The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

Background: Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results: In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Conclusions: Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication. © 2013 Morais et al.; licensee BioMed Central Ltd.

Collybistin and gephyrin are novel components of the eukaryotic translation initiation factor 3 complex

Abstract Background Collybistin (CB), a neuron-specific guanine nucleotide exchange factor, has been implicated in targeting gephyrin-GABAA receptors clusters to inhibitory postsynaptic sites. However, little is known about additional CB partners and functions. Findings Here, we identified the p40 subunit of the eukaryotic translation initiation factor 3 (eIF3H) as a novel binding partner of CB, documenting the interaction in yeast, non-neuronal cell lines, and the brain. In addition, we demonstrated that gephyrin also interacts with eIF3H in non-neuronal cells and forms a complex with eIF3 in the brain. Conclusions Together, our results suggest, for the first time, that CB and gephyrin associate with the translation initiation machinery, and lend further support to the previous evidence that gephyrin may act as a regulator of synaptic protein synthesis.

Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation.

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.

Eukaryotic translation initiation factor 3, subunit a, regulates the extracellular signal-regulated kinase pathway

The extracellular signal-regulated kinase (ERK) pathway participates in the control of numerous cellular processes, including cell proliferation. Since its activation kinetics are critical for to its biological effects, they are tightly regulated. We report that the protein translation factor, eukaryotic translation initiation factor 3, subunit a (eIF3a), binds to SHC and Raf-1, two components of the ERK pathway. The interaction of eIF3a with Raf-1 is increased by ß-arrestin2 expression and transiently decreased by epidermal growth factor (EGF) stimulation in a concentration-dependent manner. The EGF-induced decrease in Raf-1-eIF3a association kinetically correlates with the time course of ERK activation. eIF3a interferes with Raf-1 activation and eIF3a downregulation by small interfering RNA enhances ERK activation, early gene expression, DNA synthesis, expression of neuronal differentiation markers in PC12 cells, and Ras-induced focus formation in NIH 3T3 cells. Thus, eIF3a is a negative modulator of ERK pathway activation and its biological effects.

Involvement of Eukaryotic Translation Initiation Factor 5A (eIF5A) in Skeletal Muscle Stem Cell Differentiation

The eukaryotic translation initiation factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post-translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non-differentiated satellite cells and skeletal muscle. The treatment with NI-guanyl- 1,7-diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L-Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration.

Conserved sequences in the β subunit of archaeal and eukaryal translation initiation factor 2 (elF2), absent from elF5, mediate interaction with elF2γ

The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode. This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5' end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome. Here we provide a comparative sequence analysis of the β subunit of eIF2 and its archaeal counterpart (aIF2β). aIF2β differs from eIF2β in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B. The remaining sequences are highly conserved, and are shared with eIF5. Previously isolated mutations in the yeast eIF2β, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2β, but not in eIF5. We show that the sequence of eIF2B homologous to aIF2β is sufficient for binding eIF2γ, the only subunit with which it interacts, and comprises, at the most, 78 residues, eIF5 does not interact with eIF2γ, despite its similarity with eIF2β, probably because of a gap in homology in this region. These observations have implications for the evolution of the mechanism of translation initiation.

Inhibition of eukaryotic translation initiation factor 5A (eIF5A) hypusination impairs melanoma growth

The eukaryotic translation initiation factor 5A (eIF5A) undergoes a specific post-translational modification called hypusination. This modification is required for the functionality of this protein. The compound N1-guanyl-1,7-diaminoheptane (GC7) is a potent and selective inhibitor of deoxyhypusine synthase, which catalyses the first step of eIF5A hypusination process. In the present study, the effects of GC7 on cell death were investigated using two cell lines: melan-a murine melanocytes and Tm5 marine melanoma. In vitro treatment with GC7 increased by 3-fold the number of cells presenting DNA fragmentation in Tm5 cells. Exposure to GC7 also decreased viability to both cell lines. This study also describes, for the first time, the in vivo antitumour effect of GC7, as indicated by impaired melanoma growth in C57BL/6 mice. Copyright © 2006 John Wiley & Sons, Ltd.

Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

Eukaryotic Initiation Factor 4G Suppresses Nonsense-Mediated mRNA Decay by Two Genetically Separable Mechanisms

Nonsense-mediated mRNA decay (NMD), which is best known for degrading mRNAs with premature termination codons (PTCs), is thought to be triggered by aberrant translation termination at stop codons located in an environment of the mRNP that is devoid of signals necessary for proper termination. In mammals, the cytoplasmic poly(A)-binding protein 1 (PABPC1) has been reported to promote correct termination and therewith antagonize NMD by interacting with the eukaryotic release factors 1 (eRF1) and 3 (eRF3). Using tethering assays in which proteins of interest are recruited as MS2 fusions to a NMD reporter transcript, we show that the three N-terminal RNA recognition motifs (RRMs) of PABPC1 are sufficient to antagonize NMD, while the eRF3-interacting C-terminal domain is dispensable. The RRM1-3 portion of PABPC1 interacts with eukaryotic initiation factor 4G (eIF4G) and tethering of eIF4G to the NMD reporter also suppresses NMD. We identified the interactions of the eIF4G N-terminus with PABPC1 and the eIF4G core domain with eIF3 as two genetically separable features that independently enable tethered eIF4G to inhibit NMD. Collectively, our results reveal a function of PABPC1, eIF4G and eIF3 in translation termination and NMD suppression, and they provide additional evidence for a tight coupling between translation termination and initiation.

Association of a M(r) 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 alpha and premature shutoff of protein synthesis after infection with gamma 134.5- mutants of herpes simplex virus 1.

The protein encoded by the gamma 134.5 gene of herpes simplex virus precludes premature shutoff of protein synthesis in human cells triggered by stress associated with onset of viral DNA synthesis. The carboxyl terminus of the protein is essential for this function. This report indicates that the shutoff of protein synthesis is not due to mRNA degration because mRNA from wild-type or gamma 134.5- virus-infected cells directs protein synthesis. Analyses of the posttranslational modifications of translation initiation factor eIF-2 showed the following: (i) eIF-2 alpha was selectively phosphorylated by a kinase present in ribosome-enriched fraction of cells infected with gamma 134.5- virus. (ii) Endogenous eIF-2 alpha was totally phosphorylated in cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene but was not phosphorylated in mock-infected or wild-type virus-infected cells. (iii) Immune precipitates of the PKR kinase that is responsible for regulation of protein synthesis of some cells by phosphorylation of eIF-2 alpha yielded several phosphorylated polypeptides. Of particular significance were two observations. First, phosphorylation of PKR kinase was elevated in all infected cells relative to the levels in mock-infected cells. Second, the precipitates from lysates of cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene contained an additional labeled phosphoprotein of M(r) 90,000 (p90). This phosphoprotein was present in only trace amounts in the immunoprecipitate from cells infected with wild-type virus or mutants lacking a portion of the 5' domain of gamma 134.5. We conclude that in the absence of gamma 134.5 protein, PKR kinase complexes with the p90 phosphoprotein and shuts off protein synthesis by phosphorylation of the alpha subunit of translation initiation factor eIF-2.

Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure-function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aDelta tif51bDelta) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.

Evidences of a Role for Eukaryotic Translation Initiation Factor 5A (eIF5A) in Mouse Embryogenesis and Cell Differentiation

Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation. J. Cell. Physiol. 225: 500-505, 2010. (C) 2010 Wiley-Liss, Inc.

Differential expression of the eukaryotic release factor 3 (eRF3/GSPT1) according to gastric cancer histological types

Background: There are now several lines of evidence to suggest that protein synthesis and translation factors are involved in the regulation of cell proliferation and cancer development. Aims: To investigate gene expression patterns of eukaryotic releasing factor 3 (eRF3) in gastric cancer. Methods: RNA was prepared from 25 gastric tumour biopsies and adjacent non-neoplastic mucosa. Real time TaqMan reverse transcription polymerase chain reaction (RT-PCR) was performed to measure the relative gene expression levels. DNA was isolated from tumour and normal tissues and gene dosage was determined by a quantitative real time PCR using SYBR Green dye. Results: Different histological types of gastric tumours were analysed and nine of the 25 tumours revealed eRF3/GSPT1 overexpression; moreover, eight of the 12 intestinal type carcinomas analysed overexpressed the gene, whereas eRF3/GSPT1 was overexpressed in only one of the 10 diffuse type carcinomas (Kruskal-Wallis Test; p , 0.05). No correlation was found between ploidy and transcript expression levels of eRF3/GSPT1. Overexpression of eRF3/GSPT1 was not associated with increased translation rates because the upregulation of eRF3/GSPT1 did not correlate with increased eRF1 levels. Conclusions: Overexpression of eRF3/GSPT1 in intestinal type gastric tumours may lead to an increase in the translation efficiency of specific oncogenic transcripts. Alternatively, eRF3/GSPT1 may be involved in tumorigenesis as a result of its non-translational roles, namely (dis)regulating the cell cycle, apoptosis, or transcription.

Circulating insulin-like factor 3 (INSL3) in healthy and infertile women

STUDY QUESTION: How does insulin-like factor 3 (INSL3) concentration in blood vary across the menstrual cycle in women? SUMMARY ANSWER: INSL3 is secreted by the theca interna cells of growing antral follicles and is phasic in its expression. WHAT IS KNOWN ALREADY: The relaxin-like hormone INSL3 is known to be expressed in follicles of several mammal species, and was recently shown in cows to be specifically secreted into the bloodstream by growing antral follicles, corresponding to follicular waves. In males INSL3 is known to be acutely independent of the hormones of the hypothalamic-pituitary-gonadal axis, suggesting that in women INSL3 might be a novel biomarker for antral follicle recruitment and development. STUDY DESIGN, SIZE, DURATION: Two cohorts of women were studied. First, 18 healthy women of reproductive age were followed longitudinally for one and a half cycles, with blood sampling and hormone measurement every 2-3 days. A second cohort comprised a cross-sectional study of 909 women attending an infertility clinic, with a single blood sample taken at entry, together with other clinical and hormonal parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples from both retrospective cohorts were analyzed for INSL3 using a highly sensitive time-resolved fluorescent immunoassay, and data were analyzed in comparison with other clinical and hormonal parameters. MAIN RESULT AND THE ROLE OF CHANCE: For young healthy women of reproductive age, we showed a phasic expression of INSL3 corresponding to antral follicle growth in both the follicular and luteal phases of the cycle, which was significantly (P < 0.05) elevated compared with that during menses. For women attending an infertility clinic, those with diagnosed polycystic ovarian syndrome indicated significantly (P < 0.0005) greater circulating INSL3 levels and those with low ovarian reserve showed significantly (P < 0.002) decreased INSL3 values. LIMITATIONS, REASONS FOR CAUTION: These were retrospective studies and the results were obtained from natural cycles only, with their inherent variability. WIDER IMPLICATIONS OF THE FINDINGS: We show for the first time that INSL3 in women does vary across the menstrual cycle, and appears to reflect the number of growing antral follicles recruited within both follicular and luteal phases. STUDY FUNDING/COMPETING INTEREST(S): The present retrospective study was largely supported by departmental funds. There were no competing interests.

Modelling negative feedback networks for activating transcription factor 3 predicts a dominant role for miRNAs in immediate early gene regulation

Activating transcription factor 3 (Atf3) is rapidly and transiently upregulated in numerous systems, and is associated with various disease states. Atf3 is required for negative feedback regulation of other genes, but is itself subject to negative feedback regulation possibly by autorepression. In cardiomyocytes, Atf3 and Egr1 mRNAs are upregulated via ERK1/2 signalling and Atf3 suppresses Egr1 expression. We previously developed a mathematical model for the Atf3-Egr1 system. Here, we adjusted and extended the model to explore mechanisms of Atf3 feedback regulation. Introduction of an autorepressive loop for Atf3 tuned down its expression and inhibition of Egr1 was lost, demonstrating that negative feedback regulation of Atf3 by Atf3 itself is implausible in this context. Experimentally, signals downstream from ERK1/2 suppress Atf3 expression. Mathematical modelling indicated that this cannot occur by phosphorylation of pre-existing inhibitory transcriptional regulators because the time delay is too short. De novo synthesis of an inhibitory transcription factor (ITF) with a high affinity for the Atf3 promoter could suppress Atf3 expression, but (as with the Atf3 autorepression loop) inhibition of Egr1 was lost. Developing the model to include newly-synthesised miRNAs very efficiently terminated Atf3 protein expression and, with a 4-fold increase in the rate of degradation of mRNA from the mRNA/miRNA complex, profiles for Atf3 mRNA, Atf3 protein and Egr1 mRNA approximated to the experimental data. Combining the ITF model with that of the miRNA did not improve the profiles suggesting that miRNAs are likely to play a dominant role in switching off Atf3 expression post-induction.