-mTOR (2481), p-4E-BP1, and p-eEF2 were immunohistochemically confirmed to be in neurons of AD brains. This suggested that there are obvious abnormalities of elements related with translation control in AD brain and their aberrant changes may up-regulate the translation of tau mRNA, contributing to hyperphosphorylated tau accumulation in NFT-bearing neurons.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Lithium can relieve translational repression of TOP mRNAs elicited by various blocks along the cell cycle in a glycogen synthase kinase-3- and S6-kinase-independent manner.

TOP mRNAs are translationally controlled by mitogenic, growth, and nutritional stimuli through a 5'-terminal oligopyrimidine tract. Here we show that LiCl can alleviate the translational repression of these mRNAs when progression through the cell cycle is blocked at G(0), G(1)/S, or G(2)/M phases in different cell lines and by various physiological and chemical means. This derepressive effect of LiCl does not involve resumption of cell division. Unlike its efficient derepressive effect in mitotically arrested cells, LiCl alleviates inefficiently the repression of TOP mRNAs in amino acid-deprived cells and has no effect in lymphoblastoids whose TOP mRNAs are constitutively repressed even when they are proliferating. LiCl is widely used as a relatively selective inhibitor of glycogen synthase kinase-3. However, inhibition per se of this enzyme by more specific drugs failed to derepress the translation of TOP mRNAs, implying that relief of the translational repression of TOP mRNAs by LiCl is carried out in a glycogen synthase kinase-3-independent manner. Moreover, this effect is apparent, at least in some cell lines, in the absence of S6-kinase 1 activation and ribosomal protein S6 phosphorylation, thus further supporting the notion that translational control of TOP mRNAs does not rely on either of these variables.

Amino Acids↗
PubMed2006

Analysis of SAGE data in human platelets: features of the transcriptome in an anucleate cell.

A comprehensive SAGE (serial analysis of gene expression) library of purified human platelets was established. Twenty-five thousand (25,000) tags were sequenced, and after removal of mitochondrial tags, 12,609 (51%) non-mitochondrial-derived tags remained, corresponding to 2,300 different transcripts with expression levels of up to 30,000 tags per million. This new, highly purified SAGE library of platelets is enriched in specific transcripts. The complexity in terms of tag distribution is similar to cells that are still able to replenish their mRNA pool by transcription. We show that our SAGE data are consistent with recently published microarray data but show further details of the platelet transcriptome, including (i) longer UTR regions and more stable folding in the enriched mRNAs, (ii) biologically interesting new candidate mRNAs that show regulatory elements, including elements for RNA stabilization or for translational control, and (iii) significant enrichment of specific, highly transcribed mRNAs compared to a battery of SAGE libraries from other tissues. Among several regulatory mRNA elements known to be involved in mRNA localization and translational control, CPE elements are in particular enriched in the platelet transcriptome. mRNAs previously reported to be translationally regulated were found to be present in the library and were validated by real-time PCR. Furthermore, specific molecular functions such as signal transduction activity were found to be significantly enriched in the platelet transcriptome. These findings emphasize the richness and diversity of the platelet transcriptome.

3' Untranslated Regions↗
PubMed2004

Role of mTOR signalling in the control of translation initiation and elongation by nutrients.

Protein synthesis requires nutrients both as precursors (amino acids) and as a source of energy, since this process consumes a high proportion of cellular metabolic energy. Recent work has shown that both types of nutrients directly influence the activities of components of the translational machinery in mammalian cells. Amino acids positively regulate signalling through the mammalian target of the rapamycin (mTOR) pathway, although the degree of dependency on external amino acids varies between cell types. mTOR signalling modulates several key components involved in mRNA translation, in particular (via repressor proteins) the cap-binding initiation factor eIF4E, the ribosomal protein S6 kinases, and elongation factor eEF2. The branched-chain amino acid leucine is the most effective one in most cell types. It is currently unclear how mammalian cells sense prevailing amino acid levels, although this may involve intracellular amino acids. Cellular ATP levels can also influence mTOR activity. The activities of some translation factors are modulated by mTOR-independent mechanisms. Examples include the regulation of eEF2 by cellular energy levels, which may be controlled via the AMP-activated protein kinase, and the activity of the guanine nucleotide-exchange factor eIF2B, which is modulated by amino acids and metabolic fuels.

Amino Acids↗
PubMed2004

Dendritic transport and localization of protein kinase Mzeta mRNA: implications for molecular memory consolidation.

Protein kinase Mzeta (PKMzeta) is an atypical protein kinase C isoform that has been implicated in the protein synthesis-dependent maintenance of long term potentiation and memory storage in the brain. Synapse-associated kinases are uniquely positioned to promote enduring consolidation of structural and functional modifications at the synapse, provided that kinase mRNA is available on site for local input-specific translation. We now report that the mRNA encoding PKMzeta is rapidly transported and specifically localized to synaptodendritic neuronal domains. Transport of PKMzeta mRNA is specified by two cis-acting dendritic targeting elements (Mzeta DTEs). Mzeta DTE1, located at the interface of the 5'-untranslated region and the open reading frame, directs somato-dendritic export of the mRNA. Mzeta DTE2, in contrast, is located in the 3'-untranslated region and is required for delivery of the mRNA to distal dendritic segments. Colocalization with translational repressor BC1 RNA in hippocampal dendrites suggests that PKMzeta mRNA may be subject to translational control in local domains. Dendritic localization of PKMzeta mRNA provides a molecular basis for the functional integration of synaptic signal transduction and translational control pathways.

Animals↗
PubMed1997

Distinct repression of translation by wortmannin and rapamycin.

The role of phosphatidylinositol 3-kinase and FK506-binding protein rapamycin-associated protein (FRAP) in translational control has been examined by treating RD-rhabdomyosarcoma cells with wortmannin and rapamycin and studying the effects on cell-growth, translation initiation, and protein synthesis. Whereas wortmannin and rapamycin exhibit subtle effects on global translation, examination of individual mRNAs in sucrose gradients and of individual proteins in two-dimensional polyacrylamide gels reveals that wortmannin and rapamycin exhibit distinct effects on the translation of individual mRNAs. Wortmannin represses the synthesis of a third of cellular proteins, whereas rapamycin affects a subset of these proteins. Since ribosomal protein S6 was rapidly dephosphorylated following wortmannin and rapamycin treatment, and the phosphorylation status of the eukaryotic initiation factor 4E was unchanged, our data imply that the p70 signalling pathway has at least one branch-point upstream of FRAP leading to an additional route of translational control.

Androstadienes↗
PubMed2006

Mechanism of tRNA-dependent editing in translational quality control.

Protein synthesis requires the pairing of amino acids with tRNAs catalyzed by the aminoacyl-tRNA synthetases. The synthetases are highly specific, but errors in amino acid selection are occasionally made, opening the door to inaccurate translation of the genetic code. The fidelity of protein synthesis is maintained by the editing activities of synthetases, which remove noncognate amino acids from tRNAs before they are delivered to the ribosome. Although editing has been described in numerous synthetases, the reaction mechanism is unknown. To define the mechanism of editing, phenylalanyl-tRNA synthetase was used to investigate different models for hydrolysis of the noncognate product Tyr-tRNA(Phe). Deprotonation of a water molecule by the highly conserved residue betaHis-265, as proposed for threonyl-tRNA synthetase, was excluded because replacement of this and neighboring residues had little effect on editing activity. Model building suggested that, instead of directly catalyzing hydrolysis, the role of the editing site is to discriminate and properly position noncognate substrate for nucleophilic attack by water. In agreement with this model, replacement of certain editing site residues abolished substrate specificity but only reduced the catalytic efficiency of hydrolysis 2- to 10-fold. In contrast, substitution of the 3'-OH group of tRNA(Phe) severely impaired editing and revealed an essential function for this group in hydrolysis. The phenylalanyl-tRNA synthetase editing mechanism is also applicable to threonyl-tRNA synthetase and provides a paradigm for synthetase editing.

Binding Sites↗