Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase.

Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one of the most common RNA modifications; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome-wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNAMet synthetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (Ψ). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouridine for MetRS binding. Furthermore, polysomal and protein analyses revealed that Ψ1074 mediates translation. Modification of this site occurs presumably by Pus6, a pseudouridine synthetase known to modify MetRS cognate tRNA. Consistently, the deletion of Pus6 leads to a decrease in MetRS association with both tRNAMet and YEF3 mRNA. Furthermore, while global protein synthesis decreases in pus6Δ, translation of YEF3 increases. Together, our data imply that Pus6 'writes' modifications on tRNA and mRNA, and both types of RNAs are 'read' by MetRS for translation regulation purposes. This represents a novel integrated path for writing and reading modifications on both tRNA and mRNA, which may lead to coordination between global and gene-specific translational responses.

Expression, purification, crystallization and preliminary crystallographic study of the cytoplasmic domain of the mitochondrial dynamics protein MiD51.

Mitochondria play central roles in many cellular and physiological processes. They are highly dynamic organelles and continually undergo fusion and fission. Mitochondrial dynamics protein 51 kDa (MiD51), an integral mitochondrial outer membrane protein, recruits dynamin-related protein 1 (Drp1; a mitochondrial fission protein) to mitochondria and facilitates Drp1-directed mitochondrial fission. In this study, the cytoplasmic domain of MiD51 was overexpressed in Escherichia coli, purified and crystallized. An X-ray diffraction data set was collected to a resolution of 3.1 Å and the crystal belonged to space group P41212, with unit-cell parameters a = b = 90.1, c = 124.7 Å, α = ß = γ = 90°. The asymmetric unit had the highest probability of containing one molecule, with a Matthews coefficient of 3.32 Å(3) Da(-1) and a solvent content of 63.0%.

Bacterial consortium proteomics under 4-chlorosalicylate carbon-limiting conditions.

In this study, the stable consortium composed by Pseudomonas reinekei strain MT1 and Achromobacter xylosoxidans strain MT3 (cell numbers in proportion 9:1) was under investigation to reveal bacterial interactions that take place under severe nutrient-limiting conditions. The analysis of steady states in continuous cultures was carried out at the proteome, metabolic profile, and population dynamic levels. Carbon-limiting studies showed a higher metabolic versatility in the community through upregulation of parallel catabolic enzymes (salicylate 5-hydroxylase and 17-fold on 2-keto-4-pentenoate hydratase) indicating a possible alternative carbon routing in the upper degradation pathway highlighting the effect of minor proportions of strain MT3 over the major consortia component strain MT1 with a significant change in the expression levels of the enzymes of the mainly induced biodegradation pathway such as salicylate 1-hydroxylase and catechol 1,2-dioxygenase together with important changes in the outer membrane composition of P. reinekei MT1 under different culture conditions. The study has demonstrated the importance of the outer membrane as a sensing/response protective barrier caused by interspecies interactions highlighting the role of the major outer membrane proteins OprF and porin D in P. reinekei sp. MT1 under the culture conditions tested.

Cellular and subcellular localization of EFA6C, a third member of the EFA6 family, in adult mouse Purkinje cells.

EFA6C is a third member of the EFA6 family of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factor 6 (ARF6). In this study, we first demonstrated that EFA6C indeed activated ARF6 more selectively than ARF1 by ARF pull-down assay. In situ hybridization histochemistry revealed that EFA6C mRNA was expressed predominantly in mature Purkinje cells and the epithelial cells of the choroid plexus in contrast to the ubiquitous expression of ARF6 mRNA throughout the brain. EFA6C mRNA was already detectable in the Purkinje cells at embryonic day 13, increased progressively during post-natal development and peaked during post-natal second week. In Purkinje cells, the immunoreactivity for EFA6C was localized particularly in the post-synaptic density as well as the plasma membranes of the cell somata, dendritic shafts and spines, while the immunoreactivity in their axon terminals in the deep cerebellar nuclei was very faint. These findings suggest that EFA6C may be involved in the regulation of the membrane dynamics of the somatodendritic compartments of Purkinje cells through the activation of ARF6.

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.

Somatodendritic localization of the mRNA for EFA6A, a guanine nucleotide exchange protein for ARF6, in rat hippocampus and its involvement in dendritic formation.

EFA6A is a guanine nucleotide exchange protein (GEP) that can specifically activate ADP-ribosylation factor 6 (ARF6) in vitro. A recent study has demonstrated that ARF6 is involved in the dendritic formation of developing hippocampal neurons [Hernandez-Deviez et al. (2002) Nature Neurosci., 5, 623-624]. This study examined a potential role for EFA6A in hippocampal development in Wistar rats. Our results provided definitive evidence for somatodendritic localization of EFA6A mRNA in both cultured and in vivo hippocampal neurons by nonradioactive in situ hybridization. During postnatal development, EFA6A mRNA was dramatically increased and its dendritic localization was most evident between P7 and P14. In contrast, ARF6 mRNA was confined to the neuronal layers of the hippocampus throughout development. In addition, the overexpression of a GEP-defective mutant of EFA6A enhanced the dendritic formation of the primary hippocampal neurons. The present findings suggest that EFA6A is intimately involved in the regulation of the dendritic development of hippocampal neurons.

RpoS and RpoN are involved in the growth-dependent regulation of rfaH transcription and O antigen expression in Salmonella enterica serovar Typhi.

We reported earlier that the production of O antigen lipopolysaccharide (LPS) by Salmonella enterica serovar Typhi (Salmonella typhi) increases at the onset of stationary phase and correlates with a growth-regulated expression of the rfaH gene under the control of the alternative sigma factor RpoN (Microbiology 148 (2002) 3789). In this study, we demonstrate that RpoS also modulates rfaH promoter activity as revealed by the absence of growth-dependent regulation of an rfaH-lacZ transcriptional fusion and O antigen production in a S. typhi rpoS mutant. Introduction of a constitutively expressed rpoN gene into the rpoS mutant restored increased production of O antigen during stationary phase, suggesting that constitutive production of RpoN could overcome the RpoS defect. Similar results were observed when an rpoS rpoN double mutant was transformed with the intact rpoN gene. Thus, we conclude that both RpoS and RpoN control the rfaH promoter activity and concomitantly, the production of O-specific LPS in S. typhi.

Production of a functional catalytic antibody ScFv-NusA fusion protein in bacterial cytoplasm.

Functional expression of catalytic antibodies in the cytoplasm of E. coli is potentially of great interest in searching for new catalysts by genetic selection. Herein, a catalytic antibody single chain Fv (ScFv) 14D9, which catalyzes a highly enantioselective protonation, was expressed as a NusA fusion protein under the T7 promoter. A functional disulfide-containing ScFv fusion protein was obtained in the oxidizing environment of bacterial cytoplasm. The 14D9 ScFv could not be overexpressed alone without NusA fusion. The highly soluble NusA protein most likely retards aggregate formation of ScFv and indirectly supports correct folding and disulfide bridge formation in the fusion construct ScFv-NusA. The ScFv-NusA fusion product shows highly enantioselective, specific, hapten inhibited catalytic activity comparable to its parent monoclonal antibody, 14D9. The NusA fusion method might be generally helpful for functional antibody expression in vivo and for the new development of biocatalysts by genetic selection.

Localization of mRNAs for subfamily of guanine nucleotide-exchange proteins (GEP) for ARFs (ADP-ribosylation factors) in the brain of developing and mature rats under normal and postaxotomy conditions.

ADP-ribosylation factors (ARFs) play important roles in vesicular trafficking and cytoskeletal regulation and its activation depends on guanine nucleotide-exchange proteins (GEPs). By way of in situ hybridization histochemistry, the localization of mRNAs for subfamily members of low-molecular-weight ARF-GEPs in the rat brain was studied at embryonic and postnatal stages. In the embryonic brain, the gene expression for msec7-1 was distinct in the ventricular zone while that for msec7-1, -3 and EFA6 in the mantle zone. In early postnatal brain, the expression for msec7-1, -2, -3 and EFA6 was seen widely in various loci of the gray matter with different intensity, and the expression of msec7-1 and -2 mRNAs was evident in the cerebellar external granule cell layer. In the adult brain, the gene expression for the four ARF-GEPs decreased more or less in most gray matter and the distinct expression was maintained mainly in the hippocampal and dentate neuronal layers and cerebellar cortex. The expression of EFA6 mRNA was also evident in the molecular layer of the hippocampus and dentate gyrus. No obvious gene expression for cytohesin-4 and ARF-GEP100 was detected in the brain at any stages of development. The present findings suggest that ARF-GEPs are differentially involved in some processes essential to neuronal differentiation and maturation in association with ARFs.

Peptide elongation factor eEF1A-2/S1 expression in cultured differentiated myotubes and its protective effect against caspase-3-mediated apoptosis.

Peptide elongation factor eEF1A-2/S1, which shares 92% homology with eEF1A-1/EF-1alpha, is exclusively expressed in brain, heart, and skeletal muscle. In these tissues, eEF1A-2/S1 is the only type 1A elongation factor expressed in adulthood because a transition from eEF1A-1/EF-1alpha to eEF1A-2/S1 occurs in early postnatal development. In this article, we report that the expression of eEF1A-2/S1 protein is activated upon myogenic differentiation. Furthermore, we show that upon serum deprivation-induced apoptosis, eEF1A-2/S1 protein disappears and is replaced by its homolog eEF1A-1/EF-1alpha in dying myotubes; cell death is characterized by the activation of caspase-3. In addition, we show that the continuous expression of eEF1A-2/S1 resulting from adenoviral gene transfer protects differentiated myotubes from apoptosis by delaying their death, thus suggesting a prosurvival function for eEF1A-2/S1 in skeletal muscle. In contrast, myotube death is accelerated by the introduction of the homologous gene, eEF1A-1/EF-1alpha, whereas cells transfected with antisense eEF1A-1/EF-1alpha are protected from apoptosis. These results demonstrate that the two sister genes, eEF1A-1/EF-1alpha and eEF1A-2/S1, regulate myotube survival with the former exerting prodeath activity and the latter a prosurvival effect.

Elongation factor Ts can act as a steric chaperone by increasing the solubility of nucleotide binding-impaired elongation factor-Tu.

Several elongation factor (EF) Tu mutants (T25A, H22Y/T25S, D80N, D138N) that have impaired nucleotide binding show decreased solubility on overexpression in the E. coli cell, an indication that they do not fold correctly. Moreover, EF-Tu[T25A] and EF-Tu[D80N] were shown to inhibit cell growth on expression, an effect attributed to their sequestration of EF-Ts [Krab, I. M., and Parmeggiani, A. (1999) J. Biol. Chem. 274, 11132--11138; Krab, I. M., and Parmeggiani, A. (1999) Biochemistry 38, 13035--13041]. We present here results showing that the co-overexpression of EF-Ts at a 1:1 ratio dramatically improves the solubility of mutant EF-Tu, although in the case of EF-Tu[D138N]--which cannot at all bind the nucleotides available in the cell--this is a slow process. Moreover, with co-overexpression of EF-Ts, the mentioned growth inhibition is relieved. We conclude that for the formation of a correct EF-Tu structure the nucleotide plays an important role as a "folding nucleus", and also that in its absence EF-Ts can act as a folding template or steric chaperone for the correct folding of EF-Tu.

In vitro coupled transcription translation: effects of modification in lysate preparation on protein composition and biosynthesis activity.

Cell-free extracts (lysates) from Escherichia coli were used for protein synthesis in vitro. Essential steps of the lysate preparation were modified and analyzed with respect to their impact on in vitro protein synthesis capacity, using the green fluorescent protein (GFP) as a target protein. Variably manufactured lysates of low, medium and higher protein synthesis activity, were examined by high resolution two-dimensional gel electrophoresis to determine whether the modifications result in substantial alterations in protein composition of the final lysate. The total number of proteins calculated from the gel maps did not vary for lysates with different activity and thus cannot serve as an evaluation parameter. Ribosomal proteins RP-S1, RP-L9, and RP-L10 were found in stoichiometric amounts for each of these lysates and in equal concentrations in comparison among the different lysates. Conversely, depending on the activity profiles, up to 7 different isoforms of the elongation factor EF-Ts were detected in the gel maps.

Expression of elongation factor 1 beta' in Escherichia coli and its interaction with elongation factor 1 alpha from silk gland.

Silk gland elongation factor 1 (EF-1) consists of four subunits: alpha, beta, beta', and gamma. EF-1 beta beta' gamma catalyzes the exchange of GDP for GTP on EF-1 alpha and stimulates the binding of EF-1 alpha-dependent aminoacyl-tRNA to ribosomes. The carboxy-terminal regions of the EF-1 beta subunits from various species are highly conserved. We examined the region of EF-1 beta' that binds to EF-1 alpha by in vitro binding assays, and examined the GDP/GTP exchange activity using deletion mutants of a GST-EF1 beta' fusion protein. We thereby suggested a pivotal amino acid region, residues 189-222, of EF-1 beta' for binding to EF-1 alpha.

Rapamycin-resistant phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in v-SRC-transformed hamster fibroblasts.

Increased phosphorylation of the translational repressor protein 4E-BP1 was found in the cell line derived from the tumor induced in Syrian hamster by Rous sarcoma virus (RSV). This was accompanied by its dissociation from the complex with initiation factor eIF4E. The ribosomal S6 protein kinase p70S6k is supposed to be regulated by the same or a closely related rapamycin-sensitive signalling pathway to that which modulates 4E-BP1. Phosphorylation and activity of p70S6k were found to be also increased in RSV-transformed H19 cells that express significantly higher amounts of the Src protein (p60src) relative to the non-transformed hamster fibroblasts NIL-2. The increased activity and phosphorylation of p70S6k were blocked by rapamycin, indicating that the rapamycin-sensitive pathway is involved in its regulation in v-src-transformed hamster fibroblasts. In agreement with this, rapamycin reduced the expression of elongation factor eEF1alpha (whose translation is regulated by a rapamycin-sensitive mechanism thought to involve p70S6k) and did not affect the production of a housekeeping protein, alpha-tubulin, in these cells. Synthesis of Src protein was also inhibited in cells treated with rapamycin. However, treatment of cells with a concentration of rapamycin sufficient to completely inhibit the activity and phosphorylation of p70S6k resulted in only partial de-phosphorylation of 4E-BP1 and its re-association with eIF4E in the transformed cells, indicating that additional rapamycin-insensitive mechanisms/pathways are implicated in the control of 4E-BP1 phosphorylation in RSV-transformed hamster fibroblasts. Over-expression of eIF4E favours cell proliferation and can lead to a transformed phenotype, while over-expression of 4E-BP1 has the opposite effect. The altered signalling to the phosphorylation of 4E-BP1 in RSV-transformed cells, which leads to its dissociation from eIF4E and thus relief of inhibition of eIF4E function, may therefore represent an important regulatory mechanism in malignant cell growth.

Protein kinase-dependent overexpression of the nuclear protein pirin in c-JUN and RAS transformed fibroblasts.

Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of alpha-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes.

Effect of the components extracted from the needles of Taxus baccata on protein biosynthesis in a cell-free rat liver system.

Various species of Taxus contain taxanes that promote polymerization and stabilization of microtubules. They have been reported as antineoplastic compounds with highly effective chemiotherapeutic application. A decrease in incorporation of the radiolabelled precursors into DNA, RNA and proteins in vivo has been reported too. The preliminary results have shown that also the other compounds present in the aqueous extract from Taxus baccata needles, participate in the inhibition of the protein biosynthesis. The binding site of eEF-2 on the ribosome seems to be the target of this inhibition process.

A novel variant of translation elongation factor-1beta: isolation and characterization of the rice gene encoding EF-1beta2.

A rice gene encoding a novel isoform of translation elongation factor-1beta subunit (termed EF-1beta2) was isolated and characterized. The gene comprises of eight exons, and encodes a 226-amino-acid protein. Expression of EF-1beta2 mRNA is abundant in seeds and cultured cells, but is considerably low in the tissues of the rice seedling. Antiserum raised against an EF-1beta2 synthetic peptide detected a protein with a relative molecular mass of about 32 kDa, indicating the EF-1beta2 gene is actually expressed in rice tissues. EF-1beta2 showed a close similarity to the cognate subunits from plant (beta and beta').

Identification of four genes in endothelial cells whose expression is affected by tumor cells and host immune status--a study in ex vivo-isolated endothelial cells.

A spontaneously metastasizing, well-defined mouse lymphoma was chosen as an in vivo model to study the effect of tumor-host interaction on gene expression in liver sinusoidal endothelial cells. Forty-nine bovine aortic endothelial cell (BAEC) genes, recently isolated by a differential screening approach of a cDNA library enriched for tumor necrosis factor-alpha (TNF-alpha) suppressed genes, were investigated. Four of these genes were finally selected because they were affected differentially by host immuno-competence, TNF-alpha, and tumor cells. Sequence analysis showed them to encode the bovine polyubiquitin (A4), elongation factor 1alpha (B2), the acidic ribosomal phosphoprotein PO (C3), and the ribosomal protein S2 (E10). Gene expression was analyzed by dot-blot or Northern blot analysis. TNF-alpha and tumor cell conditioned supernatant suppressed the genes additive in BAEC but not in other endothelial cells except for bovine capillary endothelial cells. Ex vivo-isolated liver endothelial cells of tumor-bearing syngeneic DBA/2 mice showed strong downregulation of these four genes in comparison to normal control values. In contrast, endothelial cells of tumor-bearing immuno-incompetent Balb/c (nu/nu) mice showed no downregulation but upregulation of these genes. Consistently, all four genes were also downregulated when BAEC were incubated with supernatants derived from ex vivo-isolated liver metastases from immuno-competent but not from -incompetent mice. Thus, the expression of a group of genes involved in protein translation and processing was more profoundly altered in endothelial cells in vivo than in vitro, suggesting that microenviromental factors and cell-cell and cell-matrix interactions play an important role.

Different Sp1 family members differentially affect transcription from the human elongation factor 1 A-1 gene promoter.

The GC box is an important transcriptional regulatory element present in the promoters of many mammalian genes. In the present study we examine the effect of known GC-box-binding proteins on the promoter of the human elongation factor 1 A-1 (hEF1A-1) gene in human HeLa cells and Drosophila SL2 cells. In HeLa cells co-transfection with the GC-box-binding protein BTEB resulted in a 4-10-fold increase in hEF1A-1 promoter activity. This stimulation was dependent on a single GC box located between positions -69 and -50 of the promoter. Little or no effect was observed of other GC-box-binding proteins including Sp1, Sp3, Sp4 and BTEB2. In SL2 cells stimulation by Sp1 and Sp3 through the single GC box of the proximal promoter led to 13-fold and 21-fold increases respectively in promoter activity. Inclusion of further upstream sequences resulted in high levels of expression when Sp1 or Sp3 was co-transfected with the reporter plasmid. In this setting Sp1 stimulated transcription by 750-fold, whereas Sp3 was even more potent, yielding a 1150-fold stimulation. Mobility-shift assays performed with the promoter-proximal GC box demonstrated the binding of Sp1, Sp3 and Sp4 to this sequence. To our knowledge, the present study represents the first comparison of all known GC-box-binding proteins on a natural promoter.

Expression, purification, and spectroscopic studies of the guanine nucleotide exchange domain of human elongation factor, EF-1beta.

Two guanine nucleotide exchange domains, corresponding to the C-terminal region of the human translational elongation factor EF-1beta (which consists of 225 amino acids), were produced by DNA recombinant overexpression techniques in Escherichia coli. We describe here a fast and efficient method for purifying these two protein fragments and for concentrating their solutions rapidly to a level as high as 25 mg/ml. This technique permitted the isolation of 20-30 mg of pure, native protein per liter of bacterial culture. Both fragments were able to form a complex with their natural substrate, elongation factor EF-1alpha, as detected by gel filtration experiments. The domain of 110 residues was slightly more active than the 91-amino-acid domain in guanine nucleotide exchange assays. Folding and stability of the two C-terminal domains were explored by circular dichroism (CD) and NMR spectroscopy. In spite of optimal conditions concerning NaCl concentration, temperature, and pH, during the NMR experiments both proteins showed signs of aggregation after approximately 7 days at 303 degreesK, a time period and temperature required for future heteronuclear NMR experiments. Also, the longer fragment suffered from proteolysis in the N-terminal region, suggestive of flexibility in that part of the structure. The secondary structure content for these two EF-1beta fragments was estimated, using data from both CD and NMR. The results of both methods agree very well and indicate for each fragment the presence of approximately 20% alpha-helix and approximately 50% beta-sheet. Elucidation of the three-dimensional structure of the exchange domain of EF-1beta by NMR spectroscopy appears therefore feasible.