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Sample records for eukaryotic translation initiation

  1. The scanning mechanism of eukaryotic translation initiation.

    PubMed

    Hinnebusch, Alan G

    2014-01-01

    In eukaryotes, the translation initiation codon is generally identified by the scanning mechanism, wherein every triplet in the messenger RNA leader is inspected for complementarity to the anticodon of methionyl initiator transfer RNA (Met-tRNAi). Binding of Met-tRNAi to the small (40S) ribosomal subunit, in a ternary complex (TC) with eIF2-GTP, is stimulated by eukaryotic initiation factor 1 (eIF1), eIF1A, eIF3, and eIF5, and the resulting preinitiation complex (PIC) joins the 5' end of mRNA preactivated by eIF4F and poly(A)-binding protein. RNA helicases remove secondary structures that impede ribosome attachment and subsequent scanning. Hydrolysis of eIF2-bound GTP is stimulated by eIF5 in the scanning PIC, but completion of the reaction is impeded at non-AUG triplets. Although eIF1 and eIF1A promote scanning, eIF1 and possibly the C-terminal tail of eIF1A must be displaced from the P decoding site to permit base-pairing between Met-tRNAi and the AUG codon, as well as to allow subsequent phosphate release from eIF2-GDP. A second GTPase, eIF5B, catalyzes the joining of the 60S subunit to produce an 80S initiation complex that is competent for elongation.

  2. Crystal structure of eukaryotic translation initiation factor 2B.

    PubMed

    Kashiwagi, Kazuhiro; Takahashi, Mari; Nishimoto, Madoka; Hiyama, Takuya B; Higo, Toshiaki; Umehara, Takashi; Sakamoto, Kensaku; Ito, Takuhiro; Yokoyama, Shigeyuki

    2016-03-01

    Eukaryotic cells restrict protein synthesis under various stress conditions, by inhibiting the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, a heterotrimeric G protein consisting of α-, β- and γ-subunits. eIF2B exchanges GDP for GTP on the γ-subunit of eIF2 (eIF2γ), and is inhibited by stress-induced phosphorylation of eIF2α. eIF2B is a heterodecameric complex of two copies each of the α-, β-, γ-, δ- and ε-subunits; its α-, β- and δ-subunits constitute the regulatory subcomplex, while the γ- and ε-subunits form the catalytic subcomplex. The three-dimensional structure of the entire eIF2B complex has not been determined. Here we present the crystal structure of Schizosaccharomyces pombe eIF2B with an unprecedented subunit arrangement, in which the α2β2δ2 hexameric regulatory subcomplex binds two γε dimeric catalytic subcomplexes on its opposite sides. A structure-based in vitro analysis by a surface-scanning site-directed photo-cross-linking method identified the eIF2α-binding and eIF2γ-binding interfaces, located far apart on the regulatory and catalytic subcomplexes, respectively. The eIF2γ-binding interface is located close to the conserved 'NF motif', which is important for nucleotide exchange. A structural model was constructed for the complex of eIF2B with phosphorylated eIF2α, which binds to eIF2B more strongly than the unphosphorylated form. These results indicate that the eIF2α phosphorylation generates the 'nonproductive' eIF2-eIF2B complex, which prevents nucleotide exchange on eIF2γ, and thus provide a structural framework for the eIF2B-mediated mechanism of stress-induced translational control.

  3. Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry.

    PubMed Central

    Pestova, T V; Hellen, C U; Shatsky, I N

    1996-01-01

    Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap- and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA. PMID:8943341

  4. Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families

    NASA Technical Reports Server (NTRS)

    Kyrpides, N. C.; Woese, C. R.

    1998-01-01

    As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.

  5. Requirement of the eukaryotic translation initiation factor 4F complex in hepatitis E virus replication.

    PubMed

    Zhou, Xinying; Xu, Lei; Wang, Yijin; Wang, Wenshi; Sprengers, Dave; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

    2015-12-01

    Hepatitis E virus (HEV) infection, one of the foremost causes of acute hepatitis, is becoming a health problem of increasing magnitude. As other viruses, HEV exploits elements from host cell biochemistry, but we understand little as to which components of the human hepatocellular machinery are perverted for HEV multiplication. It is, however, known that the eukaryotic translation initiation factors 4F (eIF4F) complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, serves as an important component for the translation and replication of many viruses. Here we aim to investigate the role of three subunits of the eIF4F complex: eukaryotic translation initiation factor 4A (eIF4A), eukaryotic translation initiation factor 4G (eIF4G) and eukaryotic translation initiation factor 4E (eIF4E) in HEV replication. We found that efficient replication of HEV requires eIF4A, eIF4G and eIF4E. Consistently, the negative regulatory factors of this complex: programmed cell death 4 (PDCD4) and eIF4E-binding protein 1 (4E-BP1) exert anti-HEV activities, which further illustrates the requirement for eIF4A and eIF4E in supporting HEV replication. Notably, phosphorylation of eIF4E induced by MNK1/2 activation is not involved in HEV replication. Although ribavirin and interferon-α (IFN-α), the most often-used off-label drugs for treating hepatitis E, interact with this complex, their antiviral activities are independent of eIF4E. In contrast, eIF4E silencing provokes enhanced anti-HEV activity of these compounds. Thus, HEV replication requires eIF4F complex and targeting essential elements of this complex provides important clues for the development of novel antiviral therapy against HEV.

  6. Inter-polysomal coupling of termination and initiation during translation in eukaryotic cell-free system.

    PubMed

    Sogorin, Evgeny A; Agalarov, Sultan Ch; Spirin, Alexander S

    2016-01-01

    The recording of the luciferase-generated luminescence in the eukaryotic cell-free translation system programmed with mRNA encoding firefly luciferase (Luc-mRNA) showed that the addition of free exogenous mRNAs into the translation reactor induces the immediate release of the functionally active protein from the polyribosomes of the translation system. The phenomenon did not depend on the coding specificity of the added free mRNA. At the same time it depended on the "initiation potential" of the added mRNA (including the features that ensure the successful initiation of translation, such as the presence of the cap structure and the sufficient concentration of the added mRNA in the translation mixture). The phenomenon also strictly depended on the presence of the stop codon in the translated mRNA. As the above-mentioned features of the added mRNA imply its activity in initiation of a new translation, the experimental data are found in agreement with the scenario where the molecules of the added mRNA interact by their 5'-ends with terminating and recycling ribosomes, stimulating the release of the complete polypeptides and providing for the initiation of a new translation. PMID:27075299

  7. Inter-polysomal coupling of termination and initiation during translation in eukaryotic cell-free system

    PubMed Central

    Sogorin, Evgeny A.; Agalarov, Sultan Ch.; Spirin, Alexander S.

    2016-01-01

    The recording of the luciferase-generated luminescence in the eukaryotic cell-free translation system programmed with mRNA encoding firefly luciferase (Luc-mRNA) showed that the addition of free exogenous mRNAs into the translation reactor induces the immediate release of the functionally active protein from the polyribosomes of the translation system. The phenomenon did not depend on the coding specificity of the added free mRNA. At the same time it depended on the “initiation potential” of the added mRNA (including the features that ensure the successful initiation of translation, such as the presence of the cap structure and the sufficient concentration of the added mRNA in the translation mixture). The phenomenon also strictly depended on the presence of the stop codon in the translated mRNA. As the above-mentioned features of the added mRNA imply its activity in initiation of a new translation, the experimental data are found in agreement with the scenario where the molecules of the added mRNA interact by their 5′-ends with terminating and recycling ribosomes, stimulating the release of the complete polypeptides and providing for the initiation of a new translation. PMID:27075299

  8. Mextli is a novel eukaryotic translation initiation factor 4E-binding protein that promotes translation in Drosophila melanogaster.

    PubMed

    Hernández, Greco; Miron, Mathieu; Han, Hong; Liu, Niankun; Magescas, Jérémy; Tettweiler, Gritta; Frank, Filipp; Siddiqui, Nadeem; Sonenberg, Nahum; Lasko, Paul

    2013-08-01

    Translation is a fundamental step in gene expression, and translational control is exerted in many developmental processes. Most eukaryotic mRNAs are translated by a cap-dependent mechanism, which requires recognition of the 5'-cap structure of the mRNA by eukaryotic translation initiation factor 4E (eIF4E). eIF4E activity is controlled by eIF4E-binding proteins (4E-BPs), which by competing with eIF4G for eIF4E binding act as translational repressors. Here, we report the discovery of Mextli (Mxt), a novel Drosophila melanogaster 4E-BP that in sharp contrast to other 4E-BPs, has a modular structure, binds RNA, eIF3, and several eIF4Es, and promotes translation. Mxt is expressed at high levels in ovarian germ line stem cells (GSCs) and early-stage cystocytes, as is eIF4E-1, and we demonstrate the two proteins interact in these cells. Phenotypic analysis of mxt mutants indicates a role for Mxt in germ line stem cell (GSC) maintenance and in early embryogenesis. Our results support the idea that Mxt, like eIF4G, coordinates the assembly of translation initiation complexes, rendering Mxt the first example of evolutionary convergence of eIF4G function.

  9. Dynamic regulation of the translation initiation helicase complex by mitogenic signal transduction to eukaryotic translation initiation factor 4G.

    PubMed

    Dobrikov, Mikhail I; Dobrikova, Elena Y; Gromeier, Matthias

    2013-03-01

    Eukaryotic translation initiation factor 4F (eIF4F), comprising the cap-binding protein eIF4E, the helicase eIF4A, and the central scaffold eIF4G, is a convergence node for a complex signaling network that controls protein synthesis. Together with eIF3 and eIF4A/4B, eIF4G recruits ribosomal subunits to mRNAs and facilitates 5' untranslated region unwinding. Mammalian eIF4G contains three HEAT domains and unstructured regions involved in protein-protein interactions. Despite detailed eIF4G structure data, the mechanisms controlling initiation scaffold formation remain obscure. We found a new, highly regulated eIF4B/-3 binding site within the HEAT-1/-2 interdomain linker, harboring two phosphorylation sites that we identified as substrates for Erk1/2 and casein kinase 2. Phorbol ester-induced sequential phosphorylation of both sites detached HEAT-2 from the complex with eIF4A/-4B/-3 and stimulated the association of HEAT-3 with the mitogen-activated protein kinase signal integrating kinase Mnk1. Our results provide a mechanistic link between intracellular signal transduction and dynamic initiation complex formation coordinated by flexible eIF4G structure.

  10. Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes.

    PubMed

    Nakagawa, So; Niimura, Yoshihito; Gojobori, Takashi; Tanaka, Hiroshi; Miura, Kin-ichiro

    2008-02-01

    Understanding regulatory mechanisms of protein synthesis in eukaryotes is essential for the accurate annotation of genome sequences. Kozak reported that the nucleotide sequence GCCGCC(A/G)CCAUGG (AUG is the initiation codon) was frequently observed in vertebrate genes and that this 'consensus' sequence enhanced translation initiation. However, later studies using invertebrate, fungal and plant genes reported different 'consensus' sequences. In this study, we conducted extensive comparative analyses of nucleotide sequences around the initiation codon by using genomic data from 47 eukaryote species including animals, fungi, plants and protists. The analyses revealed that preferred nucleotide sequences are quite diverse among different species, but differences between patterns of nucleotide bias roughly reflect the evolutionary relationships of the species. We also found strong biases of A/G at position -3, A/C at position -2 and C at position +5 that were commonly observed in all species examined. Genes with higher expression levels showed stronger signals, suggesting that these nucleotides are responsible for the regulation of translation initiation. The diversity of preferred nucleotide sequences around the initiation codon might be explained by differences in relative contributions from two distinct patterns, GCCGCCAUG and AAAAAAAUG, which implies the presence of multiple molecular mechanisms for controlling translation initiation.

  11. Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3).

    PubMed

    Sun, Chaomin; Todorovic, Aleksandar; Querol-Audí, Jordi; Bai, Yun; Villa, Nancy; Snyder, Monica; Ashchyan, John; Lewis, Christopher S; Hartland, Abbey; Gradia, Scott; Fraser, Christopher S; Doudna, Jennifer A; Nogales, Eva; Cate, Jamie H D

    2011-12-20

    Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.

  12. Model of cap-dependent translation initiation in sea urchin: a step towards the eukaryotic translation regulation network.

    PubMed

    Bellé, Robert; Prigent, Sylvain; Siegel, Anne; Cormier, Patrick

    2010-03-01

    The large and rapid increase in the rate of protein synthesis following fertilization of the sea urchin egg has long been a paradigm of translational control, an important component of the regulation of gene expression in cells. This translational up-regulation is linked to physiological changes that occur upon fertilization and is necessary for entry into first cell division cycle. Accumulated knowledge on cap-dependent initiation of translation makes it suited and timely to start integrating the data into a system view of biological functions. Using a programming environment for system biology coupled with model validation (named Biocham), we have built an integrative model for cap-dependent initiation of translation. The model is described by abstract rules. It contains 51 reactions involved in 74 molecular complexes. The model proved to be coherent with existing knowledge by using queries based on computational tree logic (CTL) as well as Boolean simulations. The model could simulate the change in translation occurring at fertilization in the sea urchin model. It could also be coupled with an existing model designed for cell-cycle control. Therefore, the cap-dependent translation initiation model can be considered a first step towards the eukaryotic translation regulation network.

  13. Eukaryotic Initiation Factor 2 Phosphorylation and Translational Control in Metabolism12

    PubMed Central

    Baird, Thomas D.; Wek, Ronald C.

    2012-01-01

    Regulation of mRNA translation is a rapid and effective means to couple changes in the cellular environment with global rates of protein synthesis. In response to stresses, such as nutrient deprivation and accumulation of misfolded proteins in the endoplasmic reticulum, phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α~P) reduces general translation initiation while facilitating the preferential translation of select transcripts, such as that encoding activating transcription factor 4 (ATF4), a transcriptional activator of genes subject to the integrated stress response (ISR). In this review, we highlight the translational control processes regulated by nutritional stress, with an emphasis on the events triggered by eIF2α~P, and describe the family of eukaryotic initiation factor 2 kinases and the mechanisms by which each sense different stresses. We then address 3 questions. First, what are the mechanisms by which eIF2α~P confers preferential translation on select mRNA and what are the consequences of the gene expression induced by the ISR? Second, what are the molecular processes by which certain stresses can differentially activate eIF2α~P and ATF4 expression? The third question we address is what are the modes of cross-regulation between the ISR and other stress response pathways, such as the unfolded protein response and mammalian target of rapamycin, and how do these regulatory schemes provide for gene expression programs that are tailored for specific stresses? This review highlights recent advances in each of these areas of research, emphasizing how eIF2α~P and the ISR can affect metabolic health and disease. PMID:22585904

  14. Association of eukaryotic translation initiation factor eIF2B with fully solubilized CXCR4.

    PubMed

    Palmesino, Elena; Apuzzo, Tiziana; Thelen, Sylvia; Mueller, Bernd; Langen, Hanno; Thelen, Marcus

    2016-06-01

    Chemokine receptors are key regulators of leukocyte trafficking but also have an important role in development, tumor growth, and metastasis. Among the chemokine receptors, CXCR4 is the only one that leads to perinatal death when genetically ablated in mice, indicating a more-widespread function in development. To identify pathways that are activated downstream of CXCR4, a solubilization protocol was elaborated, which allows for the isolation of the endogenous receptor from human cells in its near-native conformation. Solubilized CXCR4 is recognized by the conformation-sensitive monoclonal antibody 12G5 and retains the ability to bind CXCL12 in solution, which was abolished in the presence of receptor antagonists. Mass spectrometry of CXCR4 immunoprecipitates revealed a specific interaction with the pentameric eukaryotic translation initiation factor 2B. The observation that the addition of CXCL12 leads to the dissociation of eukaryotic translation initiation factor 2B from CXCR4 suggests that stimulation of the receptor may trigger the local protein synthesis required for efficient cell movement. PMID:26609049

  15. Eukaryotic Initiation Factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation

    SciTech Connect

    Guo, Jianjun; Jin, Zhaoqing; Yang, Xiaohan; Li, Jian-Feng; Chen, Jay

    2011-01-01

    We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.

  16. CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

    PubMed Central

    Homma, Miwako Kato; Wada, Ikuo; Suzuki, Toshiyuki; Yamaki, Junko; Krebs, Edwin G.; Homma, Yoshimi

    2005-01-01

    Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser/Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G1 and G2/M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5/eIF2/eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation. PMID:16227438

  17. Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation

    PubMed Central

    Bordeleau, Marie-Eve; Matthews, James; Wojnar, Joanna M.; Lindqvist, Lisa; Novac, Olivia; Jankowsky, Eckhard; Sonenberg, Nahum; Northcote, Peter; Teesdale-Spittle, Paul; Pelletier, Jerry

    2005-01-01

    RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD/H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5′ m7GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEAD-box RNA helicases with small molecules. PMID:16030146

  18. Isolation and mapping of the human eukaryotic translation initiation factor 5 to chromosome 14

    SciTech Connect

    Romano, D.M.; Wasco, W.; Murell, J.

    1994-09-01

    Eukaryotic translation initiation factor 5 (eIF-5) is essential for the initiation of protein synthesis. eIF-5 catalyzes the hydrolysis of GTP on the 40S ribosomal initiation complex. Subsequent to GTP hydrolysis and the release of eIF-2-GDP, the 60S ribosomal subunit is joined to the 40S subunit to form an 80S initiation complex which can engage in peptide transfer. In an effort to isolate the major early-onset familial Alzheimer`s disease (FAD) gene on chromosome 14, we have isolated expressed sequences from this autosome in the form of exons `trapped` from chromosome 14-specific cosmids (library provided by L. Deaven, Los Alamos, NM). One cosmid yielded multiple exons displaying strong DNA and amino acid homology (>90%) with the rat eIF-5 gene. These exons were used to isolate full-length cDNAs from a human brain library. The eIF-5 message is approximately 3.6 kB in size and is ubiquitously expressed. The predicted amino acid sequence reveals multiple phosphorylation sites which may be involved in regulation of activity of eIF-5 and regions with homology to the GTPase superfamily, consistent with eIF-5`s role in GTP hydrolysis. Further studies are underway to determine whether the eIF-5 gene resides within the FAD minimal candidate region on chromosome 14q24.3.

  19. Sox2 is translationally activated by eukaryotic initiation factor 4E in human glioma-initiating cells

    SciTech Connect

    Ge, Yuqing; Zhou, Fengbiao; Chen, Hong; Cui, Chunhong; Liu, Dan; Li, Qiuping; Yang, Zhiyuan; Wu, Guoqiang; Sun, Shuhui; Gu, Jianxin; Wei, Yuanyan; Jiang, Jianhai

    2010-07-09

    Sox2, a master transcription factor, contributes to the generation of induced pluripotent stem cells and plays significant roles in sustaining the self-renewal of neural stem cells and glioma-initiating cells. Understanding the functional differences of Sox2 between glioma-initiating cells and normal neural stem cells would contribute to therapeutic approach for treatment of brain tumors. Here, we first demonstrated that Sox2 could contribute to the self-renewal and proliferation of glioma-initiating cells. The following experiments showed that Sox2 was activated at translational level in a subset of human glioma-initiating cells compared with the normal neural stem cells. Further investigation revealed there was a positive correlation between Sox2 and eukaryotic initiation factor 4E (eIF4E) in glioma tissues. Down-regulation of eIF4E decreased Sox2 protein level without altering its mRNA level in glioma-initiating cells, indicating that Sox2 was activated by eIF4E at translational level. Furthermore, eIF4E was presumed to regulate the expression of Sox2 by its 5' untranslated region (5' UTR) sequence. Our results suggest that the eIF4E-Sox2 axis is a novel mechanism of unregulated self-renewal of glioma-initiating cells, providing a potential therapeutic target for glioma.

  20. Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex.

    PubMed

    Llácer, Jose L; Hussain, Tanweer; Marler, Laura; Aitken, Colin Echeverría; Thakur, Anil; Lorsch, Jon R; Hinnebusch, Alan G; Ramakrishnan, V

    2015-08-01

    Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5' end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition.

  1. Human-Like Eukaryotic Translation Initiation Factor 3 from Neurospora crassa

    PubMed Central

    Smith, M. Duane; Gu, Yu; Querol-Audí, Jordi; Vogan, Jacob M.; Nitido, Adam; Cate, Jamie H. D.

    2013-01-01

    Eukaryotic translation initiation factor 3 (eIF3) is a key regulator of translation initiation, but its in vivo assembly and molecular functions remain unclear. Here we show that eIF3 from Neurospora crassa is structurally and compositionally similar to human eIF3. N. crassa eIF3 forms a stable 12-subunit complex linked genetically and biochemically to the 13th subunit, eIF3j, which in humans modulates mRNA start codon selection. Based on N. crassa genetic analysis, most subunits in eIF3 are essential. Subunits that can be deleted (e, h, k and l) map to the right side of the eIF3 complex, suggesting that they may coordinately regulate eIF3 function. Consistent with this model, subunits eIF3k and eIF3l are incorporated into the eIF3 complex as a pair, and their insertion depends on the presence of subunit eIF3h, a key regulator of vertebrate development. Comparisons to other eIF3 complexes suggest that eIF3 assembles around an eIF3a and eIF3c dimer, which may explain the coordinated regulation of human eIF3 levels. Taken together, these results show that Neurospora crassa eIF3 provides a tractable system for probing the structure and function of human-like eIF3 in the context of living cells. PMID:24250809

  2. Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus.

    PubMed

    Aragón, T; de la Luna, S; Novoa, I; Carrasco, L; Ortín, J; Nieto, A

    2000-09-01

    Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5' untranslated region (5' UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

  3. Posttranslational hypusination of the eukaryotic translation initiation factor-5A regulates Fusarium graminearum virulence

    PubMed Central

    Martinez-Rocha, Ana Lilia; Woriedh, Mayada; Chemnitz, Jan; Willingmann, Peter; Kröger, Cathrin; Hadeler, Birgit; Hauber, Joachim; Schäfer, Wilhelm

    2016-01-01

    Activation of eukaryotic translation initiation factor eIF5A requires a posttranslational modification, forming the unique amino acid hypusine. This activation is mediated by two enzymes, deoxyhypusine synthase, DHS, and deoxyhypusine hydroxylase, DOHH. The impact of this enzymatic complex on the life cycle of a fungal pathogen is unknown. Plant pathogenic ascomycetes possess a single copy of the eIF5A activated by hypusination. We evaluated the importance of imbalances in eIF5A hypusination in Fusarium graminearum, a devastating fungal pathogen of cereals. Overexpression of DHS leads to increased virulence in wheat, elevated production of the mycotoxin deoxynivalenol, more infection structures, faster wheat tissue invasion in plants and increases vegetatively produced conidia. In contrast, overexpression of DOHH completely prevents infection structure formation, pathogenicity in wheat and maize, leads to overproduction of ROS, reduced DON production and increased sexual reproduction. Simultaneous overexpression of both genes restores wild type-like phenotypes. Analysis of eIF5A posttranslational modification displayed strongly increased hypusinated eIF5A in DOHH overexpression mutant in comparison to wild type, and the DHS overexpression mutants. These are the first results pointing to different functions of differently modified eIF5A. PMID:27098988

  4. Functional role of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in NSCLC

    PubMed Central

    Cao, Yueyu; Wei, Mengdan; Li, Bing; Liu, Yali; Lu, Ying; Tang, Zhipeng; Lu, Tianbao; Yin, Yujiao; Qin, Zhiqiang; Xu, Zengguang

    2016-01-01

    Eukaryotic translation initiation factor 4 gamma 1(EIF4G1) is related to tumorigenesis and tumor progression. However, its role and the underlying mechanisms in the regulation of tumor development in non–small cell lung cancers (NSCLC) remain largely unknown. Here we report that the levels of EIF4G1 expression are much higher in NSCLC cell lines and tumor tissues than those in the normal lung cells and adjacent normal tissues from the same patients. Using shRNA to knock down EIF4G1 expression stably, we found EIF4G1 required for NSCLC cell proliferation, anchorage-independent growth, migration and invasion. Furthermore, silencing of EIF4G1 induces NSCLC cell apoptosis and causes G0/G1 cell cycle arrest. To identify the partner protein network of EIF4G1 in NSCLC cells, we found that Ubiquitin-specific protease 10 (USP10) can directly interacts with EIF4G1, while acting as a negative regulator for EIF4G1-mediated functions. Together, our results indicate that EIF4G1 functions as an oncoprotein during NSCLC development, which may represent a novel and promising therapeutic target in lung cancer. PMID:27003362

  5. Functional role of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in NSCLC.

    PubMed

    Cao, Yueyu; Wei, Mengdan; Li, Bing; Liu, Yali; Lu, Ying; Tang, Zhipeng; Lu, Tianbao; Yin, Yujiao; Qin, Zhiqiang; Xu, Zengguang

    2016-04-26

    Eukaryotic translation initiation factor 4 gamma 1(EIF4G1) is related to tumorigenesis and tumor progression. However, its role and the underlying mechanisms in the regulation of tumor development in non-small cell lung cancers (NSCLC) remain largely unknown. Here we report that the levels of EIF4G1 expression are much higher in NSCLC cell lines and tumor tissues than those in the normal lung cells and adjacent normal tissues from the same patients. Using shRNA to knock down EIF4G1 expression stably, we found EIF4G1 required for NSCLC cell proliferation, anchorage-independent growth, migration and invasion. Furthermore, silencing of EIF4G1 induces NSCLC cell apoptosis and causes G0/G1 cell cycle arrest. To identify the partner protein network of EIF4G1 in NSCLC cells, we found that Ubiquitin-specific protease 10 (USP10) can directly interacts with EIF4G1, while acting as a negative regulator for EIF4G1-mediated functions. Together, our results indicate that EIF4G1 functions as an oncoprotein during NSCLC development, which may represent a novel and promising therapeutic target in lung cancer.

  6. The Structure of Eukaryotic Translation Initiation Factor-4E from Wheat Reveals a Novel Disulfide Bond

    SciTech Connect

    Monzingo,A.; Dhaliwal, S.; Dutt-Chaudhuri, A.; Lyon, A.; Sadow, J.; Hoffman, D.; Robertus, J.; Browning, K.

    2007-01-01

    Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m{sup 7} guanosine nucleotide at the 5' end of eukaryotic messenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4E from wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance (NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight {beta}-strands, three {alpha}-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m{sup 7}GTP in its binding site, despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usual cap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into the cavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that are conserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallized with m{sup 7}GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMR spectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced to form the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raises the possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that in oxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based binding assay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein each bind m{sup 7}GTP in a similar and labile manner, with dissociation rates in the range of 20

  7. The Yeast Eukaryotic Translation Initiation Factor 2B Translation Initiation Complex Interacts with the Fatty Acid Synthesis Enzyme YBR159W and Endoplasmic Reticulum Membranes

    PubMed Central

    Browne, Christopher M.; Samir, Parimal; Fites, J. Scott; Villarreal, Seth A.

    2013-01-01

    Using affinity purifications coupled with mass spectrometry and yeast two-hybrid assays, we show the Saccharomyces cerevisiae translation initiation factor complex eukaryotic translation initiation factor 2B (eIF2B) and the very-long-chain fatty acid (VLCFA) synthesis keto-reductase enzyme YBR159W physically interact. The data show that the interaction is specifically between YBR159W and eIF2B and not between other members of the translation initiation or VLCFA pathways. A ybr159wΔ null strain has a slow-growth phenotype and a reduced translation rate but a normal GCN4 response to amino acid starvation. Although YBR159W localizes to the endoplasmic reticulum membrane, subcellular fractionation experiments show that a fraction of eIF2B cofractionates with lipid membranes in a YBR159W-independent manner. We show that a ybr159wΔ yeast strain and other strains with null mutations in the VLCFA pathway cause eIF2B to appear as numerous foci throughout the cytoplasm. PMID:23263984

  8. Phosphorylation of eukaryotic translation initiation factor 4E and eukaryotic translation initiation factor 4E-binding protein (4EBP) and their upstream signaling components undergo diurnal oscillation in the mouse hippocampus: implications for memory persistence.

    PubMed

    Saraf, Amit; Luo, Jie; Morris, David R; Storm, Daniel R

    2014-07-18

    Translation of mRNA plays a critical role in consolidation of long-term memory. Here, we report that markers of initiation of mRNA translation are activated during training for contextual memory and that they undergo diurnal oscillation in the mouse hippocampus with maximal activity observed during the daytime (zeitgeber time 4-8 h). Phosphorylation and activation of eukaryotic translation initiation factor 4E (eIF4E), eIF4E-binding protein 1 (4EBP1), ribosomal protein S6, and eIF4F cap-complex formation, all of which are markers for translation initiation, were higher in the hippocampus during the daytime compared with night. The circadian oscillation in markers of mRNA translation was lost in memory-deficient transgenic mice lacking calmodulin-stimulated adenylyl cyclases. Moreover, disruption of the circadian rhythm blocked diurnal oscillations in eIF4E, 4EBP1, rpS6, Akt, and ERK1/2 phosphorylation and impaired memory consolidation. Furthermore, repeated inhibition of translation in the hippocampus 48 h after contextual training with the protein synthesis inhibitor anisomycin impaired memory persistence. We conclude that repeated activation of markers of translation initiation in hippocampus during the circadian cycle might be critical for memory persistence.

  9. The N-terminal region of eukaryotic translation initiation factor 5A signals to nuclear localization of the protein

    SciTech Connect

    Parreiras-e-Silva, Lucas T.; Gomes, Marcelo D.; Oliveira, Eduardo B.; Costa-Neto, Claudio M.

    2007-10-19

    The eukaryotic translation initiation factor 5A (eIF5A) is a ubiquitous protein of eukaryotic and archaeal organisms which undergoes hypusination, a unique post-translational modification. We have generated a polyclonal antibody against murine eIF5A, which in immunocytochemical assays in B16-F10 cells revealed that the endogenous protein is preferentially localized to the nuclear region. We therefore analyzed possible structural features present in eIF5A proteins that could be responsible for that characteristic. Multiple sequence alignment analysis of eIF5A proteins from different eukaryotic and archaeal organisms showed that the former sequences have an extended N-terminal segment. We have then performed in silico prediction analyses and constructed different truncated forms of murine eIF5A to verify any possible role that the N-terminal extension might have in determining the subcellular localization of the eIF5A in eukaryotic organisms. Our results indicate that the N-terminal extension of the eukaryotic eIF5A contributes in signaling this protein to nuclear localization, despite of bearing no structural similarity with classical nuclear localization signals.

  10. Molecular characterization and phylogenetic analysis of the eukaryotic translation initiation factor 4A gene in Antheraea pernyi (Lepdoptera: Saturniidae).

    PubMed

    Chen, Miao-Miao; Liu, Yan-Qun; Li, Yan; Yao, Rui; Chen, Mo; Xia, Run-Xi; Li, Qun; Qin, Li

    2014-01-01

    Eukaryotic initiation factor 4A (eIF-4A) is an essential component for protein translation in eukaryotes. The eIF-4A gene (ApeIF-4A) was isolated and characterized from Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae). The obtained cDNA sequence was 1,435-bp long with an open reading frame of 1,266 bp encoding 421 amino acids. The predicted amino acid sequence shared several conserved features as found in known eIF-4As and revealed 74 and 78% identities with eIF-4As of Homo sapiens L. and Drosophila melanogaster (Meigen), respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that ApeIF-4A was transcribed at four developmental stages and in all tissues tested, suggesting that it plays an important role in development of A. pernyi. Homologous alignment suggested that eIF-4As are highly conserved throughout evolution of eukaryote organisms. Phylogenetic trees based on the amino acid and nucleotide sequences of eIF-4A demonstrated a similar topology with the classical systematics, suggesting that it has the potential value in phylogenetic inference of eukaryotes.

  11. Inactivation of the mTORC1-Eukaryotic Translation Initiation Factor 4E Pathway Alters Stress Granule Formation

    PubMed Central

    Fournier, Marie-Josée; Coudert, Laetitia; Mellaoui, Samia; Adjibade, Pauline; Gareau, Cristina; Côté, Marie-France; Sonenberg, Nahum; Gaudreault, René C.

    2013-01-01

    Stress granules (SG) are cytoplasmic multimeric RNA bodies that form under stress conditions known to inhibit cap-dependent translation. SG contain translation initiation factors, RNA binding proteins, and signaling molecules. SG are known to inhibit apoptotic pathways, thus contributing to chemo- and radioresistance in tumor cells. However, whether stress granule formation involves oncogenic signaling pathways is currently unknown. Here, we report a novel role of the mTORC1-eukaryotic translation initiation factor 4E (eIF4E) pathway, a key regulator of cap-dependent translation initiation of oncogenic factors, in SG formation. mTORC1 specifically drives the eIF4E-mediated formation of SG through the phosphorylation of 4E-BP1, a key factor known to inhibit formation of the mTORC1-dependent eIF4E-eIF4GI interactions. Disrupting formation of SG by inactivation of mTOR with its specific inhibitor pp242 or by depletion of eIF4E or eIF4GI blocks the SG-associated antiapoptotic p21 pathway. Finally, pp242 sensitizes cancer cells to death in vitro and inhibits the growth of chemoresistant tumors in vivo. This work therefore highlights a novel role of the oncogenic mTORC1-eIF4E pathway, namely, the promotion of formation of antiapoptotic SG. PMID:23547259

  12. Eukaryotic translation initiation factor 5 (eIF5) acts as a classical GTPase-activator protein.

    PubMed

    Paulin, F E; Campbell, L E; O'Brien, K; Loughlin, J; Proud, C G

    2001-01-01

    GTP hydrolysis occurs at several specific stages during the initiation, elongation, and termination stages of mRNA translation. However, it is unclear how GTP hydrolysis occurs; it has previously been suggested to involve a GTPase active center in the ribosome, although proof for this is lacking. Alternatively, it could involve the translation factors themselves, e.g., be similar to the situation for small G in which the GTPase active site involves arginine residues contributed by a further protein termed a GTPase-activator protein (GAP). During translation initiation in eukaryotes, initiation factor eIF5 is required for hydrolysis of GTP bound to eIF2 (the protein which brings the initiator Met-tRNA(i) to the 40S subunit). Here we show that eIF5 displays the hallmarks of a classical GAP (e.g., RasGAP). Firstly, its interaction with eIF2 is enhanced by AlF(4)(-). Secondly, eIF5 possesses a conserved arginine (Arg15) which, like the "arginine fingers" of classical GAPs, is flanked by hydrophobic residues. Mutation of Arg15 to methionine abolishes the ability of eIF5 either to stimulate GTP hydrolysis or to support mRNA translation in vitro. Mutation studies suggest that a second conserved arginine (Arg48) also contributes to the GTPase active site of the eIF2.eIF5 complex. Our data thus show that eIF5 behaves as a classical GAP and that GTP hydrolysis during translation involves proteins extrinsic to the ribosome. Indeed, inspection of their sequences suggests that other translation factors may also act as GAPs. PMID:11166181

  13. Translation Initiation on mRNAs Bound by Nuclear Cap-binding Protein Complex CBP80/20 Requires Interaction between CBP80/20-dependent Translation Initiation Factor and Eukaryotic Translation Initiation Factor 3g*

    PubMed Central

    Choe, Junho; Oh, Nara; Park, Sungjin; Lee, Ye Kyung; Song, Ok-Kyu; Locker, Nicolas; Chi, Sung-Gil; Kim, Yoon Ki

    2012-01-01

    In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET. PMID:22493286

  14. Lentivirus-mediated knockdown of eukaryotic translation initiation factor 3 subunit D inhibits proliferation of HCT116 colon cancer cells.

    PubMed

    Yu, Xiaojun; Zheng, Bo'an; Chai, Rui

    2014-12-12

    Dysregulation of protein synthesis is emerging as a major contributory factor in cancer development. eIF3D (eukaryotic translation initiation factor 3 subunit D) is one member of the eIF3 (eukaryotic translation initiation factor 3) family, which is essential for initiation of protein synthesis in eukaryotic cells. Acquaintance with eIF3D is little since it has been identified as a dispensable subunit of eIF3 complex. Recently, eIF3D was found to embed somatic mutations in human colorectal cancers, indicating its importance for tumour progression. To further probe into its action in colon cancer, we utilized lentivirus-mediated RNA interference to knock down eIF3D expression in one colon cancer cell line HCT116. Knockdown of eIF3D in HCT116 cells significantly inhibited cell proliferation and colony formation in vitro. Flow cytometry analysis indicated that depletion of eIF3D led to cell-cycle arrest in the G2/M phase, and induced an excess accumulation of HCT116 cells in the sub-G1 phase representing apoptotic cells. Signalling pathways responsible for cell growth and apoptosis have also been found altered after eIF3D silencing, such as AMPKα (AMP-activated protein kinase alpha), Bad, PRAS40 [proline-rich Akt (PKB) substrate of 40 kDa], SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase), GSK3β and PARP [poly(ADP-ribose) polymerase]. Taken together, these findings suggest that eIF3D might play an important role in colon cancer progression.

  15. Intra-axonal synthesis of eukaryotic translation initiation factors regulates local protein synthesis and axon growth in rat sympathetic neurons.

    PubMed

    Kar, Amar N; MacGibeny, Margaret A; Gervasi, Noreen M; Gioio, Anthony E; Kaplan, Barry B

    2013-04-24

    Axonal protein synthesis is a complex process involving selective mRNA localization and translational regulation. In this study, using in situ hybridization and metabolic labeling, we show that the mRNAs encoding eukaryotic translation initiation factors eIF2B2 and eIF4G2 are present in the axons of rat sympathetic neurons and are locally translated. We also report that a noncoding microRNA, miR16, modulates the axonal expression of eIF2B2 and eIF4G2. Transfection of axons with precursor miR16 and anti-miR16 showed that local miR16 levels modulated axonal eIF2B2 and eIF4G2 mRNA and protein levels, as well as axon outgrowth. siRNA-mediated knock-down of axonal eIF2B2 and eIF4G2 mRNA also resulted in a significant decrease in axonal eIF2B2 and eIF4G2 protein. Moreover, results of metabolic labeling studies showed that downregulation of axonal eIF2B2 and eIF4G2 expression also inhibited local protein synthesis and axon growth. Together, these data provide evidence that miR16 mediates axonal growth, at least in part, by regulating the local protein synthesis of eukaryotic translation initiation factors eIF2B2 and eIF4G2 in the axon.

  16. Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists

    PubMed Central

    Jagus, Rosemary; Bachvaroff, Tsvetan R.; Joshi, Bhavesh; Place, Allen R.

    2012-01-01

    The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in “text-book” model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed. PMID:22778692

  17. Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists.

    PubMed

    Jagus, Rosemary; Bachvaroff, Tsvetan R; Joshi, Bhavesh; Place, Allen R

    2012-01-01

    The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in "text-book" model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed.

  18. Global gene expression profiling reveals widespread yet distinctive translational responses to different eukaryotic translation initiation factor 2B-targeting stress pathways.

    PubMed

    Smirnova, Julia B; Selley, Julian N; Sanchez-Cabo, Fatima; Carroll, Kathleen; Eddy, A Alan; McCarthy, John E G; Hubbard, Simon J; Pavitt, Graham D; Grant, Chris M; Ashe, Mark P

    2005-11-01

    Global inhibition of protein synthesis is a hallmark of many cellular stress conditions. Even though specific mRNAs defy this (e.g., yeast GCN4 and mammalian ATF4), the extent and variation of such resistance remain uncertain. In this study, we have identified yeast mRNAs that are translationally maintained following either amino acid depletion or fusel alcohol addition. Both stresses inhibit eukaryotic translation initiation factor 2B, but via different mechanisms. Using microarray analysis of polysome and monosome mRNA pools, we demonstrate that these stress conditions elicit widespread yet distinct translational reprogramming, identifying a fundamental role for translational control in the adaptation to environmental stress. These studies also highlight the complex interplay that exists between different stages in the gene expression pathway to allow specific preordained programs of proteome remodeling. For example, many ribosome biogenesis genes are coregulated at the transcriptional and translational levels following amino acid starvation. The transcriptional regulation of these genes has recently been connected to the regulation of cellular proliferation, and on the basis of our results, the translational control of these mRNAs should be factored into this equation.

  19. Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells

    PubMed Central

    Decarlo, Lindsey; Mestel, Celine; Barcellos-Hoff, Mary-Helen

    2015-01-01

    Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity. PMID:25986608

  20. Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells.

    PubMed

    Decarlo, Lindsey; Mestel, Celine; Barcellos-Hoff, Mary-Helen; Schneider, Robert J

    2015-08-01

    Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity.

  1. OGFOD1, a novel modulator of eukaryotic translation initiation factor 2alpha phosphorylation and the cellular response to stress.

    PubMed

    Wehner, Karen A; Schütz, Sylvia; Sarnow, Peter

    2010-04-01

    Cells possess mechanisms that permit survival and recovery from stress, several of which regulate the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). We identified the human OGFOD1 protein as a novel stress granule component that regulates the phosphorylation of eIF2alpha and the resumption of translation in cells recovering from arsenite-induced stress. Coimmunoprecipitation studies revealed that OGFOD1 associates with a small subset of stress granule proteins (G3BP1, USP10, Caprin1, and YB-1) and the ribosome in both unstressed and stressed cells. Overexpression of OGFOD1 led to increased abundance of phosphorylated eIF2alpha, both in unstressed cells and in cells exposed to arsenite-induced stress, and to accelerated apoptosis during stress. Conversely, knockdown of OGFOD1 resulted in smaller amounts of phosphorylated eIF2alpha and a faster accumulation of polyribosomes in cells recovering from stress. Finally, OGFOD1 interacted with both eIF2alpha and the eIF2alpha kinase heme-regulated inhibitor (HRI), which was identified as a novel stress granule resident. These findings argue that OGFOD1 plays important proapoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2alpha in cells subjected to arsenite-induced stress.

  2. The eukaryotic translation initiation factor eIF4E in the nucleus: taking the road less traveled

    PubMed Central

    Osborne, Michael J.; Borden, Katherine L.B.

    2014-01-01

    Summary The eukaryotic translation initiation factor eIF4E is a potent oncogene. Although eIF4E has traditional roles in translation initiation in the cytoplasm, it is also found in the nucleus, suggesting that it has activities beyond its role in protein synthesis. The road less traveled has been taken to study these nuclear activities and to understand their contribution to the oncogenic potential of eIF4E. The molecular features and biological pathways underpinning eIF4E’s nuclear mRNA export are described. New classes of eIF4E regulators have been identified and their relevance to cancer shown. The studies presented here reveal the molecular, biophysical, and structural bases for eIF4E regulation. Finally, recent clinical work targeting eIF4E in acute myeloid leukemia patients with ribavirin is discussed. In summary, these findings provide a novel paradigm for eIF4E function and the molecular basis for targeting it in leukemia patients. PMID:25510279

  3. The Rice Eukaryotic Translation Initiation Factor 3 Subunit e (OseIF3e) Influences Organ Size and Pollen Maturation

    PubMed Central

    Wang, Wenyi; Xu, Mengyun; Liu, Xuejiao; Tu, Jumin

    2016-01-01

    Eukaryotic translation initiation factor 3 (eIF3) is a large protein complex that participates in most translation initiation processes. While eIF3 has been well characterized, less is known about the roles of individual eIF3 subunits, particularly in plants. Here, we identified and characterized OseIF3e in rice (Oryza sativa L.). OseIF3e was constitutively expressed in various tissues, but most strongly in vigorously growing organs. Transgenic OseIF3e-silenced rice plants showed inhibited growth in seedling and vegetative stages. Repression of OseIF3e led to defects in pollen maturation but did not affect pollen mitosis. In rice, eIF3e interacted with eIF3 subunits b, d, e, f, h, and k, and with eIF6, forming homo- and heterodimers to initiate translation. Furthermore, OseIF3e was shown by yeast two-hybrid assay to specifically bind to inhibitors of cyclin-dependent kinases 1, 5, and 6. This interaction was mediated by the sequence of amino acid residues at positions 118–138, which included a conserved motif (IGPEQIETLYQFAKF). These results suggested although OseIF3e is not a “functional core” subunit of eIF3, it still plays crucial roles in rice growth and development, in combination with other factors. We proposed a pathway by which OseIF3e influence organ size and pollen maturation in rice, providing an opportunity to optimize plant architecture for crop breeding. PMID:27703462

  4. Translation initiation requires cell division cycle 123 (Cdc123) to facilitate biogenesis of the eukaryotic initiation factor 2 (eIF2).

    PubMed

    Perzlmaier, Angelika F; Richter, Frank; Seufert, Wolfgang

    2013-07-26

    The eukaryotic translation initiation factor 2 (eIF2) is central to the onset of protein synthesis and its modulation in response to physiological demands. eIF2, a heterotrimeric G-protein, is activated by guanine nucleotide exchange to deliver the initiator methionyl-tRNA to the ribosome. Here we report that assembly of the eIF2 complex in vivo depends on Cdc123, a cell proliferation protein conserved among eukaryotes. Mutations of CDC123 in budding yeast reduced the association of eIF2 subunits, diminished polysome levels, and increased GCN4 expression indicating that Cdc123 is critical for eIF2 activity. Cdc123 bound the unassembled eIF2γ subunit, but not the eIF2 complex, and the C-terminal domain III region of eIF2γ was both necessary and sufficient for Cdc123 binding. Alterations of the binding site revealed a strict correlation between Cdc123 binding, the biological function of eIF2γ, and its ability to assemble with eIF2α and eIF2β. Interestingly, high levels of Cdc123 neutralized the assembly defect and restored the biological function of an eIF2γ mutant. Moreover, the combined overexpression of eIF2 subunits rescued an otherwise inviable cdc123 deletion mutant. Thus, Cdc123 is a specific eIF2 assembly factor indispensable for the onset of protein synthesis. Human Cdc123 is encoded by a disease risk locus, and, therefore, eIF2 biogenesis control by Cdc123 may prove relevant for normal cell physiology and human health. This work identifies a novel step in the eukaryotic translation initiation pathway and assigns a biochemical function to a protein that is essential for growth and viability of eukaryotic cells.

  5. HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus

    PubMed Central

    Guerrero, Santiago; Batisse, Julien; Libre, Camille; Bernacchi, Serena; Marquet, Roland; Paillart, Jean-Christophe

    2015-01-01

    Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1) uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication. PMID:25606970

  6. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation.

    PubMed

    Martin, Franck; Ménétret, Jean-François; Simonetti, Angelita; Myasnikov, Alexander G; Vicens, Quentin; Prongidi-Fix, Lydia; Natchiar, S Kundhavai; Klaholz, Bruno P; Eriani, Gilbert

    2016-08-24

    Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon.

  7. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation.

    PubMed

    Martin, Franck; Ménétret, Jean-François; Simonetti, Angelita; Myasnikov, Alexander G; Vicens, Quentin; Prongidi-Fix, Lydia; Natchiar, S Kundhavai; Klaholz, Bruno P; Eriani, Gilbert

    2016-01-01

    Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon. PMID:27554013

  8. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation

    PubMed Central

    Martin, Franck; Ménétret, Jean-François; Simonetti, Angelita; Myasnikov, Alexander G.; Vicens, Quentin; Prongidi-Fix, Lydia; Natchiar, S. Kundhavai; Klaholz, Bruno P.; Eriani, Gilbert

    2016-01-01

    Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon. PMID:27554013

  9. Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.

    PubMed Central

    Rousseau, D; Kaspar, R; Rosenwald, I; Gehrke, L; Sonenberg, N

    1996-01-01

    The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8577715

  10. Eukaryotic Translation Initiation Factor 3a (eIF3a) Promotes Cell Proliferation and Motility in Pancreatic Cancer

    PubMed Central

    2016-01-01

    Identifying a target molecule that is crucially involved in pancreatic tumor growth and metastasis is necessary in developing an effective treatment. The study aimed to investigate the role of the eukaryotic translation initiation factor 3a (eIF3a) in the cell proliferation and motility in pancreatic cancer. Our data showed that the expression of eIF3a was upregulated in pancreatic ductal adenocarcinoma as compared with its expression in normal pancreatic tissues. Knockdown of eIF3a by a specific shRNA caused significant decreases in cell proliferation and clonogenic abilities in pancreatic cancer SW1990 and Capan-1 cells. Consistently, the pancreatic cancer cell growth rates were also impaired in xenotransplanted mice. Moreover, wound-healing assay showed that depletion of eIF3a significantly slowed down the wound recovery processes in SW1990 and Capan-1 cells. Transwell migration and invasion assays further showed that cell migration and invasion abilities were significantly inhibited by knockdown of eIF3a in SW1990 and Capan-1 cells. Statistical analysis of eIF3a expression in 140 cases of pancreatic ductal adenocarcinoma samples revealed that eIF3a expression was significantly associated with tumor metastasis and TNM staging. These analyses suggest that eIF3a contributes to cell proliferation and motility in pancreatic ductal adenocarcinoma. PMID:27550487

  11. Investigation of the conserved glutamate immediately following the DEAD box in eukaryotic translation initiation factor 4AI.

    PubMed

    Patel, Krishnaben; Shah, Grishma K; Kommaraju, Sai Shilpa; Low, Woon-Kai

    2014-02-01

    The DExD-box family (DEAD-box) of proteins was surveyed for eukaryotic translation initiation factor 4A-specific sequences surrounding the DEAD box. An eIF4A-unique glutamate residue (E186 in eIF4AI) was identified immediately following the D-E-A-D sequence in eIF4AI, II, and III that was found to be conserved from yeast to Man. Mutation to a selection of alternative amino acids was performed within recombinant eIF4AI expressed in Escherichia coli and mutant proteins were surveyed for RNA-dependent ATPase activity. The mutants were also investigated for changes in activity in the presence of the two eIF4AI-binding domains of eIF4GI as well as for co-purification ability to these two domains. The E186 residue was found to be of significance for RNA-dependent ATPase activity for eIF4AI alone and in the presence of eIF4AI-binding domains of eIF4GI through point-mutation analysis. Furthermore, binding interactions between eIF4AI and eIF4GI domains were also significantly influenced by mutation of E186, as observed through co-purification assays. Thus, this residue appears to be of functional significance for eIF4A. PMID:24471916

  12. Externalization and recognition by macrophages of large subunit of eukaryotic translation initiation factor 3 in apoptotic cells

    SciTech Connect

    Nakai, Yuji; Shiratsuchi, Akiko; Manaka, Junko; Nakayama, Hiroshi; Takio, Koji; Zhang Jianting; Suganuma, Tatsuo; Nakanishi, Yoshinobu . E-mail: nakanaka@kenroku.kanazawa-u.ac.jp

    2005-09-10

    We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages [C. Fujii, A. Shiratsuchi, J. Manaka, S. Yonehara, Y. Nakanishi. Cell Death Differ. 8 (2001) 1113-1122]. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis.

  13. Investigation of the conserved glutamate immediately following the DEAD box in eukaryotic translation initiation factor 4AI.

    PubMed

    Patel, Krishnaben; Shah, Grishma K; Kommaraju, Sai Shilpa; Low, Woon-Kai

    2014-02-01

    The DExD-box family (DEAD-box) of proteins was surveyed for eukaryotic translation initiation factor 4A-specific sequences surrounding the DEAD box. An eIF4A-unique glutamate residue (E186 in eIF4AI) was identified immediately following the D-E-A-D sequence in eIF4AI, II, and III that was found to be conserved from yeast to Man. Mutation to a selection of alternative amino acids was performed within recombinant eIF4AI expressed in Escherichia coli and mutant proteins were surveyed for RNA-dependent ATPase activity. The mutants were also investigated for changes in activity in the presence of the two eIF4AI-binding domains of eIF4GI as well as for co-purification ability to these two domains. The E186 residue was found to be of significance for RNA-dependent ATPase activity for eIF4AI alone and in the presence of eIF4AI-binding domains of eIF4GI through point-mutation analysis. Furthermore, binding interactions between eIF4AI and eIF4GI domains were also significantly influenced by mutation of E186, as observed through co-purification assays. Thus, this residue appears to be of functional significance for eIF4A.

  14. Eukaryotic Translation Initiation Factor 3a (eIF3a) Promotes Cell Proliferation and Motility in Pancreatic Cancer.

    PubMed

    Wang, Shu Qian; Liu, Yu; Yao, Min Ya; Jin, Jing

    2016-10-01

    Identifying a target molecule that is crucially involved in pancreatic tumor growth and metastasis is necessary in developing an effective treatment. The study aimed to investigate the role of the eukaryotic translation initiation factor 3a (eIF3a) in the cell proliferation and motility in pancreatic cancer. Our data showed that the expression of eIF3a was upregulated in pancreatic ductal adenocarcinoma as compared with its expression in normal pancreatic tissues. Knockdown of eIF3a by a specific shRNA caused significant decreases in cell proliferation and clonogenic abilities in pancreatic cancer SW1990 and Capan-1 cells. Consistently, the pancreatic cancer cell growth rates were also impaired in xenotransplanted mice. Moreover, wound-healing assay showed that depletion of eIF3a significantly slowed down the wound recovery processes in SW1990 and Capan-1 cells. Transwell migration and invasion assays further showed that cell migration and invasion abilities were significantly inhibited by knockdown of eIF3a in SW1990 and Capan-1 cells. Statistical analysis of eIF3a expression in 140 cases of pancreatic ductal adenocarcinoma samples revealed that eIF3a expression was significantly associated with tumor metastasis and TNM staging. These analyses suggest that eIF3a contributes to cell proliferation and motility in pancreatic ductal adenocarcinoma. PMID:27550487

  15. A unique binding mode of the eukaryotic translation initiation factor 4E for guiding the design of novel peptide inhibitors.

    PubMed

    Di Marino, Daniele; D'Annessa, Ilda; Tancredi, Holly; Bagni, Claudia; Gallicchio, Emilio

    2015-09-01

    The interaction between the eukaryotic translation initiation factor 4E (eIF4E) and eIF4E binding proteins (4E-BP) is a promising template for the inhibition of eIF4E and the treatment of diseases such as cancer and a spectrum of autism disorders, including the Fragile X syndrome (FXS). Here, we report an atomically detailed model of the complex between eIF4E and a peptide fragment of a 4E-BP, the cytoplasmic Fragile X interacting protein (CYFIP1). This model was generated using computer simulations with enhanced sampling from an alchemical replica exchange approach and validated using long molecular dynamics simulations. 4E-BP proteins act as post-transcriptional regulators by binding to eIF4E and preventing mRNA translation. Dysregulation of eIF4E activity has been linked to cancer, FXS, and autism spectrum disorders. Therefore, the study of the mechanism of inhibition of eIF4E by 4E-BPs is key to the development of drug therapies targeting this regulatory pathways. The results obtained in this work indicate that CYFIP1 interacts with eIF4E by an unique mode not shared by other 4E-BP proteins and elucidate the mechanism by which CYFIP1 interacts with eIF4E despite having a sequence binding motif significantly different from most 4E-BPs. Our study suggests an alternative strategy for the design of eIF4E inhibitor peptides with superior potency and specificity than currently available.

  16. A mouse model for eukaryotic translation initiation factor 2B-leucodystrophy reveals abnormal development of brain white matter.

    PubMed

    Geva, Michal; Cabilly, Yuval; Assaf, Yaniv; Mindroul, Nina; Marom, Liraz; Raini, Gali; Pinchasi, Dalia; Elroy-Stein, Orna

    2010-08-01

    Eukaryotic translation initiation factor 2B is a major housekeeping complex that governs the rate of global protein synthesis under normal and stress conditions. Mutations in any of its five subunits lead to leucoencephalopathy with vanishing white matter, an inherited chronic-progressive fatal brain disease with unknown aetiology, which is among the most prevalent childhood white matter disorders. We generated the first animal model for the disease by introducing a point mutation into the mouse Eif2b5 gene locus, leading to R132H replacement corresponding to the clinically significant human R136H mutation in the catalytic subunit. In contrast to human patients, mice homozygous for the mutant Eif2b5 allele (Eif2b5(R132H/R132H) mice) enable multiple analyses under a defined genetic background during the pre-symptomatic stages and during recovery from a defined brain insult. Time-course magnetic resonance imaging revealed for the first time the delayed development of the brain white matter due to the mutation. Electron microscopy demonstrated a higher proportion of small-calibre nerve fibres. Immunohistochemistry detected an abnormal abundance of oligodendrocytes and astrocytes in the brain of younger animals, as well as an abnormal level of major myelin proteins. Most importantly, mutant mice failed to recover from cuprizone-induced demyelination, reflecting an increased sensitivity to brain insults. The anomalous development of white matter in Eif2b5(R132H/R132H) mice underscores the importance of tight translational control to normal myelin formation and maintenance.

  17. The Structure of Eukaryotic Translation Initiation Factor-4E from Wheat Reveals a Novel Disulfide Bond1[OA

    PubMed Central

    Monzingo, Arthur F.; Dhaliwal, Simrit; Dutt-Chaudhuri, Anirvan; Lyon, Angeline; Sadow, Jennifer H.; Hoffman, David W.; Robertus, Jon D.; Browning, Karen S.

    2007-01-01

    Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m7 guanosine nucleotide at the 5′ end of eukaryotic messenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4E from wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance (NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight β-strands, three α-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m7GTP in its binding site, despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usual cap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into the cavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that are conserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallized with m7GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMR spectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced to form the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raises the possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that in oxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based binding assay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein each bind m7GTP in a similar and labile manner, with dissociation rates in the range of 20 to 100 s−1. PMID:17322339

  18. Integrin β6 can be translationally regulated by eukaryotic initiation factor 4E: Contributing to colonic tumor malignancy.

    PubMed

    Enyu, Liu; Zhengchuan, Niu; Jiayong, Wang; Benjia, Liang; Qi, Sun; Ruixi, Qin; Cheng, Peng; Khan, Abdul Qadir; Wei, Song; Jun, Niu

    2015-08-01

    It is well known that both eukaryotic initiation factor 4E (eIF4E) and integrin αvβ6 can contribute to malignant behavior of colon cancer. We have found that integrin αvβ6 and eIF4E were co-expressed and positively correlated in colon cancer tissues. Recently, deregulation of the protein synthesis apparatus has begun to gain attention as a major participant in cancer development and progression. However, the regulation of integrin β6 expression at translational level has never been investigated before. In present study, gene-silencing technique for eIF4E by small interfering RNA (siRNA) was used in all the subsequent experiments, in order to investigate whether eIF4E could translationally regulate expression of integrin β6 in colon cancer SW480 and HT-29 cell lines. Additionally, the subsequent effects of eIF4E knockdown on cellular malignant behavior were observed. siRNA in SW480 and HT-29 transfectants. Subsequently, protein expression of β6 was markedly suppressed, while mRNA expression of β6 showed no significant variation before and after eIF4E RNA interfering. Therefore, it could be seen that eIF4E could upregulate the expression of β6, without effect on β6 mRNA expression. More importantly, after treated with eIF4E siRNA, cellular migratory capacity on fibronectin of HT-29 and β6-transfected SW480 as well as their survival to 5-FU was decreased distinctly. Expression of integrin β6 could be translationally regulated by eIF4E, which subsequently contributed to tumor malignancy through enhancing cellular migration, survival, anti-apoptosis, and chemoresistance of colon cancer in vitro. Thus, targeting eIF4E in integrin αvβ6 expressing tumors can be a potential therapeutic strategy for patients with colon cancer.

  19. The eukaryotic translation initiation regulator CDC123 defines a divergent clade of ATP-grasp enzymes with a predicted role in novel protein modifications.

    PubMed

    Burroughs, A Maxwell; Zhang, Dapeng; Aravind, L

    2015-05-15

    Deciphering the origin of uniquely eukaryotic features of sub-cellular systems, such as the translation apparatus, is critical in reconstructing eukaryogenesis. One such feature is the highly conserved, but poorly understood, eukaryotic protein CDC123, which regulates the abundance of the eukaryotic translation initiation eIF2 complex and binds one of its components eIF2γ. We show that the eukaryotic protein CDC123 defines a novel clade of ATP-grasp enzymes distinguished from all other members of the superfamily by a RAGNYA domain with two conserved lysines (henceforth the R2K clade). Combining the available biochemical and genetic data on CDC123 with the inferred enzymatic function, we propose that the eukaryotic CDC123 proteins are likely to function as ATP-dependent protein-peptide ligases which modify proteins by ribosome-independent addition of an oligopeptide tag. We also show that the CDC123 family emerged first in bacteria where it appears to have diversified along with the two other families of the R2K clade. The bacterial CDC123 family members are of two distinct types, one found as part of type VI secretion systems which deliver polymorphic toxins and the other functioning as potential effectors delivered to amoeboid eukaryotic hosts. Representatives of the latter type have also been independently transferred to phylogenetically unrelated amoeboid eukaryotes and their nucleo-cytoplasmic large DNA viruses. Similarly, the two other prokaryotic R2K clade families are also proposed to participate in biological conflicts between bacteriophages and their hosts. These findings add further evidence to the recently proposed hypothesis that the horizontal transfer of enzymatic effectors from the bacterial endosymbionts of the stem eukaryotes played a fundamental role in the emergence of the characteristically eukaryotic regulatory systems and sub-cellular structures.

  20. Characterization of a eukaryotic translation initiation factor 5A homolog from Tamarix androssowii involved in plant abiotic stress tolerance

    PubMed Central

    2012-01-01

    Background The eukaryotic translation initiation factor 5A (eIF5A) promotes formation of the first peptide bond at the onset of protein synthesis. However, the function of eIF5A in plants is not well understood. Results In this study, we characterized the function of eIF5A (TaeIF5A1) from Tamarix androssowii. The promoter of TaeIF5A1 with 1,486 bp in length was isolated, and the cis-elements in the promoter were identified. A WRKY (TaWRKY) and RAV (TaRAV) protein can specifically bind to a W-box motif in the promoter of TaeIF5A1 and activate the expression of TaeIF5A1. Furthermore, TaeIF5A1, TaWRKY and TaRAV share very similar expression pattern and are all stress-responsive gene that functions in the abscisic acid (ABA) signaling pathway, indicating that they are components of a single regulatory pathway. Transgenic yeast and poplar expressing TaeIF5A1 showed elevated protein levels combined with improved abiotic stresses tolerance. Furthermore, TaeIF5A1-transformed plants exhibited enhanced superoxide dismutase (SOD) and peroxidase (POD) activities, lower electrolyte leakage and higher chlorophyll content under salt stress. Conclusions These results suggested that TaeIF5A1 is involved in abiotic stress tolerance, and is likely regulated by transcription factors TaWRKY and TaRAV both of which can bind to the W-box motif. In addition, TaeIF5A1 may mediate stress tolerance by increasing protein synthesis, enhancing ROS scavenging by improving SOD and POD activities, and preventing chlorophyll loss and membrane damage. Therefore, eIF5A may play an important role in plant adaptation to changing environmental conditions. PMID:22834699

  1. Eukaryotic Initiation Factor 4E Binding Protien Family of Protiens: Sentinels at a Translational Control Checkpoint in Lung Tumor Defense

    PubMed Central

    Kim, Yong Y; Von Weymarn, Linda; Larsson, Ola; Fan, Danhua; Underwood, Jon M; Hecht, Stephen S; Polunovsky, Vitaly A; Bitterman, Peter B

    2009-01-01

    The usurping of translational control by sustained activation of translation initiation factors is oncogenic. Here we show that the primary negative regulators of these oncogenic initiation factors - the 4E-BP protein family - operate as guardians of a translational control checkpoint in lung tumor defense. When challenged with the tobacco carcinogen NNK, 4ebp1−/−/4ebp2−/− mice showed increased sensitivity to tumorigenesis compared to their wild type counterparts. The 4E-BP deficient state per se creates pro-oncogenic, genome-wide skewing of the molecular landscape - with translational activation of genes governing angiogenesis, growth and proliferation; and translational activation of the precise cytochrome p450 enzyme isoform (CYP2A5) that bioactivates NNK into mutagenic metabolites. Our study provides in vivo proof for a translational control checkpoint in lung tumor defense. PMID:19843855

  2. The Rice Eukaryotic Translation Initiation Factor 3 Subunit f (OseIF3f) Is Involved in Microgametogenesis

    PubMed Central

    Li, Qi; Deng, Zhuyun; Gong, Chunyan; Wang, Tai

    2016-01-01

    Microgametogenesis is the post-meiotic pollen developmental phase when unicellular microspores develop into mature tricellular pollen. In rice, microgametogenesis can influence grain yields to a great degree because pollen abortion occurs more easily during microgametogenesis than during other stages of pollen development. However, our knowledge of the genes involved in microgametogenesis in rice remains limited. Due to the dependence of pollen development on the regulatory mechanisms of protein expression, we identified the encoding gene of the eukaryotic translation initiation factor 3, subunit f in Oryza sativa (OseIF3f). Immunoprecipitation combined with mass spectrometry confirmed that OseIF3f was a subunit of rice eIF3, which consisted of at least 12 subunits including eIF3a, eIF3b, eIF3c, eIF3d, eIF3e, eIF3f, eIF3g, eIF3h, eIF3i, eIF3k, eIF3l, and eIF3m. OseIF3f showed high mRNA levels in immature florets and is highly abundant in developing anthers. Subcellular localization analysis showed that OseIF3f was localized to the cytosol and the endoplasmic reticulum in rice root cells. We further analyzed the biological function of OseIF3f using the double-stranded RNA-mediated interference (RNAi) approach. The OseIF3f-RNAi lines grew normally at the vegetative stage but displayed a large reduction in seed production and pollen viability, which is associated with the down-regulation of OseIF3f. Further cytological observations of pollen development revealed that the OseIF3f-RNAi lines showed no obvious abnormalities at the male meiotic stage and the unicellular microspore stage. However, compared to the wild-type, OseIF3f-RNAi lines contained a higher percentage of arrested unicellular pollen at the bicellular stage and a higher percentage of arrested unicellular and bicellular pollen, and aborted pollen at the tricellular stage. These results indicate that OseIF3f plays a role in microgametogenesis. PMID:27200010

  3. Overexpression of eukaryotic initiation factor 5 rescues the translational defect of tpk1w in a manner that necessitates a novel phosphorylation site.

    PubMed

    Bavli-Kertselli, Ira; Melamed, Daniel; Bar-Ziv, Lavi; Volf, Hila; Arava, Yoav

    2015-02-01

    Cells respond to changes in their environment through mechanisms that often necessitate reprogramming of the translation machinery. The fastest and strongest of all tested responses is the translation inhibition observed following abrupt depletion of glucose from the media of yeast cells. The speed of the response suggests a post-translational modification of a key component of the translation machinery. This translation factor is as yet unknown. A cAMP-dependent protein kinase mutant yeast strain (tpk1(w)) that does not respond properly to glucose depletion and maintains translation was described previously. We hypothesized that the inability of tpk1(w) to arrest translation results from abnormal expression of key translation mediators. Genome-wide analysis of steady-state mRNA levels in tpk1(w) revealed underexpression of several candidates. Elevating the cellular levels of eukaryotic initiation factor (eIF) 5 by overexpression rescued the translational defect of tpk1(w). Restoring ribosomal dissociation by eIF5 necessitated an active GAP domain and multiple regions throughout this protein. Phosphoproteomics analysis of wild-type cells overexpressing eIF5 revealed increased phosphorylation in a novel site (Thr191) upon glucose depletion. Mutating this residue and introducing it into tpk1(w) abolished the ability of eIF5 to rescue the translational defect. Intriguingly, introducing this mutation into the wild-type strain did not hamper its translational response. We further show that Thr191 is phosphorylated in vitro by Casein Kinase II (CKII), and yeast cells with a mutated CKII have a reduced response to glucose depletion. These results implicate phosphorylation of eIF5 at Thr191 by CKII as one of the pathways for regulating translation upon glucose depletion.

  4. Eukaryotic initiation factor (eIF) 4F binding to barley yellow dwarf virus (BYDV) 3'-untranslated region correlates with translation efficiency.

    PubMed

    Banerjee, Bidisha; Goss, Dixie J

    2014-02-14

    Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley yellow dwarf virus (BYDV) employs a cap-independent mechanism of translation initiation that is mediated by a structural BYDV translation element (BTE) located in the 3'-UTR of its mRNA. eIF4F bound the BTE and a translationally inactive mutant with high affinity, thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5 to 164% compared with WT were studied. Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity (complex stability) correlated well with translation efficiency, whereas the "on" rate of binding did not; 2) other unidentified proteins or small molecules in wheat germ extract prevented eIF4F binding to mutant BTE but not WT BTE; 3) BTE mutant-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52-90% to ΔG° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding. Further, the eIF4F bound to the 3' BTE with higher affinity than for either m(7)G cap or tobacco etch virus internal ribosome entry site, suggesting that the 3' BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation. PMID:24379412

  5. rRNA suppressor of a eukaryotic translation initiation factor 5B/initiation factor 2 mutant reveals a binding site for translational GTPases on the small ribosomal subunit.

    PubMed

    Shin, Byung-Sik; Kim, Joo-Ran; Acker, Michael G; Maher, Kathryn N; Lorsch, Jon R; Dever, Thomas E

    2009-02-01

    The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.

  6. Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins.

    PubMed

    Nguyen, Suong; Leija, Chrisopher; Kinch, Lisa; Regmi, Sandesh; Li, Qiong; Grishin, Nick V; Phillips, Margaret A

    2015-08-01

    The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens.

  7. Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins*

    PubMed Central

    Nguyen, Suong; Leija, Chrisopher; Kinch, Lisa; Regmi, Sandesh; Li, Qiong; Grishin, Nick V.; Phillips, Margaret A.

    2015-01-01

    The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens. PMID:26082486

  8. Interaction between 25S rRNA A loop and eukaryotic translation initiation factor 5B promotes subunit joining and ensures stringent AUG selection.

    PubMed

    Hiraishi, Hiroyuki; Shin, Byung-Sik; Udagawa, Tsuyoshi; Nemoto, Naoki; Chowdhury, Wasimul; Graham, Jymie; Cox, Christian; Reid, Megan; Brown, Susan J; Asano, Katsura

    2013-09-01

    In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes.

  9. Cleavage of Eukaryotic Translation Initiation Factor 4G by Exogenously Added Hybrid Proteins Containing Poliovirus 2Apro in HeLa Cells: Effects on Gene Expression

    PubMed Central

    Novoa, Isabel; Carrasco, Luis

    1999-01-01

    Efficient cleavage of both forms of eukaryotic initiation factor 4G (eIF4G-1 and eIF4G-2) has been achieved in HeLa cells by incubation with hybrid proteins containing poliovirus 2Apro. Entry of these proteins into cells is promoted by adenovirus particles. Substantial levels of ongoing translation on preexisting cellular mRNAs still continue for several hours after eIF4G degradation. Treatment of control HeLa cells with hypertonic medium causes an inhibition of translation that is reversed upon restoration of cells to normal medium. Protein synthesis is not restored in cells lacking intact eIF4G after hypertonic treatment. Notably, induction of synthesis of heat shock proteins still occurs in cells pretreated with poliovirus 2Apro, suggesting that transcription and translation of these mRNAs takes place even in the presence of cleaved eIF4G. Finally, the synthesis of luciferase was examined in a HeLa cell line bearing the luciferase gene under control of a tetracycline-regulated promoter. Transcription of the luciferase gene and transport of the mRNA to the cytoplasm occurs at control levels in eIF4G-deficient cells. However, luciferase synthesis is strongly inhibited in these cells. These findings indicate that intact eIF4G is necessary for the translation of mRNAs not engaged in translation with the exception of heat shock mRNAs but is not necessary for the translation of mRNAs that are being translated. PMID:10082510

  10. NIa-Pro of Papaya ringspot virus interacts with Carica papaya eukaryotic translation initiation factor 3 subunit G (CpeIF3G).

    PubMed

    Gao, Le; Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2015-02-01

    The interaction of papaya eukaryotic translation initiation factor 3 subunit G (CpeIF3G) with Papaya ringspot virus (PRSV) NIa-Pro was validated using a bimolecular fluorescence complementation assay in papaya protoplasts based on the previous yeast two-hybrid assay results. The C-terminal (residues 133-239) fragment of PRSV NIa-Pro and the central domain (residues 59-167) of CpeIF3G were required for effective interaction between NIa-Pro and CpeIF3G as shown by a Sos recruitment yeast two-hybrid system with several deletion mutants of NIa-Pro and CpeIF3G. The central domain of CpeIF3G, which contains a C2HC-type zinc finger motif, is required to bind to other eIFs of the translational machinery. In addition, quantitative real-time reverse transcription PCR assay confirmed that PRSV infection leads to a 2- to 4.5-fold up-regulation of CpeIF3G mRNA in papaya. Plant eIF3G is involved in various stress response by enhancing the translation of resistance-related proteins. It is proposed that the NIa-Pro-CpeIF3G interaction may impair translation preinitiation complex assembly of defense proteins and interfere with host defense.

  11. Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII.

    PubMed

    Li, Q; Imataka, H; Morino, S; Rogers, G W; Richter-Cook, N J; Merrick, W C; Sonenberg, N

    1999-11-01

    Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions.

  12. Eukaryotic Translation Initiation Factor 4G Is Targeted for Proteolytic Cleavage by Caspase 3 during Inhibition of Translation in Apoptotic Cells

    PubMed Central

    Marissen, Wilfred E.; Lloyd, Richard E.

    1998-01-01

    Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity. PMID:9819442

  13. Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis.

    PubMed

    Gallie, Daniel R

    2016-01-15

    The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5'-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation.

  14. Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis.

    PubMed

    Gallie, Daniel R

    2016-01-15

    The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5'-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation. PMID:26578519

  15. Apoptosis-inducing factor (AIF) inhibits protein synthesis by interacting with the eukaryotic translation initiation factor 3 subunit p44 (eIF3g).

    PubMed

    Kim, Jong-Tae; Kim, Kwang Dong; Song, Eun Young; Lee, Hee Gu; Kim, Jae Wha; Kim, Jung Woo; Chae, Suhn-Kee; Kim, Eunhee; Lee, Myeong-Sok; Yang, Young; Lim, Jong-Seok

    2006-11-27

    Apoptosis-inducing factor (AIF) is a ubiquitous FAD-binding flavoprotein comprised of 613 amino acids and plays an important role in caspase-independent apoptosis. During apoptotic induction, AIF is translocated from the mitochondrial intermembrane space to the nucleus, where it interacts with DNA and activates a nuclear endonuclease. By performing a yeast two-hybrid screen with mature AIF, we have isolated the eukaryotic translation initiation factor 3 subunit p44 (eIF3g). Our deletion mutant analysis revealed that the eIF3g N-terminus interacts with the C-terminal region of AIF. The direct interaction between AIF and eIF3g was confirmed in a GST pull-down assay and also verified by the results of co-immunoprecipitation and confocal microscopy studies. Using an in vitro TNT coupled transcription-translation system, we found that mature AIF could inhibit newly-translated protein synthesis and this inhibition was significantly blocked by eIF3g competitively. These results were also confirmed in cells. In addition, mature AIF overexpression specifically resulted in the activation of caspase-7, thereby amplifying the inhibition of protein synthesis including eIF3g cleavage. Our data suggest that eIF3g is one of the cytosolic targets that interacts with mature AIF, and provide insight into the AIF's cellular functions of the inhibition of protein synthesis during apoptosis.

  16. Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling.

    PubMed Central

    Valentini, Sandro R; Casolari, Jason M; Oliveira, Carla C; Silver, Pamela A; McBride, Anne E

    2002-01-01

    The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIF5A domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIF5A may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes. PMID:11861547

  17. Antisense oligonucleotide targeting eukaryotic translation initiation factor 4E reduces growth and enhances chemosensitivity of non-small-cell lung cancer cells.

    PubMed

    Thumma, S C; Jacobson, B A; Patel, M R; Konicek, B W; Franklin, M J; Jay-Dixon, J; Sadiq, A; De, A; Graff, J R; Kratzke, R A

    2015-08-01

    Elevated levels of eukaryotic translation initiation factor 4E (eIF4E) enhance translation of many malignancy-related proteins, such as vascular endothelial growth factor (VEGF), c-Myc and osteopontin. In non-small-cell lung cancer (NSCLC), levels of eIF4E are significantly increased compared with normal lung tissue. Here, we used an antisense oligonucleotide (ASO) to inhibit the expression of eIF4E in NSCLC cell lines. eIF4E levels were significantly reduced in a dose-dependent manner in NSCLC cells treated with eIF4E-specific ASO (4EASO) compared with control ASO. Treatment of NSCLC cells with the 4EASO resulted in decreased cap-dependent complex formation, decreased cell proliferation and increased sensitivity to gemcitabine. At the molecular level, repression of eIF4E with ASO resulted in decreased expression of the oncogenic proteins VEGF, c-Myc and osteopontin, whereas expression of β-actin was unaffected. Based on these findings, we conclude that eIF4E-silencing therapy alone or in conjunction with chemotherapy represents a promising approach deserving of further investigation in future NSCLC clinical trials.

  18. Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses

    SciTech Connect

    Costa-Neto, Claudio M. . E-mail: claudio@fmrp.usp.br; Parreiras-e-Silva, Lucas T.; Ruller, Roberto; Oliveira, Eduardo B.; Miranda, Antonio; Oliveira, Laerte; Ward, Richard J.

    2006-09-01

    The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance.

  19. An eukaryotic translation initiation factor, AteIF5A-2, affects cadmium accumulation and sensitivity in Arabidopsis.

    PubMed

    Xu, Xiao-Yan; Ding, Zhong-Jie; Chen, Lei; Yan, Jin-Ying; Li, Gui-Xin; Zheng, Shao-Jian

    2015-10-01

    Cadmium (Cd) is one of the most toxic elements and can be accumulated in plants easily; meanwhile, eIF5A is a highly conserved protein in all eukaryotic organisms. The present work tried to investigate whether eIF5A is involved in Cd accumulation and sensitivity in Arabidopsis (Arabidopsis thaliana L.) by comparing the wild-type Columbia-0 (Col-0) with a knockdown mutant of AteIF5A-2, fbr12-3 under Cd stress conditions. The results showed that the mutant fbr12-3 accumulated more Cd in roots and shoots and had significantly lower chlorophyll content, shorter root length, and smaller biomass, suggesting that downregulation of AteIF5A-2 makes the mutant more Cd sensitive. Real-time polymerase chain reaction revealed that the expressions of metal transporters involved in Cd uptake and translocation including IRT1, ZIP1, AtNramp3, and AtHMA4 were significantly increased but the expressions of PCS1 and PCS2 related to Cd detoxification were decreased notably in fbr12-3 compared with Col-0. As a result, an increase in MDA and H2 O2 content but decrease in root trolox, glutathione and proline content under Cd stress was observed, indicating that a severer oxidative stress occurs in the mutant. All these results demonstrated for the first time that AteIF5A influences Cd sensitivity by affecting Cd uptake, accumulation, and detoxification in Arabidopsis.

  20. An eukaryotic translation initiation factor, AteIF5A-2, affects cadmium accumulation and sensitivity in Arabidopsis.

    PubMed

    Xu, Xiao-Yan; Ding, Zhong-Jie; Chen, Lei; Yan, Jin-Ying; Li, Gui-Xin; Zheng, Shao-Jian

    2015-10-01

    Cadmium (Cd) is one of the most toxic elements and can be accumulated in plants easily; meanwhile, eIF5A is a highly conserved protein in all eukaryotic organisms. The present work tried to investigate whether eIF5A is involved in Cd accumulation and sensitivity in Arabidopsis (Arabidopsis thaliana L.) by comparing the wild-type Columbia-0 (Col-0) with a knockdown mutant of AteIF5A-2, fbr12-3 under Cd stress conditions. The results showed that the mutant fbr12-3 accumulated more Cd in roots and shoots and had significantly lower chlorophyll content, shorter root length, and smaller biomass, suggesting that downregulation of AteIF5A-2 makes the mutant more Cd sensitive. Real-time polymerase chain reaction revealed that the expressions of metal transporters involved in Cd uptake and translocation including IRT1, ZIP1, AtNramp3, and AtHMA4 were significantly increased but the expressions of PCS1 and PCS2 related to Cd detoxification were decreased notably in fbr12-3 compared with Col-0. As a result, an increase in MDA and H2 O2 content but decrease in root trolox, glutathione and proline content under Cd stress was observed, indicating that a severer oxidative stress occurs in the mutant. All these results demonstrated for the first time that AteIF5A influences Cd sensitivity by affecting Cd uptake, accumulation, and detoxification in Arabidopsis. PMID:25559189

  1. Influenza A Virus-Induced Degradation of Eukaryotic Translation Initiation Factor 4B Contributes to Viral Replication by Suppressing IFITM3 Protein Expression

    PubMed Central

    Wang, Song; Chi, Xiaojuan; Wei, Haitao; Chen, Yuhai; Chen, Zhilong; Huang, Shile

    2014-01-01

    ABSTRACT Although alteration in host cellular translation machinery occurs in virus-infected cells, the role of such alteration and the precise pathogenic processes are not well understood. Influenza A virus (IAV) infection shuts off host cell gene expression at transcriptional and translational levels. Here, we found that the protein level of eukaryotic translation initiation factor 4B (eIF4B), an integral component of the translation initiation apparatus, was dramatically reduced in A549 cells as well as in the lung, spleen, and thymus of mice infected with IAV. The decrease in eIF4B level was attributed to lysosomal degradation of eIF4B, which was induced by viral NS1 protein. Silencing eIF4B expression in A549 cells significantly promoted IAV replication, and conversely, overexpression of eIF4B markedly inhibited the viral replication. Importantly, we observed that eIF4B knockdown transgenic mice were more susceptible to IAV infection, exhibiting faster weight loss, shorter survival time, and more-severe organ damage. Furthermore, we demonstrated that eIF4B regulated the expression of interferon-induced transmembrane protein 3 (IFITM3), a critical protein involved in immune defense against a variety of RNA viruses, including influenza virus. Taken together, our findings reveal that eIF4B plays an important role in host defense against IAV infection at least by regulating the expression of IFITM3, which restricts viral entry and thereby blocks early stages of viral production. These data also indicate that influenza virus has evolved a strategy to overcome host innate immunity by downregulating eIF4B protein. IMPORTANCE Influenza A virus (IAV) infection stimulates the host innate immune system, in part, by inducing interferons (IFNs). Secreted IFNs activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, leading to elevated transcription of a large group of IFN-stimulated genes that have antiviral function. To circumvent

  2. MicroRNA-216a inhibits the growth and metastasis of oral squamous cell carcinoma by targeting eukaryotic translation initiation factor 4B.

    PubMed

    Li, Lei; Ma, Hui-Qiang

    2015-08-01

    There is increasing evidence to suggest that microRNAs (miRNAs; miRs) are involved in the development of oral squamous cell carcinoma (OSCC). miR-216a has been identified as being involved in tumorigenesis, however, the mechanisms of miR-216a in various types of cancer, either as a tumor suppressor or as an oncogenic miRNA, and the specific regulatory role of miR-216a in OSCC remain to be elucidated. The present study demonstrated that the expression of miR-216a was significantly reduced in OSCC tissues and cell lines. Overexpression of miR-216a significantly suppressed the proliferation, colony formation, migration and invasion of the OSCC cells. In addition, eukaryotic translation initiation factor 4B (EIF4B) was identified as a direct target of miR-216a, which was observed to be upregulated in the OSCC tissues. Furthermore, overexpression of EIF4B significantly attenuated the antitumor effect of miR-216a, and a negative correlation was observed between miR-216a and EIF4B in the OSCC tissues. Taken together, these findings indicated that miR-216a has a suppressive role in OSCC cells by directly targeting EIF4B, and may function as a potential prognostic biomarker and novel therapeutic target.

  3. Skeletal muscle-specific eukaryotic translation initiation factor 2α phosphorylation controls amino acid metabolism and fibroblast growth factor 21-mediated non-cell-autonomous energy metabolism.

    PubMed

    Miyake, Masato; Nomura, Akitoshi; Ogura, Atsushi; Takehana, Kenji; Kitahara, Yoshihiro; Takahara, Kazuna; Tsugawa, Kazue; Miyamoto, Chinobu; Miura, Naoko; Sato, Ryosuke; Kurahashi, Kiyoe; Harding, Heather P; Oyadomari, Miho; Ron, David; Oyadomari, Seiichi

    2016-02-01

    The eukaryotic translation initiation factor 2α (eIF2α) phosphorylation-dependent integrated stress response (ISR), a component of the unfolded protein response, has long been known to regulate intermediary metabolism, but the details are poorly worked out. We report that profiling of mRNAs of transgenic mice harboring a ligand-activated skeletal muscle-specific derivative of the eIF2α protein kinase R-like ER kinase revealed the expected up-regulation of genes involved in amino acid biosynthesis and transport but also uncovered the induced expression and secretion of a myokine, fibroblast growth factor 21 (FGF21), that stimulates energy consumption and prevents obesity. The link between the ISR and FGF21 expression was further reinforced by the identification of a small-molecule ISR activator that promoted Fgf21 expression in cell-based screens and by implication of the ISR-inducible activating transcription factor 4 in the process. Our findings establish that eIF2α phosphorylation regulates not only cell-autonomous proteostasis and amino acid metabolism, but also affects non-cell-autonomous metabolic regulation by induced expression of a potent myokine.

  4. The Arabidopsis Eukaryotic Translation Initiation Factor eIF5A-2 Regulates Root Protoxylem Development by Modulating Cytokinin Signaling[W

    PubMed Central

    Ren, Bo; Chen, Qingguo; Hong, Sulei; Zhao, Wenming; Feng, Jian; Feng, Haizhong; Zuo, Jianru

    2013-01-01

    The phytohormone cytokinin regulates various aspects of plant growth and development, including root vascular development. In Arabidopsis thaliana, mutations in the cytokinin signaling components cause misspecification of protoxylem cell files. Auxin antagonizes cytokinin-regulated root protoxylem differentiation by inducing expression of ARABIDOPSIS PHOSPHOTRANSFER PROTEIN6 (AHP6), a negative regulator of cytokinin signaling. However, the molecular mechanism of cytokinin-regulated protoxylem differentiation is not fully understood. Here, we show that a mutation in Arabidopsis FUMONISIN B1-RESISTANT12 (FBR12), which encodes a eukaryotic translation initiation factor 5A, causes defective protoxylem development and reduced sensitivity to cytokinin. FBR12 genetically interacts with the cytokinin receptor CYTOKININ RESPONSE1 (CRE1) and downstream AHP genes, as double mutants show enhanced phenotypes. FBR12 forms a protein complex with CRE1 and AHP1, and cytokinin regulates formation of this protein complex. Intriguingly, ahp6 partially suppresses the fbr12 mutant phenotype, and the fbr12 mutation causes increased expression of AHP6, indicating that FBR12 negatively regulates AHP6. Consistent with this, ectopic expression of FBR12 in the CRE1-expressing domain partially rescues defective protoxylem development in fbr12, and overexpression of AHP6 causes an fbr12-like phenotype. These results define a regulatory role of the highly conserved FBR12 in cytokinin-mediated root protoxylem specification. PMID:24163315

  5. Aerosol delivery of eukaryotic translation initiation factor 4E-binding protein 1 effectively suppresses lung tumorigenesis in K-rasLA1 mice.

    PubMed

    Chang, S-H; Kim, J-E; Lee, J-H; Minai-Tehrani, A; Han, K; Chae, C; Cho, Y-H; Yun, J-H; Park, K; Kim, Y-S; Cho, M-H

    2013-06-01

    Conventional radiotherapy or chemotherapy for the long-term survival of patients with lung cancer is still difficult for treatment in metastatic and advanced tumors. Therefore, the safe and effective approaches to the treatment of lung cancer are needed. In this study, the effect of delivered eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) on lung cancer progression was evaluated. Recombinant adeno-associated virus (rAAV)-M3/4E-BP1 was delivered into 6-week-old K-rasLA1 lung cancer model mice through a nose-only inhalation system twice a week for 4 weeks. Long-term repeated delivery of 4E-BP1 effectively reduced tumor progression in the lungs of K-rasLA1 mice. Reduction of eIF4E by overexpression of 4E-BP1 resulted in suppression of cap-dependent protein expression of basic fibroblast growth factor (bFGF or FGF-2) and vascular endothelial growth factor (VEGF). In addition, delivered 4E-BP1 inhibited the proliferation of lung cancer cells in K-rasLA1 mice model. Our results suggest that long-term repeated viral delivery of 4E-BP1 may provide a useful tool for designing lung cancer treatment. PMID:23640516

  6. Myxoma Virus Immunomodulatory Protein M156R is a Structural Mimic of Eukaryotic Translation Initiation Factor eIF2 alpha

    SciTech Connect

    Ramelot, Theresa A.; Cort, John R.; Yee, Adelinda; Liu, Furong; Goshe, Michael B.; Edwards, Aled M.; Smith, Richard D.; Arrowsmith, Cheryl H.; Dever, Thomas E.; Kennedy, Michael A.

    2002-10-04

    M156R, the product of the myxoma virus M156R open reading frame, is a protein of unknown function. However, several homologs of M156R from other viruses are immunomodulatory proteins that bind to interferon-induced protein kinase PKR and inhibit phosphorylation of the eukaryotic translation initiation factor eIF2a. In this study, we have determined the nuclear magnetic resonance (NMR) structure of M156R, the first structure of a myxoma virus protein. The fold consists of a five-stranded antiparallel b-barrel with two of the strands connected by a long loop and a short a-helix. The similarity between M156R and the predicted S1 motif structure of eIF2a suggests that the viral homologs are pseudosubstrate inhibitors of PKR that mimic eIF2a in order to compete for binding to PKR. A homology modeled structure of the well studied vaccinia virus K3L was generated based on alignment with M156R. Residues important for binding to PKR are conserved residues on the surface of the b-barrel and in the mobile loop, identifying the putative PKR recognition motif.

  7. Multiple copies of eukaryotic translation initiation factors in Brassica rapa facilitate redundancy, enabling diversification through variation in splicing and broad-spectrum virus resistance.

    PubMed

    Nellist, Charlotte F; Qian, Wei; Jenner, Carol E; Moore, Jonathan D; Zhang, Shujiang; Wang, Xiaowu; Briggs, William H; Barker, Guy C; Sun, Rifei; Walsh, John A

    2014-01-01

    Recessive strain-specific resistance to a number of plant viruses in the Potyvirus genus has been found to be based on mutations in the eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E. We identified three copies of eIF(iso)4E in a number of Brassica rapa lines. Here we report broad-spectrum resistance to the potyvirus Turnip mosaic virus (TuMV) due to a natural mechanism based on the mis-splicing of the eIF(iso)4E allele in some TuMV-resistant B. rapa var. pekinensis lines. Of the splice variants, the most common results in a stop codon in intron 1 and a much truncated, non-functional protein. The existence of multiple copies has enabled redundancy in the host plant's translational machinery, resulting in diversification and emergence of the resistance. Deployment of the resistance is complicated by the presence of multiple copies of the gene. Our data suggest that in the B. rapa subspecies trilocularis, TuMV appears to be able to use copies of eIF(iso)4E at two loci. Transformation of different copies of eIF(iso)4E from a resistant B. rapa line into an eIF(iso)4E knockout line of Arabidopsis thaliana proved misleading because it showed that, when expressed ectopically, TuMV could use multiple copies which was not the case in the resistant B. rapa line. The inability of TuMV to access multiple copies of eIF(iso)4E in B. rapa and the broad spectrum of the resistance suggest it may be durable.

  8. Multiple copies of eukaryotic translation initiation factors in Brassica rapa facilitate redundancy, enabling diversification through variation in splicing and broad-spectrum virus resistance.

    PubMed

    Nellist, Charlotte F; Qian, Wei; Jenner, Carol E; Moore, Jonathan D; Zhang, Shujiang; Wang, Xiaowu; Briggs, William H; Barker, Guy C; Sun, Rifei; Walsh, John A

    2014-01-01

    Recessive strain-specific resistance to a number of plant viruses in the Potyvirus genus has been found to be based on mutations in the eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E. We identified three copies of eIF(iso)4E in a number of Brassica rapa lines. Here we report broad-spectrum resistance to the potyvirus Turnip mosaic virus (TuMV) due to a natural mechanism based on the mis-splicing of the eIF(iso)4E allele in some TuMV-resistant B. rapa var. pekinensis lines. Of the splice variants, the most common results in a stop codon in intron 1 and a much truncated, non-functional protein. The existence of multiple copies has enabled redundancy in the host plant's translational machinery, resulting in diversification and emergence of the resistance. Deployment of the resistance is complicated by the presence of multiple copies of the gene. Our data suggest that in the B. rapa subspecies trilocularis, TuMV appears to be able to use copies of eIF(iso)4E at two loci. Transformation of different copies of eIF(iso)4E from a resistant B. rapa line into an eIF(iso)4E knockout line of Arabidopsis thaliana proved misleading because it showed that, when expressed ectopically, TuMV could use multiple copies which was not the case in the resistant B. rapa line. The inability of TuMV to access multiple copies of eIF(iso)4E in B. rapa and the broad spectrum of the resistance suggest it may be durable. PMID:24274163

  9. Eukaryotic translation initiation factor 5A2 regulates the migration and invasion of hepatocellular carcinoma cells via pathways involving reactive oxygen species.

    PubMed

    Liu, Rong-Rong; Lv, Ya-Su; Tang, Yue-Xiao; Wang, Yan-Fang; Chen, Xiao-Ling; Zheng, Xiao-Xiao; Xie, Shang-Zhi; Cai, Ying; Yu, Jun; Zhang, Xian-Ning

    2016-04-26

    Eukaryotic translation initiation factor 5A2 (eIF5A2) has been identified as a critical gene in tumor metastasis. Research has suggested that reactive oxygen species (ROS) serve as signaling molecules in cancer cell proliferation and migration. However, the mechanisms linking eIF5A2 and ROS are not fully understood. Here, we investigated the effects of ROS on the eIF5A2-induced epithelial-mesenchymal transition (EMT) and migration in six hepatocellular carcinoma (HCC) cell lines. Western hybridization, siRNA transfection, transwell migration assays, wound-healing assays, and immunofluorescence analysis were used. The protein levels of eIF5A2 in tumor and adjacent tissue samples from 90 HCC patients with detailed clinical, pathological, and clinical follow-up data were evaluated. Overexpression of eIF5A2 was found in cancerous tissues compared with adjacent tissues. We found that eIF5A2 overexpression in HCC was associated with reduced overall survival. Knockdown of eIF5A2 and intracellular reduction of ROS significantly suppressed the invasion and metastasis of HCC cells. Interestingly, N1-guanyl-1, 7-diaminoheptane (GC7) suppressed the intracellular ROS levels. After blocking the EMT, administration of GC7 or N-acetyl-L-cysteine did not reduce cell migration further. Based on the experimental data, we concluded that inhibition of eIF5A2 alters progression of the EMT to decrease the invasion and metastasis of HCC cells via ROS-related pathways. PMID:27028999

  10. Fluorofenidone attenuates bleomycin-induced pulmonary fibrosis by inhibiting eukaryotic translation initiation factor 3a (eIF3a) in rats.

    PubMed

    Wu, Yue-Han; Li, Xian-Wei; Li, Wen-Qun; Li, Xiao-Hui; Li, Yuan-Jian; Hu, Gao-Yun; Liu, Zhao-Qian; Li, Dai

    2016-02-15

    Fluorofenidone is a novel derivative of l-mimosine. It has remarkable anti-fibrotic properties. In this study, we established that fluorofenidone ameliorates pulmonary fibrosis (PF) both in vivo and in vitro by specifically inhibiting the expression of eukaryotic translation initiation factor 3a (eIF3a). eIF3a plays an important role in the development and progression of PF. An animal model of PF was induced by intratracheal instillation of bleomycin (5mg/kg) in rats. Rats were orally administered with fluorofenidone (250, 500 mg/kg/d·[i.g.]) and pirfenidone (500 mg/kg/d·[i.g.]) for 28 days. Primary pulmonary fibroblasts were cultured to determine the effect of fluorofenidone on TGF-β1-induced (5 ng/ml) proliferation and differentiation of fibroblasts. The expression/level of eIF3a, TGF-β1, α-SMA, collagen I, and collagen III were analyzed by ELISA, real-time PCR, and western blot. The cell proliferation rate was determined by MTS assay. The results indicate that fluorofenidone significantly improves the pathological changes in lung tissues and reduces the deposition of collagen by inhibiting eIF3a in rats with bleomycin-induced PF. Moreover, in a culture of pulmonary fibroblasts, fluorofenidone decreased the up-regulation of TGF-β1-induced eIF3a by inhibiting the proliferation of cells and reducing the expression of α-SMA, collagen I, and collagen III. These findings suggest that eIF3a is a new and special target of fluorofenidone, which could be potentially used in the development of a drug that treats PF.

  11. Eukaryotic translation initiation factor 5A2 regulates the migration and invasion of hepatocellular carcinoma cells via pathways involving reactive oxygen species

    PubMed Central

    Tang, Yue-Xiao; Wang, Yan-Fang; Chen, Xiao-Ling; Zheng, Xiao-Xiao; Xie, Shang-Zhi; Cai, Ying; Yu, Jun; Zhang, Xian-Ning

    2016-01-01

    Eukaryotic translation initiation factor 5A2 (eIF5A2) has been identified as a critical gene in tumor metastasis. Research has suggested that reactive oxygen species (ROS) serve as signaling molecules in cancer cell proliferation and migration. However, the mechanisms linking eIF5A2 and ROS are not fully understood. Here, we investigated the effects of ROS on the eIF5A2-induced epithelial-mesenchymal transition (EMT) and migration in six hepatocellular carcinoma (HCC) cell lines. Western hybridization, siRNA transfection, transwell migration assays, wound-healing assays, and immunofluorescence analysis were used. The protein levels of eIF5A2 in tumor and adjacent tissue samples from 90 HCC patients with detailed clinical, pathological, and clinical follow-up data were evaluated. Overexpression of eIF5A2 was found in cancerous tissues compared with adjacent tissues. We found that eIF5A2 overexpression in HCC was associated with reduced overall survival. Knockdown of eIF5A2 and intracellular reduction of ROS significantly suppressed the invasion and metastasis of HCC cells. Interestingly, N1-guanyl-1, 7-diaminoheptane (GC7) suppressed the intracellular ROS levels. After blocking the EMT, administration of GC7 or N-acetyl-L-cysteine did not reduce cell migration further. Based on the experimental data, we concluded that inhibition of eIF5A2 alters progression of the EMT to decrease the invasion and metastasis of HCC cells via ROS-related pathways. PMID:27028999

  12. The eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NF-κB and is aberrantly regulated in Acute Myeloid Leukemia

    PubMed Central

    Hariri, Fadi; Arguello, Meztli; Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Nielsen, Torsten Holm; Hiscott, John; Mann, Koren K.; Borden, Katherine L.B.

    2015-01-01

    The eukaryotic translation initiation factor eIF4E is a potent oncogene elevated in many cancers including the M4 and M5 subtypes of acute myeloid leukemia (AML). While eIF4E RNA levels are elevated 3–10 fold in M4/M5 AML, the molecular underpinnings of this dysregulation were unknown. Here, we demonstrate that EIF4E is a direct transcriptional target of NF-κB that is dysregulated preferentially in M4 and M5 AML. In primary hematopoietic cells and in cell lines, eIF4E levels are induced by NF-κB activating stimuli. Pharmacological or genetic inhibition of NF-κB represses activation. The endogenous human EIF4E promoter recruits p65 and cRel to evolutionarily conserved κB sites in vitro and in vivo following NF-κB activation. Transcriptional activation is demonstrated by recruitment of p300 to the κB sites and phosphorylated Pol II to the transcriptional start site. In primary AML specimens, generally we observe that substantially more NF-κB complexes associate with eIF4E promoter elements in M4 and M5 AML specimens examined than in other subtypes or unstimulated normal primary hematopoietic cells. Consistently, genetic inhibition of NF-κB abrogates eIF4E RNA levels in this same population. These findings provide novel insights into the transcriptional control of eIF4E and a novel molecular basis for its dysregulation in at least a subset of M4/M5 AML specimens. PMID:23467026

  13. Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E, and the mRNA 5' cap.

    PubMed

    Modrak-Wojcik, Anna; Gorka, Michal; Niedzwiecka, Katarzyna; Zdanowski, Konrad; Zuberek, Joanna; Niedzwiecka, Anna; Stolarski, Ryszard

    2013-12-11

    Initiation is the rate-limiting step during mRNA 5' cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.

  14. Translational control of intron splicing in eukaryotes.

    PubMed

    Jaillon, Olivier; Bouhouche, Khaled; Gout, Jean-François; Aury, Jean-Marc; Noel, Benjamin; Saudemont, Baptiste; Nowacki, Mariusz; Serrano, Vincent; Porcel, Betina M; Ségurens, Béatrice; Le Mouël, Anne; Lepère, Gersende; Schächter, Vincent; Bétermier, Mireille; Cohen, Jean; Wincker, Patrick; Sperling, Linda; Duret, Laurent; Meyer, Eric

    2008-01-17

    Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.

  15. Eukaryotic initiation factor 3 (eIF3) and 5’ mRNA leader sequences as agents of translational regulation in Arabidopsis. Final report

    SciTech Connect

    von Arnim, Albrecht G.

    2015-02-04

    Protein synthesis, or translation, consumes a sizable fraction of the cell’s energy budget, estimated at 5% and up to 50% in differentiated and growing cells, respectively. Plants also invest significant energy and biomass to construct and maintain the translation apparatus. Translation is regulated by a variety of external stimuli. Compared to transcriptional control, attributes of translational control include reduced sensitivity to stochastic fluctuation, a finer gauge of control, and more rapid responsiveness to environmental stimuli. Yet, our murky understanding of translational control allows few generalizations. Consequently, translational regulation is underutilized in the context of transgene regulation, although synthetic biologists are now beginning to appropriate RNA-level gene regulation into their regulatory circuits. We also know little about how translational control contributes to the diversity of plant form and function. This project explored how an emerging regulatory mRNA sequence element, upstream open reading frames (uORFs), is integrated with the general translation initiation machinery to permit translational regulation on specific mRNAs.

  16. Overexpression of eukaryotic translation initiation factor 5A2 (EIF5A2) correlates with cell aggressiveness and poor survival in gastric cancer.

    PubMed

    Meng, Qing-Bin; Kang, Wei-Ming; Yu, Jian-Chun; Liu, Yu-Qin; Ma, Zhi-Qiang; Zhou, Li; Cui, Quan-Cai; Zhou, Wei-Xun

    2015-01-01

    Eukaryotic translation initiation factor 5A2 (EIF5A2) plays an important role in tumor progression and prognosis evaluation. However, little information is available about its potential role in gastric cancer. This study aimed to investigate the function of EIF5A2 in tumor progression and its potential mechanisms. EIF5A2 expression was measured in human gastric cancer cell lines, the immortalized gastric mucosal epithelial cell line (GES-1) and human gastric cancer tissues and knocked down by RNA interference or upregulated by EIF5A2 plasmid transfection. Cell proliferation, migration and invasion were assessed in vitro. The downstream targets of EIF5A2 were examined by western blotting. EIF5A2 and its potential target metastasis-associated protein 1 (MTA1) expression were examined in 160 pairs of human gastric cancer and adjacent non-tumor specimens using immunohistochemistry (IHC) staining, and its correlation with clinicopathological features and survival was investigated. Knockdown of EIF5A2 or MTA1 caused an apparent suppression of HGC27 cell proliferation, migration and invasion. After knockdown of EIF5A2 in HGC27 cells, E-cadherin levels were upregulated and vimentin, cyclin D1, cyclin D3, C-MYC and MTA1 levels were downregulated. Upregulation of EIF5A2 in MKN45 cells resulted in the converse. IHC results showed a positive correlation between EIF5A2 and MTA1 expression in gastric cancers (P<0.001). Both EIF5A2 and MTA1 overexpression were correlated with pT stage (P=0.018 and P=0.042), pN stage (P=0.037 and P=0.020) and lymphovascular invasion (P=0.016 and P=0.044). EIF5A2 or MTA1 overexpression was significantly associated with poor overall survival and disease-free survival (All P<0.05). Multivariate analyses identified EIF5A2 as an independent predictor for both overall survival (P=0.012) and disease-free survival (P=0.008) in gastric cancer patients. Our findings indicate that EIF5A2 upregulation plays an important oncogenic role in gastric cancer. EIF5A

  17. Homozygous mutation in the eukaryotic translation initiation factor 2alpha phosphatase gene, PPP1R15B, is associated with severe microcephaly, short stature and intellectual disability

    PubMed Central

    Kernohan, Kristin D.; Tétreault, Martine; Liwak-Muir, Urszula; Geraghty, Michael T.; Qin, Wen; Venkateswaran, Sunita; Davila, Jorge; Holcik, Martin; Majewski, Jacek; Richer, Julie; Boycott, Kym M.

    2015-01-01

    Protein translation is an essential cellular process initiated by the association of a methionyl–tRNA with the translation initiation factor eIF2. The Met-tRNA/eIF2 complex then associates with the small ribosomal subunit, other translation factors and mRNA, which together comprise the translational initiation complex. This process is regulated by the phosphorylation status of the α subunit of eIF2 (eIF2α); phosphorylated eIF2α attenuates protein translation. Here, we report a consanguineous family with severe microcephaly, short stature, hypoplastic brainstem and cord, delayed myelination and intellectual disability in two siblings. Whole-exome sequencing identified a homozygous missense mutation, c.1972G>A; p.Arg658Cys, in protein phosphatase 1, regulatory subunit 15b (PPP1R15B), a protein which functions with the PPP1C phosphatase to maintain dephosphorylated eIF2α in unstressed cells. The p.R658C PPP1R15B mutation is located within the PPP1C binding site. We show that patient cells have greatly diminished levels of PPP1R15B–PPP1C interaction, which results in increased eIF2α phosphorylation and resistance to cellular stress. Finally, we find that patient cells have elevated levels of PPP1R15B mRNA and protein, suggesting activation of a compensatory program aimed at restoring cellular homeostasis which is ineffective due to PPP1R15B alteration. PPP1R15B now joins the expanding list of translation-associated proteins which when mutated cause rare genetic diseases. PMID:26307080

  18. Engineering of ribosomal shunt-modulating eukaryotic ON riboswitches by using a cell-free translation system.

    PubMed

    Ogawa, Atsushi

    2015-01-01

    A number of natural and artificial bacterial riboswitches have been reported thus far. However, they generally function only in bacteria, not in eukaryotes. This is because of the differences of expression mechanisms (transcription, translation, and so on) between these two main types of organisms. For example, the mechanism of translation initiation is quite different between bacteria and eukaryotes, especially in ribosome loading on mRNA. While the bacterial ribosome binds to a well-conserved, internal sequence some bases before the start codon to initiate translation, the eukaryotic one is loaded on the 5' terminus with the help of certain eukaryotic initiation factors. This means not only that bacterial riboswitches regulating translation initiation are not available in eukaryotic translation systems, but also that it is physically difficult to construct eukaryotic ON riboswitches that regulate the eukaryotic canonical translation initiation, because an aptamer cannot be inserted upstream of the ribosome loading site. However, the mechanism of noncanonical translation initiation via "ribosomal shunt" enables us to design translation initiation-modulating (specifically, ribosomal shunt-modulating) eukaryotic ON riboswitches. This chapter describes a facile method for engineering these ribosomal shunt-modulating eukaryotic ON riboswitches by using a cell-free translation system. Because these riboswitches do not require hybridization switching thanks to a unique shunting mechanism, they have the major advantages of a low energy requirement for upregulation and relatively straightforward design over common hybridization switch-based ON riboswitches.

  19. Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.

    PubMed

    Papadopoulos, Evangelos; Jenni, Simon; Kabha, Eihab; Takrouri, Khuloud J; Yi, Tingfang; Salvi, Nicola; Luna, Rafael E; Gavathiotis, Evripidis; Mahalingam, Poornachandran; Arthanari, Haribabu; Rodriguez-Mias, Ricard; Yefidoff-Freedman, Revital; Aktas, Bertal H; Chorev, Michael; Halperin, Jose A; Wagner, Gerhard

    2014-08-01

    The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between β-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.

  20. Up-regulation of Translation Eukaryotic Initiation Factor 4E in Nucleophosmin 1 Haploinsufficient Cells Results in Changes in CCAAT Enhancer-binding Protein α Activity

    PubMed Central

    Khanna-Gupta, Arati; Abayasekara, Nirmalee; Levine, Michelle; Sun, Hong; Virgilio, Maria; Nia, Navid; Halene, Stephanie; Sportoletti, Paolo; Jeong, Jee-Yeong; Pandolfi, Pier Paolo; Berliner, Nancy

    2012-01-01

    NPM1 is a ubiquitously expressed nucleolar phosphoprotein, the gene for which maps to chromosome 5q35 in close proximity to a commonly deleted region associated with (del)5q, a type of myelodysplastic syndrome (MDS). This region is also a frequent target of deletions in de novo and therapy-related MDS/acute myeloid leukemia. Previous studies have shown that Npm1+/− mice develop an MDS-like disease that transforms to acute myeloid leukemia over time. To better understand the mechanism by which NPM1 haploinsufficiency causes an MDS phenotype, we generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice and demonstrated compromised neutrophil-specific gene expression in the MNPM1+/− cells. We attribute these observations to increased levels of the shorter, dominant negative leukemogenic isoform (p30) of CCAAT enhancer-binding protein α (C/EBPα). We show that this increase is caused, in part, by elevated levels of the activated translation initiation factor eIF4E, overexpression of which also increases translation of C/EBPαp30 in HEK293 cells. In a positive feedback loop, eIF4E expression is further elevated both at the mRNA and protein levels by C/EBPαp30 but not by the full-length C/EBPαp42. Re-expression of C/EBPαp42 or NPM1 but not C/EBPαp30 in MNPM1+/− cells partially rescues the myeloid phenotype. Our observations suggest that the aberrant feed-forward pathway that keeps eIF4E and C/EBPαp30 elevated in NPM1+/− cells contributes to the MDS phenotype associated with NPM1 deficiency. PMID:22851180

  1. Translational Control of Viral Gene Expression in Eukaryotes

    PubMed Central

    Gale, Michael; Tan, Seng-Lai; Katze, Michael G.

    2000-01-01

    As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell. PMID:10839817

  2. Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G

    PubMed Central

    Papadopoulos, Evangelos; Jenni, Simon; Kabha, Eihab; Takrouri, Khuloud J.; Yi, Tingfang; Salvi, Nicola; Luna, Rafael E.; Gavathiotis, Evripidis; Mahalingam, Poornachandran; Arthanari, Haribabu; Rodriguez-Mias, Ricard; Yefidoff-Freedman, Revital; Aktas, Bertal H.; Chorev, Michael; Halperin, Jose A.; Wagner, Gerhard

    2014-01-01

    The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5′ end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5′ UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer–biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between β-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E. PMID:25049413

  3. Eukaryotic translation initiation factor 5A inhibition alters physiopathology and immune responses in a “humanized” transgenic mouse model of type 1 diabetes

    PubMed Central

    Imam, Shahnawaz; Mirmira, Raghavendra G.

    2014-01-01

    Therapeutic options for treatment of type 1 diabetes (T1D) are still missing. New avenues for immune modulation need to be developed. Here we attempted at altering the diabetes outcome of our humanized model of T1D by inhibiting translation-initiation factor eIF5A hypusination in vivo. Double-transgenic (DQ8-GAD65) mice were immunized with adenoviral vectors carrying GAD65 for diabetes induction. Animals were subsequently treated with deoxyhypusine synthase (DHS) inhibitor GC7 and monitored for diabetes development over time. On one hand, helper CD4+ T cells were clearly affected by the downregulation of the eIF5A not just at the pancreas level but overall. On the other hand, the T regulatory cell component of CD4 responded with activation and proliferation significantly higher than in the non-GC7-treated controls. Female mice seemed to be more susceptible to these effects. All together, our results show for the first time that downregulation of eIF5A through inhibition of DHS altered the physiopathology and observed immune outcome of diabetes in an animal model that closely resembles human T1D. Although the development of diabetes could not be abrogated by DHS inhibition, the immunomodulatory capacity of this approach may supplement other interventions directed at increasing regulation of autoreactive T cells in T1D. PMID:24496311

  4. Coordinated movements of eukaryotic translation initiation factors eIF1, eIF1A, and eIF5 trigger phosphate release from eIF2 in response to start codon recognition by the ribosomal preinitiation complex.

    PubMed

    Nanda, Jagpreet S; Saini, Adesh K; Muñoz, Antonio M; Hinnebusch, Alan G; Lorsch, Jon R

    2013-02-22

    Accurate recognition of the start codon in an mRNA by the eukaryotic translation preinitiation complex (PIC) is essential for proper gene expression. The process is mediated by eukaryotic translation initiation factors (eIFs) in conjunction with the 40 S ribosomal subunit and (initiator) tRNA(i). Here, we provide evidence that the C-terminal tail (CTT) of eIF1A, which we previously implicated in start codon recognition, moves closer to the N-terminal domain of eIF5 when the PIC encounters an AUG codon. Importantly, this movement is coupled to dissociation of eIF1 from the PIC, a critical event in start codon recognition, and is dependent on the scanning enhancer elements in the eIF1A CTT. The data further indicate that eIF1 dissociation must be accompanied by the movement of the eIF1A CTT toward eIF5 in order to trigger release of phosphate from eIF2, which converts the latter to its GDP-bound state. Our results also suggest that release of eIF1 from the PIC and movement of the CTT of eIF1A are triggered by the same event, most likely accommodation of tRNA(i) in the P site of the 40 S subunit driven by base pairing between the start codon in the mRNA and the anticodon in tRNA(i). Finally, we show that the C-terminal domain of eIF5 is responsible for the factor's activity in antagonizing eIF1 binding to the PIC. Together, our data provide a more complete picture of the chain of molecular events that is triggered when the scanning PIC encounters an AUG start codon in the mRNA.

  5. Skeletal muscle–specific eukaryotic translation initiation factor 2α phosphorylation controls amino acid metabolism and fibroblast growth factor 21–mediated non–cell-autonomous energy metabolism

    PubMed Central

    Miyake, Masato; Nomura, Akitoshi; Ogura, Atsushi; Takehana, Kenji; Kitahara, Yoshihiro; Takahara, Kazuna; Tsugawa, Kazue; Miyamoto, Chinobu; Miura, Naoko; Sato, Ryosuke; Kurahashi, Kiyoe; Harding, Heather P.; Oyadomari, Miho; Ron, David; Oyadomari, Seiichi

    2016-01-01

    The eukaryotic translation initiation factor 2α (eIF2α) phosphorylation-dependent integrated stress response (ISR), a component of the unfolded protein response, has long been known to regulate intermediary metabolism, but the details are poorly worked out. We report that profiling of mRNAs of transgenic mice harboring a ligand-activated skeletal muscle–specific derivative of the eIF2α protein kinase R-like ER kinase revealed the expected up-regulation of genes involved in amino acid biosynthesis and transport but also uncovered the induced expression and secretion of a myokine, fibroblast growth factor 21 (FGF21), that stimulates energy consumption and prevents obesity. The link between the ISR and FGF21 expression was further reinforced by the identification of a small-molecule ISR activator that promoted Fgf21 expression in cell-based screens and by implication of the ISR-inducible activating transcription factor 4 in the process. Our findings establish that eIF2α phosphorylation regulates not only cell-autonomous proteostasis and amino acid metabolism, but also affects non–cell-autonomous metabolic regulation by induced expression of a potent myokine.—Miyake, M., Nomura, A., Ogura, A., Takehana, K., Kitahara, Y., Takahara, K., Tsugawa, K., Miyamoto, C., Miura, N., Sato, R., Kurahashi, K., Harding, H. P., Oyadomari, M., Ron, D., Oyadomari, S. Skeletal muscle–specific eukaryotic translation initiation factor 2α phosphorylation controls amino acid metabolism and fibroblast growth factor 21–mediated non–cell-autonomous energy metabolism. PMID:26487695

  6. Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2

    PubMed Central

    Gaussand, Gwénaël M. D. J.-M.; Jia, Qi; van der Graaff, Eric; Lamers, Gerda E. M.; Fransz, Paul F.; Hooykaas, Paul J. J.; de Pater, Sylvia

    2011-01-01

    From a pool of transgenic Arabidopsis (Arabidopsis thaliana) plants harboring an activator T-DNA construct, one mutant was identified that developed spontaneous necrotic spots (sns-D) on the rosette leaves under aseptic conditions. The sns-D mutation is dominant and homozygous plants are embryo lethal. The mutant produced smaller rosettes with a different number of stomata than the wild-type. DNA fragmentation in the nuclei of cells in the necrotic spots and a significant increase of caspase-3 and caspase-6 like activities in sns-D leaf extracts indicated that the sns-D mutation caused programmed cell death (PCD). The integration of the activator T-DNA caused an increase of the expression level of At1g13020, which encodes the eukaryotic translation initiation factor eIF4B2. The expression level of eIF4B2 was positively correlated with the severity of sns-D mutant phenotype. Overexpression of the eIF4B2 cDNA mimicked phenotypic traits of the sns-D mutant indicating that the sns-D mutant phenotype is indeed caused by activation tagging of eIF4B2. Thus, incorrect regulation of translation initiation may result in PCD. PMID:22639576

  7. Translational control by 5'-untranslated regions of eukaryotic mRNAs.

    PubMed

    Hinnebusch, Alan G; Ivanov, Ivaylo P; Sonenberg, Nahum

    2016-06-17

    The eukaryotic 5' untranslated region (UTR) is critical for ribosome recruitment to the messenger RNA (mRNA) and start codon choice and plays a major role in the control of translation efficiency and shaping the cellular proteome. The ribosomal initiation complex is assembled on the mRNA via a cap-dependent or cap-independent mechanism. We describe various mechanisms controlling ribosome scanning and initiation codon selection by 5' upstream open reading frames, translation initiation factors, and primary and secondary structures of the 5'UTR, including particular sequence motifs. We also discuss translational control via phosphorylation of eukaryotic initiation factor 2, which is implicated in learning and memory, neurodegenerative diseases, and cancer.

  8. Translational control by 5'-untranslated regions of eukaryotic mRNAs.

    PubMed

    Hinnebusch, Alan G; Ivanov, Ivaylo P; Sonenberg, Nahum

    2016-06-17

    The eukaryotic 5' untranslated region (UTR) is critical for ribosome recruitment to the messenger RNA (mRNA) and start codon choice and plays a major role in the control of translation efficiency and shaping the cellular proteome. The ribosomal initiation complex is assembled on the mRNA via a cap-dependent or cap-independent mechanism. We describe various mechanisms controlling ribosome scanning and initiation codon selection by 5' upstream open reading frames, translation initiation factors, and primary and secondary structures of the 5'UTR, including particular sequence motifs. We also discuss translational control via phosphorylation of eukaryotic initiation factor 2, which is implicated in learning and memory, neurodegenerative diseases, and cancer. PMID:27313038

  9. Second-generation derivatives of the eukaryotic translation initiation inhibitor pateamine A targeting eIF4A as potential anticancer agents.

    PubMed

    Low, Woon-Kai; Li, Jing; Zhu, Mingzhao; Kommaraju, Sai Shilpa; Shah-Mittal, Janki; Hull, Ken; Liu, Jun O; Romo, Daniel

    2014-01-01

    A series of pateamine A (1) derivatives were synthesized for structure/activity relationship (SAR) studies and a selection of previous generation analogs were re-evaluated based on current information regarding the mechanism of action of these translation inhibitors. Structural modifications in the new generation of derivatives focused on alterations to the C19-C22 Z,E-diene and the trienyl side chain of the previously described simplified, des-methyl, des-amino pateamine A (DMDAPatA, 2). Derivatives were tested for anti-proliferative activity in cell culture and for inhibition of mammalian cap-dependent translation in vitro. Activity was highly dependent on the rigidity and conformation of the macrolide and the functionality of the side chain. The only well tolerated substitutions were replacement of the N,N-dimethyl amino group found on the side chain of 2 with other tertiary amine groups. SAR reported here suggests that this site may be modified in future studies to improve serum stability, cell-type specificity, and/or specificity towards rapidly proliferating cells. PMID:24359706

  10. Second-generation derivatives of the eukaryotic translation initiation inhibitor pateamine A targeting eIF4A as potential anticancer agents.

    PubMed

    Low, Woon-Kai; Li, Jing; Zhu, Mingzhao; Kommaraju, Sai Shilpa; Shah-Mittal, Janki; Hull, Ken; Liu, Jun O; Romo, Daniel

    2014-01-01

    A series of pateamine A (1) derivatives were synthesized for structure/activity relationship (SAR) studies and a selection of previous generation analogs were re-evaluated based on current information regarding the mechanism of action of these translation inhibitors. Structural modifications in the new generation of derivatives focused on alterations to the C19-C22 Z,E-diene and the trienyl side chain of the previously described simplified, des-methyl, des-amino pateamine A (DMDAPatA, 2). Derivatives were tested for anti-proliferative activity in cell culture and for inhibition of mammalian cap-dependent translation in vitro. Activity was highly dependent on the rigidity and conformation of the macrolide and the functionality of the side chain. The only well tolerated substitutions were replacement of the N,N-dimethyl amino group found on the side chain of 2 with other tertiary amine groups. SAR reported here suggests that this site may be modified in future studies to improve serum stability, cell-type specificity, and/or specificity towards rapidly proliferating cells.

  11. Hsp90 binds and regulates Gcn2, the ligand-inducible kinase of the alpha subunit of eukaryotic translation initiation factor 2 [corrected].

    PubMed

    Donzé, O; Picard, D

    1999-12-01

    The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2. PMID:10567567

  12. Human inducible nitric oxide synthase (iNOS) expression depends on chromosome region maintenance 1 (CRM1)- and eukaryotic translation initiation factor 4E (elF4E)-mediated nucleocytoplasmic mRNA transport.

    PubMed

    Bollmann, Franziska; Fechir, Katrin; Nowag, Sebastian; Koch, Kathrin; Art, Julia; Kleinert, Hartmut; Pautz, Andrea

    2013-04-01

    Human inducible nitric oxide synthase (iNOS) is regulated on the expressional level mostly by post-transcriptional mechanisms modulating the mRNA stability. Another important step in the control of eukaryotic gene expression is the nucleocytoplasmic mRNA transport. Most cellular mRNAs are exported via the TAP/Nxt complex of proteins. However, some mRNAs are transported by a different mechanism involving the nuclear export receptor CRM1. Treatment of DLD-1 cells with the CRM1 inhibitor leptomycin B (LMB) or anti-CRM1 siRNAs reduced cytokine-induced iNOS expression. We could demonstrate that the iNOS mRNA is exported from the nucleus in a CRM1-dependent manner. Since CRM1 itself does not possess any RNA binding affinity, an adapter protein is needed to mediate CRM1-dependent mRNA export. Western blot experiments showed that the eukaryotic translation initiation factor eIF4E is retained in the nucleus after LMB treatment. Blockade of eIF4E by ribavirin or overexpression of the promyelocytic leukemia protein (PML) decreased iNOS expression due to reduced iNOS mRNA export from the nucleus. Transfection experiments provide evidence that the 3'-untranslated region of the iNOS mRNA is involved in eIF4E-mediated iNOS mRNA transport. In summary, CRM1 and eIF4E seem to play an important role in the nucleocytoplasmic export of human iNOS mRNA.

  13. The small molecule '1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate' and its derivatives regulate global protein synthesis by inactivating eukaryotic translation initiation factor 2-alpha.

    PubMed

    Hong, Mi-Na; Nam, Ky-Youb; Kim, Kyung Kon; Kim, So-Young; Kim, InKi

    2016-05-01

    By environmental stresses, cells can initiate a signaling pathway in which eukaryotic translation initiation factor 2-alpha (eIF2-α) is involved to regulate the response. Phosphorylation of eIF2-α results in the reduction of overall protein neogenesis, which allows cells to conserve resources and to reprogram energy usage for effective stress control. To investigate the role of eIF2-α in cell stress responses, we conducted a viability-based compound screen under endoplasmic reticulum (ER) stress condition, and identified 1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate (AMC-01) and its derivatives as eIF2-α-inactivating chemical. Molecular characterization of this signaling pathway revealed that AMC-01 induced inactivation of eIF2-α by phosphorylating serine residue 51 in a dose- and time-dependent manner, while the negative control compounds did not affect eIF2-α phosphorylation. In contrast with ER stress induction by thapsigargin, phosphorylation of eIF2-α persisted for the duration of incubation with AMC-01. By pathway analysis, AMC-01 clearly induced the activation of protein kinase RNA-activated (PKR) kinase and nuclear factor-κB (NF-κB), whereas it did not modulate the activity of PERK or heme-regulated inhibitor (HRI). Finally, we could detect a lower protein translation rate in cells incubated with AMC-01, establishing AMC-01 as a potent chemical probe that can regulate eIF2-α activity. We suggest from these data that AMC-01 and its derivative compounds can be used as chemical probes in future studies of the role of eIF2-α in protein synthesis-related cell physiology.

  14. Novel RNA-binding protein P311 binds eukaryotic translation initiation factor 3 subunit b (eIF3b) to promote translation of transforming growth factor β1-3 (TGF-β1-3).

    PubMed

    Yue, Michael M; Lv, Kaosheng; Meredith, Stephen C; Martindale, Jennifer L; Gorospe, Myriam; Schuger, Lucia

    2014-12-01

    P311, a conserved 8-kDa intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas, represents the first documented stimulator of TGF-β1-3 translation in vitro and in vivo. Here we initiated efforts to define the mechanism underlying P311 function. PONDR® (Predictor Of Naturally Disordered Regions) analysis suggested and CD confirmed that P311 is an intrinsically disordered protein, therefore requiring an interacting partner to acquire tertiary structure and function. Immunoprecipitation coupled with mass spectroscopy identified eIF3 subunit b (eIF3b) as a novel P311 binding partner. Immunohistochemical colocalization, GST pulldown, and surface plasmon resonance studies revealed that P311-eIF3b interaction is direct and has a Kd of 1.26 μm. Binding sites were mapped to the non-canonical RNA recognition motif of eIF3b and a central 11-amino acid-long region of P311, here referred to as eIF3b binding motif. Disruption of P311-eIF3b binding inhibited translation of TGF-β1, 2, and 3, as indicated by luciferase reporter assays, polysome fractionation studies, and Western blot analysis. RNA precipitation assays after UV cross-linking and RNA-protein EMSA demonstrated that P311 binds directly to TGF-β 5'UTRs mRNAs through a previously unidentified RNA recognition motif-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that, by interacting with TGF-βs 5'UTRs and eIF3b, stimulates the translation of TGF-β1, 2, and 3.

  15. Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.

    PubMed

    Saini, Adesh K; Nanda, Jagpreet S; Martin-Marcos, Pilar; Dong, Jinsheng; Zhang, Fan; Bhardwaj, Monika; Lorsch, Jon R; Hinnebusch, Alan G

    2014-09-01

    eIF5 is the GTPase activating protein (GAP) for the eIF2 · GTP · Met-tRNAi (Met) ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2 · GDP · Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui(-) mutations in numerous factors. We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.

  16. N1-guanyl-1,7-diaminoheptane sensitizes bladder cancer cells to doxorubicin by preventing epithelial-mesenchymal transition through inhibition of eukaryotic translation initiation factor 5A2 activation.

    PubMed

    Yang, Jinsong; Yu, Haogang; Shen, Mo; Wei, Wei; Xia, Lihong; Zhao, Peng

    2014-02-01

    Drug resistance greatly reduces the efficacy of doxorubicin-based chemotherapy in bladder cancer treatment; however, the underlying mechanisms are poorly understood. We aimed to investigate whether N1-guanyl-1,7-diaminoheptane (GC7), which inhibits eukaryotic translation initiation factor 5A2 (eIF5A2) activation, exerts synergistic cytotoxicity with doxorubicin in bladder cancer, and whether eIF5A2 is involved in chemoresistance to doxorubicin-based bladder cancer treatment. BIU-87, J82, and UM-UC-3 bladder cancer cells were transfected with eIF5A2 siRNA or negative control siRNA before incubation with doxorubicin alone or doxorubicin plus GC7 for 48 h. Doxorubicin cytotoxicity was enhanced by GC7 in BIU-87, J82, and UM-UC-3 cells. It significantly inhibited activity of eIF5A2, suppressed doxorubicin-induced epithelial-mesenchymal transition in BIU-87 cells, and promoted mesenchymal-epithelial transition in J82 and UM-UC-3 cells. Knockdown of eIF5A2 sensitized bladder cancer cells to doxorubicin, prevented doxorubicin-induced EMT in BIU-87 cells, and encouraged mesenchymal-epithelial transition in J82 and UM-UC-3 cells. Combination therapy with GC7 may enhance the therapeutic efficacy of doxorubicin in bladder cancer by inhibiting eIF5A2 activation and preventing epithelial-mesenchymal transition.

  17. Specificity of the deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A.

    PubMed

    Kang, Kee Ryeon; Kim, Yeon Sook; Wolff, Edith C; Park, Myung Hee

    2007-03-16

    Deoxyhypusine hydroxylase (DOHH) is a novel metalloenzyme that catalyzes the final step of the post-translational synthesis of hypusine (Nepsilon-(4-amino-2-hydroxybutyl)lysine) in the eukaryotic translation initiation factor 5A (eIF5A). Hypusine synthesis is unique in that it occurs in only one protein, denoting the strict specificity of the modification enzymes toward the substrate protein. The specificity of the interaction between eIF5A and DOHH was investigated using human eIF5A (eIF5A-1 isoform) and human recombinant DOHH. DOHH displayed a strong preference for binding the deoxyhypusine-containing form of eIF5A, over the eIF5A precursor or the hypusine-containing eIF5A, indicating a role for the deoxyhypusine residue in binding. In addition to the deoxyhypusine residue, a large portion of the eIF5A polypeptide (>20-90 amino acids) is required for effective modification by DOHH. We have identified the amino acid residues of DOHH that are critical for substrate binding by alanine substitution of 36 conserved amino acid residues. Of these, alanine substitution at Glu57, Glu90, Glu208, Glu241, Gly63, or Gly214 caused a severe impairment in eIF5A(Dhp) binding, with a complete loss of binding and activity in the E57A and E208A mutant enzymes. Only aspartate substitution mutants, E57D or E208D, retained partial activity and substrate binding, whereas alanine, glutamine, or asparagine mutants did not. These findings support a proposed model of DOHH-eIF5A binding in which the amino group(s) of the deoxyhypusine side chain of the substrate is primarily anchored by gamma-carboxyl groups of Glu57 and Glu208 at the DOHH active site. PMID:17213197

  18. Conformation transitions of eukaryotic polyribosomes during multi-round translation.

    PubMed

    Afonina, Zhanna A; Myasnikov, Alexander G; Shirokov, Vladimir A; Klaholz, Bruno P; Spirin, Alexander S

    2015-01-01

    Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic polyribosomes formed in a long-term cell-free translation system were analyzed over all the active system lifetime (20-30 translation rounds during 6-8 h in wheat germ extract at 25°C). Three distinct types of the conformations were observed: (i) circular polyribosomes, varying from ring-shaped forms to circles collapsed into double rows, (ii) linear polyribosomes, tending to acquire planar zigzag-like forms and (iii) densely packed 3D helices. At the start, during the first two rounds of translation mostly the circular (ring-shaped and double-row) polyribosomes and the linear (free-shaped and zigzag-like) polyribosomes were formed ('juvenile phase'). The progressive loading of the polyribosomes with translating ribosomes induced the opening of the circular polyribosomes and the transformation of a major part of the linear polyribosomes into the dense 3D helices ('transitional phase'). After 2 h from the beginning (about 8-10 rounds of translation) this compact form of polyribosomes became predominant, whereas the circular and linear polyribosome fractions together contained less than half of polysomal ribosomes ('steady-state phase'). The latter proportions did not change for several hours. Functional tests showed a reduced translational activity in the fraction of the 3D helical polyribosomes.

  19. Knockdown of eukaryotic translation initiation factor 4E suppresses cell growth and invasion, and induces apoptosis and cell cycle arrest in a human lung adenocarcinoma cell line.

    PubMed

    Chen, Baofu; Zhang, Bo; Xia, Lilong; Zhang, Jian; Chen, Yu; Hu, Quanteng; Zhu, Chengchu

    2015-12-01

    Eukaryotic translation initiation factor 4E (eIF4E) was shown to be upregulated in malignant human tumors. To assess the effect of downregulation of eIF4E on the proliferation and invasiveness of a human lung adenocarcinoma cell line, a short hairpin (sh)RNA targeting eIF4E was constructed and transfected into A549 human lung adenocarcinoma cells. The expression of eIF4E was determined by reverse transcription‑quantitative polymerase chain reaction and western blotting. Cell viability was assessed using a Cell Counting kit‑8, and apoptosis levels and cell cycle distribution were assessed by flow cytometry. Invasiveness was assessed using Transwell chambers. Transfection of the A549 cells with eIF4E targeting shRNA reduced the mRNA and protein expression levels of eIF4E by >70% 48 and 72 h following transfection, and eIF4E targeting shRNA‑transfected cells were significantly less viable compared with the cells transfected with scrambled shRNA. The rate of apoptosis was also significantly increased, significantly more cells were in the G0/G1 phase and fewer were in the S phase, indicating cell cycle arrest. The fraction of transfected cells migrating across Transwell inserts were also reduced. In conclusion, inhibition of eIF4E suppressed cell growth and invasion, induced apoptosis and cell cycle arrest, suggesting that eIF4E may be a potential therapeutic target in lung adenocarcinoma.

  20. Synaptic Plasticity and Translation Initiation

    ERIC Educational Resources Information Center

    Klann, Eric; Antion, Marcia D.; Banko, Jessica L.; Hou, Lingfei

    2004-01-01

    It is widely accepted that protein synthesis, including local protein synthesis at synapses, is required for several forms of synaptic plasticity. Local protein synthesis enables synapses to control synaptic strength independent of the cell body via rapid protein production from pre-existing mRNA. Therefore, regulation of translation initiation is…

  1. Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches.

    PubMed

    Anzalone, Andrew V; Lin, Annie J; Zairis, Sakellarios; Rabadan, Raul; Cornish, Virginia W

    2016-05-01

    Protein synthesis in eukaryotes is regulated by diverse reprogramming mechanisms that expand the coding capacity of individual genes. Here, we exploit one such mechanism, termed -1 programmed ribosomal frameshifting (-1 PRF), to engineer ligand-responsive RNA switches that regulate protein expression. First, efficient -1 PRF stimulatory RNA elements were discovered by in vitro selection; then, ligand-responsive switches were constructed by coupling -1 PRF stimulatory elements to RNA aptamers using rational design and directed evolution in Saccharomyces cerevisiae. We demonstrate that -1 PRF switches tightly control the relative stoichiometry of two distinct protein outputs from a single mRNA, exhibiting consistent ligand response across whole populations of cells. Furthermore, -1 PRF switches were applied to build single-mRNA logic gates and an apoptosis module in yeast. Together, these results showcase the potential for harnessing translation-reprogramming mechanisms for synthetic biology, and they establish -1 PRF switches as powerful RNA tools for controlling protein synthesis in eukaryotes. PMID:26999002

  2. Reprogramming Eukaryotic Translation with Ligand-Responsive Synthetic RNA Switches

    PubMed Central

    Anzalone, Andrew V.; Lin, Annie J.; Zairis, Sakellarios; Rabadan, Raul; Cornish, Virginia W.

    2016-01-01

    Protein synthesis in eukaryotes is regulated by diverse reprogramming mechanisms that expand the coding capacity of individual genes. Here, we exploit one such mechanism termed −1 programmed ribosomal frameshifting (−1 PRF) to engineer ligand-responsive RNA switches that regulate protein expression. First, efficient −1 PRF stimulatory RNA elements were discovered by in vitro selection; then, ligand-responsive switches were constructed by coupling −1 PRF stimulatory elements to RNA aptamers using rational design and in vivo directed evolution. We demonstrate that −1 PRF switches tightly control the relative stoichiometry of two distinct protein outputs from a single mRNA, exhibiting consistent ligand response across whole populations of cells. Furthermore, −1 PRF switches were applied to build single-mRNA logic gates and an apoptosis module in yeast. Together, these results showcase the potential for harnessing translation-reprogramming mechanisms for synthetic biology, and establish −1 PRF switches as powerful RNA tools for controlling protein synthesis in eukaryotes. PMID:26999002

  3. Archaeal aIF2B interacts with eukaryotic translation initiation factors eIF2α and eIF2Bα: implications for aIF2B function and eIF2B regulation

    PubMed Central

    Dev, Kamal; Santangelo, Thomas J.; Rothenburg, Stefan; Neculai, Dante; Dey, Madhusudan; Sicheri, Frank; Dever, Thomas E.; Reeve, John N.; Hinnebusch, Alan G.

    2009-01-01

    Translation initiation is down-regulated in eukaryotes by phosphorylation of the α subunit of eIF2, which inhibits its guanine nucleotide exchange factor eIF2B. The N-terminal S1 domain of phosphorylated eIF2α interacts with a subcomplex of eIF2B formed by the three regulatory subunits, α/GCN3, β/GCD7, and δ/GCD2, blocking the GDP-GTP exchange activity of the catalytic ε-subunit of eIF2B. These regulatory subunits have related sequences and also have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO2 fixation. Our sequence analyses predicted however that members of one phylogenetically distinct and coherent group of these archaeal proteins (designated aIF2Bs) are functional homologues of the α, β and δ subunits of eIF2B. Three of these proteins, from different Archaea, have been shown to bind in vitro to the α subunit of the archaeal aIF2 from the cognate Archaeon. In once case, the aIF2B protein was shown further to bind to the S1 domain of the α subunit of yeast eIF2 in vitro and to interact with eIF2Bα/GCN3 in vivo in yeast. The aIF2B-eIF2α interaction was however independent of eIF2α phosphorylation. Mass spectrometry has identified several proteins that copurify with aIF2B from Thermococcus kodakaraensis and these include aIF2α, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2Bε, and several large subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2α. PMID:19616556

  4. The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E.

    PubMed Central

    Ohlmann, T; Rau, M; Pain, V M; Morley, S J

    1996-01-01

    The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N-terminal (Nt) and C-terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products themselves may directly promote cap-dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES-driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap-independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule. Images PMID:8635470

  5. Eukaryotic elongation factor 2 kinase regulates the cold stress response by slowing translation elongation.

    PubMed

    Knight, John R P; Bastide, Amandine; Roobol, Anne; Roobol, Jo; Jackson, Thomas J; Utami, Wahyu; Barrett, David A; Smales, C Mark; Willis, Anne E

    2015-01-15

    Cells respond to external stress conditions by controlling gene expression, a process which occurs rapidly via post-transcriptional regulation at the level of protein synthesis. Global control of translation is mediated by modification of translation factors to allow reprogramming of the translatome and synthesis of specific proteins that are required for stress protection or initiation of apoptosis. In the present study, we have investigated how global protein synthesis rates are regulated upon mild cooling. We demonstrate that although there are changes to the factors that control initiation, including phosphorylation of eukaryotic translation initiation factor 2 (eIF2) on the α-subunit, the reduction in the global translation rate is mediated by regulation of elongation via phosphorylation of eukaryotic elongation factor 2 (eEF2) by its specific kinase, eEF2K (eukaryotic elongation factor 2 kinase). The AMP/ATP ratio increases following cooling, consistent with a reduction in metabolic rates, giving rise to activation of AMPK (5'-AMP-activated protein kinase), which is upstream of eEF2K. However, our data show that the major trigger for activation of eEF2K upon mild cooling is the release of Ca2+ ions from the endoplasmic reticulum (ER) and, importantly, that it is possible to restore protein synthesis rates in cooled cells by inhibition of this pathway at multiple points. As cooling has both therapeutic and industrial applications, our data provide important new insights into how the cellular responses to this stress are regulated, opening up new possibilities to modulate these responses for medical or industrial use at physiological or cooler temperatures.

  6. Analysis of translation initiation during stress conditions by polysome profiling.

    PubMed

    Coudert, Laëtitia; Adjibade, Pauline; Mazroui, Rachid

    2014-01-01

    Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

  7. Evolution of eukaryotic translation elongation and termination factors: variations of evolutionary rate and genetic code deviations.

    PubMed

    Moreira, David; Kervestin, Stéphanie; Jean-Jean, Olivier; Philippe, Hervé

    2002-02-01

    Translation is carried out by the ribosome and several associated protein factors through three consecutive steps: initiation, elongation, and termination. Termination remains the least understood of them, partly because of the nonuniversality of the factors involved. To get some insights on the evolution of eukaryotic translation termination, we have compared the phylogeny of the release factors eRF1 and eRF3 to that of the elongation factors EF-1alpha and EF-2, with special focus on ciliates. Our results show that these four translation proteins have experienced different modes of evolution. This is especially evident for the EF-1alpha, EF-2, and eRF1 ciliate sequences. Ciliates appear as monophyletic in the EF-2 phylogenetic tree but not in the EF-1alpha and eRF1 phylogenetic trees. This seems to be mainly because of phylogeny reconstruction artifacts (the long-branch attraction) produced by the acceleration of evolutionary rate of ciliate EF-1alpha and eRF1 sequences. Interaction with the highly divergent actin found in ciliates, or on the contrary, loss of interaction, could explain the acceleration of the evolutionary rate of the EF-1alpha sequences. In the case of ciliate eRF1 sequences, their unusually high evolutionary rate may be related to the deviations in the genetic code usage found in diverse ciliates. These deviations involve a relaxation (or even abolition) of the recognition of one or two stop codons by eRF1. To achieve this, structural changes in eRF1 are needed, and this may affect its evolutionary rate. Eukaryotic translation seems to have followed a mosaic evolution, with its different elements governed by different selective pressures. However, a correlation analysis shows that, beneath the disagreement shown by the different translation proteins, their concerted evolution can still be made apparent when they are compared with other proteins that are not involved in translation.

  8. Eukaryotic translation initiator protein 1A isoform, CCS-3, enhances the transcriptional repression of p21CIP1 by proto-oncogene FBI-1 (Pokemon/ZBTB7A).

    PubMed

    Choi, Won-Il; Kim, Youngsoo; Kim, Yuri; Yu, Mi-young; Park, Jungeun; Lee, Choong-Eun; Jeon, Bu-Nam; Koh, Dong-In; Hur, Man-Wook

    2009-01-01

    FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors. PMID:19471103

  9. Eukaryotic translation initiator protein 1A isoform, CCS-3, enhances the transcriptional repression of p21CIP1 by proto-oncogene FBI-1 (Pokemon/ZBTB7A).

    PubMed

    Choi, Won-Il; Kim, Youngsoo; Kim, Yuri; Yu, Mi-young; Park, Jungeun; Lee, Choong-Eun; Jeon, Bu-Nam; Koh, Dong-In; Hur, Man-Wook

    2009-01-01

    FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors.

  10. Sequential eukaryotic translation initiation factor 5 (eIF5) binding to the charged disordered segments of eIF4G and eIF2β stabilizes the 48S preinitiation complex and promotes its shift to the initiation mode.

    PubMed

    Singh, Chingakham Ranjit; Watanabe, Ryosuke; Chowdhury, Wasimul; Hiraishi, Hiroyuki; Murai, Marcelo J; Yamamoto, Yasufumi; Miles, David; Ikeda, Yuka; Asano, Masayo; Asano, Katsura

    2012-10-01

    During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2β bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2β-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2β-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2β mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2β and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA(i)(Met):mRNA binding to the P site.

  11. Hippuristanol - A potent steroid inhibitor of eukaryotic initiation factor 4A.

    PubMed

    Cencic, Regina; Pelletier, Jerry

    2016-01-01

    Protein synthesis and its regulatory signaling pathways play essential roles in the initiation and maintenance of the cancer phenotype. Insight obtained over the last 3 decades on the mechanisms regulating translation in normal and transformed cells have revealed that perturbed control in cancer cells may offer an Achilles' heel for the development of novel anti-neoplastic agents. Several small molecule inhibitors have been identified and characterized that target translation initiation - more specifically, the rate-limiting step where ribosomes are recruited to mRNA templates. Among these, hippuristanol, a polyhydroxysteroid from the gorgonian Isis hippuris has been found to inhibit translation initiation by blocking the activity of eukaryotic initiation factor (eIF) 4A, an essential RNA helicase involved in this process. Herein, we highlight the biological properties of this compound, its potential development as an anti-cancer agent, and its use to validate eIF4A as an anti-neoplastic target. PMID:27335721

  12. Two Arabidopsis loci encode novel eukaryotic initiation factor 4E isoforms that are functionally distinct from the conserved plant eukaryotic initiation factor 4E.

    PubMed

    Patrick, Ryan M; Mayberry, Laura K; Choy, Grace; Woodard, Lauren E; Liu, Joceline S; White, Allyson; Mullen, Rebecca A; Tanavin, Toug M; Latz, Christopher A; Browning, Karen S

    2014-04-01

    Canonical translation initiation in eukaryotes begins with the Eukaryotic Initiation Factor 4F (eIF4F) complex, made up of eIF4E, which recognizes the 7-methylguanosine cap of messenger RNA, and eIF4G, which serves as a scaffold to recruit other translation initiation factors that ultimately assemble the 80S ribosome. Many eukaryotes have secondary EIF4E genes with divergent properties. The model plant Arabidopsis (Arabidopsis thaliana) encodes two such genes in tandem loci on chromosome 1, EIF4E1B (At1g29550) and EIF4E1C (At1g29590). This work identifies EIF4E1B/EIF4E1C-type genes as a Brassicaceae-specific diverged form of EIF4E. There is little evidence for EIF4E1C gene expression; however, the EIF4E1B gene appears to be expressed at low levels in most tissues, though microarray and RNA Sequencing data support enrichment in reproductive tissue. Purified recombinant eIF4E1b and eIF4E1c proteins retain cap-binding ability and form functional complexes in vitro with eIF4G. The eIF4E1b/eIF4E1c-type proteins support translation in yeast (Saccharomyces cerevisiae) but promote translation initiation in vitro at a lower rate compared with eIF4E. Findings from surface plasmon resonance studies indicate that eIF4E1b and eIF4E1c are unlikely to bind eIF4G in vivo when in competition with eIF4E. This study concludes that eIF4E1b/eIF4E1c-type proteins, although bona fide cap-binding proteins, have divergent properties and, based on apparent limited tissue distribution in Arabidopsis, should be considered functionally distinct from the canonical plant eIF4E involved in translation initiation.

  13. Two Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E1[W][OPEN

    PubMed Central

    Patrick, Ryan M.; Mayberry, Laura K.; Choy, Grace; Woodard, Lauren E.; Liu, Joceline S.; White, Allyson; Mullen, Rebecca A.; Tanavin, Toug M.; Latz, Christopher A.; Browning, Karen S.

    2014-01-01

    Canonical translation initiation in eukaryotes begins with the Eukaryotic Initiation Factor 4F (eIF4F) complex, made up of eIF4E, which recognizes the 7-methylguanosine cap of messenger RNA, and eIF4G, which serves as a scaffold to recruit other translation initiation factors that ultimately assemble the 80S ribosome. Many eukaryotes have secondary EIF4E genes with divergent properties. The model plant Arabidopsis (Arabidopsis thaliana) encodes two such genes in tandem loci on chromosome 1, EIF4E1B (At1g29550) and EIF4E1C (At1g29590). This work identifies EIF4E1B/EIF4E1C-type genes as a Brassicaceae-specific diverged form of EIF4E. There is little evidence for EIF4E1C gene expression; however, the EIF4E1B gene appears to be expressed at low levels in most tissues, though microarray and RNA Sequencing data support enrichment in reproductive tissue. Purified recombinant eIF4E1b and eIF4E1c proteins retain cap-binding ability and form functional complexes in vitro with eIF4G. The eIF4E1b/eIF4E1c-type proteins support translation in yeast (Saccharomyces cerevisiae) but promote translation initiation in vitro at a lower rate compared with eIF4E. Findings from surface plasmon resonance studies indicate that eIF4E1b and eIF4E1c are unlikely to bind eIF4G in vivo when in competition with eIF4E. This study concludes that eIF4E1b/eIF4E1c-type proteins, although bona fide cap-binding proteins, have divergent properties and, based on apparent limited tissue distribution in Arabidopsis, should be considered functionally distinct from the canonical plant eIF4E involved in translation initiation. PMID:24501003

  14. cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

    PubMed Central

    Graves, L M; Bornfeldt, K E; Argast, G M; Krebs, E G; Kong, X; Lin, T A; Lawrence, J C

    1995-01-01

    Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7638171

  15. Protein phylogeny of translation elongation factor EF-1 alpha suggests microsporidians are extremely ancient eukaryotes.

    PubMed

    Kamaishi, T; Hashimoto, T; Nakamura, Y; Nakamura, F; Murata, S; Okada, N; Okamoto, K; Shimizu, M; Hasegawa, M

    1996-02-01

    Partial regions of the mRNA encoding a major part of translation elongation factor 1 alpha (EF-1 alpha) from a mitochondrion-lacking protozoan, Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1 alpha's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed, G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. PMID:8919877

  16. Alcohol myopathy: impairment of protein synthesis and translation initiation.

    PubMed

    Lang, C H; Kimball, S R; Frost, R A; Vary, T C

    2001-05-01

    Alcohol consumption leads to numerous morphological, biochemical and functional changes in skeletal and cardiac muscle. One such change observed in both tissues after either acute alcohol intoxication or chronic alcohol consumption is a characteristic decrease in the rate of protein synthesis. A decrease in translation efficiency appears to be responsible for at least part of the reduction. This review highlights advances in determining the molecular mechanisms by which alcohol impairs protein synthesis and places these observations in context of earlier studies on alcoholic myopathy. Both acute and chronic alcohol administration impairs translational control by modulating various aspects of peptide-chain initiation. Moreover, this alcohol-induced impairment in initiation is associated with a decreased availability of eukaryotic initiation factor (eIF) 4E in striated muscle, as evidenced by an increase in the amount of the inactive eIF4E.4E-BP1 complex and decrease in the active eIF4E.eIF4G complex. In contrast, alcohol does not produce consistent alterations in the control of translation initiation by the eIF2 system. The etiology of these changes remain unresolved. However, defects in the availability or effectiveness of various anabolic hormones, particularly insulin-like growth factor-I, are consistent with the alcohol-induced decrease in protein synthesis and translation initiation.

  17. Reinitiation and other unconventional posttermination events during eukaryotic translation.

    PubMed

    Skabkin, Maxim A; Skabkina, Olga V; Hellen, Christopher U T; Pestova, Tatyana V

    2013-07-25

    During ribosome recycling, posttermination complexes are dissociated by ABCE1 and eRF1 into 60S and tRNA/mRNA-associated 40S subunits, after which tRNA and mRNA are released by eIF1/eIF1A, Ligatin, or MCT-1/DENR. Occasionally, 40S subunits remain associated with mRNA and reinitiate at nearby AUGs. We recapitulated reinitiation using a reconstituted mammalian translation system. The presence of eIF2, eIF3, eIF1, eIF1A, and Met-tRNAi(Met) was sufficient for recycled 40S subunits to remain on mRNA, scan bidirectionally, and reinitiate at upstream and downstream AUGs if mRNA regions flanking the stop codon were unstructured. Imposition of 3' directionality additionally required eIF4F. Strikingly, posttermination ribosomes were not stably anchored on mRNA and migrated bidirectionally to codons cognate to the P site tRNA. Migration depended on the mode of peptide release (puromycin > eRF1⋅eRF3) and nature of tRNA and was enhanced by eEF2. The mobility of posttermination ribosomes suggests that some reinitiation events could involve 80S ribosomes rather than 40S subunits.

  18. Evolutionarily Conserved Binding of Translationally Controlled Tumor Protein to Eukaryotic Elongation Factor 1B*

    PubMed Central

    Wu, Huiwen; Gong, Weibin; Yao, Xingzhe; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2015-01-01

    Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP. PMID:25635048

  19. Nitric oxide: a regulator of eukaryotic initiation factor 2 kinases.

    PubMed

    Tong, Lingying; Heim, Rachel A; Wu, Shiyong

    2011-06-15

    Generation of nitric oxide (NO(•)) can upstream induce and downstream mediate the kinases that phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2α), which plays a critical role in regulating gene expression. There are four known eIF2α kinases (EIF2AKs), and NO(•) affects each one uniquely. Whereas NO(•) directly activates EIF2AK1 (HRI), it indirectly activates EIF2AK3 (PERK). EIF2AK4 (GCN2) is activated by depletion of l-arginine, which is used by nitric oxide synthase (NOS) during the production of NO(•). Finally EIF2AK2 (PKR), which can mediate inducible NOS expression and therefore NO(•) production, can also be activated by NO(•). The production of NO(•) and activation of EIF2AKs coordinately regulate physiological and pathological events such as innate immune response and cell apoptosis. PMID:21463677

  20. Crystal Structure of Hypusine-Containing Translation Factor eIF5A Bound to a Rotated Eukaryotic Ribosome.

    PubMed

    Melnikov, Sergey; Mailliot, Justine; Shin, Byung-Sik; Rigger, Lukas; Yusupova, Gulnara; Micura, Ronald; Dever, Thomas E; Yusupov, Marat

    2016-09-11

    Eukaryotic translation initiation factor eIF5A promotes protein synthesis by resolving polyproline-induced ribosomal stalling. Here, we report a 3.25-Å resolution crystal structure of eIF5A bound to the yeast 80S ribosome. The structure reveals a previously unseen conformation of an eIF5A-ribosome complex and highlights a possible functional link between conformational changes of the ribosome during protein synthesis and the eIF5A-ribosome association.

  1. Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome

    PubMed Central

    Schmidt, Christian; Becker, Thomas; Heuer, André; Braunger, Katharina; Shanmuganathan, Vivekanandan; Pech, Markus; Berninghausen, Otto; Wilson, Daniel N.; Beckmann, Roland

    2016-01-01

    During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis. PMID:26715760

  2. Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome.

    PubMed

    Schmidt, Christian; Becker, Thomas; Heuer, André; Braunger, Katharina; Shanmuganathan, Vivekanandan; Pech, Markus; Berninghausen, Otto; Wilson, Daniel N; Beckmann, Roland

    2016-02-29

    During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis. PMID:26715760

  3. The emerging role of rectified thermal fluctuations in initiator aa-tRNA- and start codon selection during translation initiation

    PubMed Central

    Caban, Kelvin; Gonzalez, Ruben L.

    2016-01-01

    Decades of genetic, biochemical, biophysical, and structural studies suggest that the conformational dynamics of the translation machinery (TM), of which the ribosome is the central component, play a fundamental role in the mechanism and regulation of translation. More recently, single-molecule fluorescence resonance energy transfer (smFRET) studies have provided a unique and powerful approach for directly monitoring the real-time dynamics of the TM. Indeed, smFRET studies of the elongation stage of translation have significantly enriched our understanding of the mechanisms through which stochastic, thermally driven conformational fluctuations of the TM are exploited to drive and regulate the individual steps of translation elongation1. Beyond translation elongation, smFRET studies of the conformational dynamics of the initiation stage of translation offer great potential for providing mechanistic information that has thus far remained difficult or impossible to obtain using traditional methods. This is particularly true of the mechanisms through which the accuracy of initiator tRNA- and start codon selection is established during translation initiation. Given that translation initiation is a major checkpoint for regulating the translation of mRNAs, obtaining such mechanistic information holds great promise for our understanding of the translational regulation of gene expression. Here, we provide an overview of the bacterial translation initiation pathway, summarize what is known regarding the biochemical functions of the IFs, and discuss various new and exciting mechanistic insights that have emerged from several recently published smFRET studies of the mechanisms that guide initiator tRNA- and start codon selection during translation initiation. These studies provide a springboard for future investigations of the conformational dynamics of the more complex eukaryotic translation initiation pathway and mechanistic studies of the role of translational regulation of

  4. Eukaryotic RNAse H shares a conserved domain with caulimovirus proteins that facilitate translation of polycistronic RNA.

    PubMed Central

    Mushegian, A R; Edskes, H K; Koonin, E V

    1994-01-01

    RNAse H (RNH1 protein) from the trypanosomatid Crithidia fasciculata has a functionally uncharacterized N-terminal domain dispensable for the RNAse H activity. Using computer methods for database search and multiple alignment, we show that the N-terminal domains of RNH1 and its homologue encoded by a cDNA from chicken lens are related to the conserved domain in caulimovirus ORF VI product that facilitates translation of polycistronic virus RNA in plant cells. We hypothesize that the N-terminal domain of eukaryotic RNAse H performs an as yet uncharacterized regulatory function, possibly in mRNA translation or turnover. PMID:7937142

  5. Granzyme B Inhibits Vaccinia Virus Production through Proteolytic Cleavage of Eukaryotic Initiation Factor 4 Gamma 3

    PubMed Central

    Marcet-Palacios, Marcelo; Duggan, Brenda Lee; Shostak, Irene; Barry, Michele; Geskes, Tracy; Wilkins, John A.; Yanagiya, Akiko; Sonenberg, Nahum; Bleackley, R. Chris

    2011-01-01

    Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD1408S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery. PMID:22194691

  6. Cap-independent translation initiation in Xenopus oocytes.

    PubMed Central

    Keiper, B D; Rhoads, R E

    1997-01-01

    Eukaryotic cellular mRNAs contain a cap at their 5'-ends, but some viral and cellular mRNAs bypass the cap-dependent mechanism of translation initiation in favor of internal entry of ribosomes at specific RNA sequences. Cap-dependent initiation requires intact initiation factor eIF4G (formerly eIF-4gamma, eIF-4Fgamma or p220), whereas internal initiation can proceed with eIF4G cleaved by picornaviral 2A or L proteases. Injection of recombinant coxsackievirus B4 protease 2A into Xenopus oocytes led to complete cleavage of endogenous eIF4G, but protein synthesis decreased by only 35%. Co-injection of edeine reduced synthesis by >90%, indicating that eIF4G-independent synthesis involved ongoing initiation. The spectrum of endogenous proteins synthesized was very similar in the presence or absence of intact eIF4G. Translation of exogenous rabbit globin mRNA, by contrast, was drastically inhibited by eIF4G cleavage. The N-terminal cleavage product of eIF4G (cpN), which binds eIF4E, was completely degraded within 6-12 h, while the C-terminal cleavage product (cpC), which binds to eIF3 and eIF4A, was more stable over the same period. Thus, translation initiation of most endogenous mRNAs inXenopusoocytes requires no eIF4G, or perhaps only cpC, suggesting a cap-independent mechanism. PMID:9016570

  7. The C-terminal region of eukaryotic translation initiation factor 3a (eIF3a) promotes mRNA recruitment, scanning, and, together with eIF3j and the eIF3b RNA recognition motif, selection of AUG start codons.

    PubMed

    Chiu, Wen-Ling; Wagner, Susan; Herrmannová, Anna; Burela, Laxminarayana; Zhang, Fan; Saini, Adesh K; Valásek, Leos; Hinnebusch, Alan G

    2010-09-01

    The C-terminal domain (CTD) of the a/Tif32 subunit of budding yeast eukaryotic translation initiation factor 3 (eIF3) interacts with eIF3 subunits j/Hcr1 and b/Prt1 and can bind helices 16 to 18 of 18S rRNA, suggesting proximity to the mRNA entry channel of the 40S subunit. We have identified substitutions in the conserved Lys-Glu-Arg-Arg (KERR) motif and in residues of the nearby box6 element of the a/Tif32 CTD that impair mRNA recruitment by 43S preinitiation complexes (PICs) and confer phenotypes indicating defects in scanning and start codon recognition. The normally dispensable CTD of j/Hcr1 is required for its binding to a/Tif32 and to mitigate the growth defects of these a/Tif32 mutants, indicating physical and functional interactions between these two domains. The a/Tif32 CTD and the j/Hcr1 N-terminal domain (NTD) also interact with the RNA recognition motif (RRM) in b/Prt1, and mutations in both subunits that disrupt their interactions with the RRM increase leaky scanning of an AUG codon. These results, and our demonstration that the extreme CTD of a/Tif32 binds to Rps2 and Rps3, lead us to propose that the a/Tif32 CTD directly stabilizes 43S subunit-mRNA interaction and that the b/Prt1-RRM-j/Hcr1-a/Tif32-CTD module binds near the mRNA entry channel and regulates the transition between scanning-conducive and initiation-competent conformations of the PIC.

  8. Molecular characterization and functional analysis of subunit 7 of eukaryotic initiation factor 3 from Eimeria tenella.

    PubMed

    Han, Hongyu; Kong, Chunlin; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Zhai, Qi; Liang, Siting; Li, Sha; Yang, Shihan; Huang, Bing

    2015-07-01

    The initiation of translation in eukaryotic cells is stimulated by proteins known as initiation factors (eIFs). A structurally complex eIF composed of multiple subunits, eIF3 has been shown to have various functions in translation in a variety of eukaryotes. Until now, little is known about eIF3 in Eimeria tenella. Based on a previously identified expressed sequence tag(EST), we cloned the eIF3 subunit 7 gene (EteIF3s7) from E. tenella by rapid amplification of the cDNA ends(RACE). The 2278-bp full-length complementary DNA of EteIF3s7 contained a 1716-bp open reading frame (ORF) that encoded a 571-amino acid (aa) polypeptide. The EteIF3s7 protein contained the subunit 7 domain that is characteristic of members of the eIF3 zeta superfamily. The levels of EteIF3s7 messenger RNA and protein were higher in second generation merozoites than in sporulated oocysts, unsporulated oocysts, or sporozoites, and the EteIF3s7 protein was barely detectable in unsporulated oocysts. Our immunofluorescence analysis showed that the EteIF3s7 protein was uniformly distributed throughout the cytoplasm of sporozoites. After sporozoites were incubated in complete medium, the EteIF3s7 protein localized to the anterior region of the parasite. Following the first schizogenous division, the protein was uniformly dispersed in trophozoites, immature schizonts, and mature schizonts, and the EteIF3s7 protein was observed to be closely associated with the parasitophorous vacuole membrane. An anti-rEteIF3s7 polyclonal antibody inhibited the ability of E. tenella to invade DF-1 cells, which suggested that EteIF3s7 might be involved in host cell invasion and required for the growth of the parasite in the host.

  9. A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells

    PubMed Central

    Cao, Jicong; Arha, Manish; Sudrik, Chaitanya; Mukherjee, Abhirup; Wu, Xia; Kane, Ravi S.

    2015-01-01

    We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein–RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5′ untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells. PMID:25845589

  10. A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells.

    PubMed

    Cao, Jicong; Arha, Manish; Sudrik, Chaitanya; Mukherjee, Abhirup; Wu, Xia; Kane, Ravi S

    2015-04-30

    We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein-RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5' untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells.

  11. Cordycepin as a sensitizer to tumour necrosis factor (TNF)-α-induced apoptosis through eukaryotic translation initiation factor 2α (eIF2α)- and mammalian target of rapamycin complex 1 (mTORC1)-mediated inhibition of nuclear factor (NF)-κB

    PubMed Central

    Kadomatsu, M; Nakajima, S; Kato, H; Gu, L; Chi, Y; Yao, J; Kitamura, M

    2012-01-01

    Cordycepin (3′-deoxyadenosine) is one of the major bioactive substances produced by Cordyceps militaris, a traditional medicinal mushroom. Cordycepin possesses several biological activities, including both pro-apoptotic and anti-apoptotic properties. In the present report, we investigated an effect of cordycepin on the survival of cells exposed to tumour necrosis factor (TNF)-α. We found that subtoxic doses of cordycepin increased susceptibility of cells to TNF-α-induced apoptosis. It was associated with suppression of nuclear factor-κB (NF-κB), a major prosurvival component involved in TNF-α signalling. The adenosine transporter and A3 adenosine receptor, but not A1 and A2 adenosine receptors, mediated both anti-NF-κB and pro-apoptotic effects. We found that cordycepin had the potential to phosphorylate eukaryotic translation initiation factor 2α (eIF2α) and that activation of eIF2α mimicked the suppressive effect of cordycepin on the NF-κB pathway. Furthermore, activation of eIF2α sensitized cells to TNF-α-induced apoptosis. To identify molecular events downstream of eIF2α, the role of mammalian target of rapamycin complex 1 (mTORC1) was examined. Selective activation of 3eIF2α, as well as treatment with cordycepin, caused phosphorylation of mTORC1. Rapamycin, an inhibitor of mTORC1, significantly reversed the suppressive effects of eIF2α on NF-κB. These results suggest that cordycepin sensitizes cells to TNF-α-induced apoptosis, at least in part, via induction of the eIF2α–mTORC1 pathway and consequent suppression of NF-κB. PMID:22519596

  12. Initiator Recognition in a Primitive Eukaryote: IBP39, an Initiator-Binding Protein from Trichomonas vaginalis

    PubMed Central

    Liston, David R.; Lau, Audrey O. T.; Ortiz, Diana; Smale, Stephen T.; Johnson, Patricia J.

    2001-01-01

    While considerable progress has been made in understanding the mechanisms of transcription in higher eukaryotes, transcription in single-celled, primitive eukaryotes remains poorly understood. Promoters of protein-encoding genes in the parasitic protist Trichomonas vaginalis, which represents one of the deepest-branching eukaryotic lineages, have a bipartite structure with gene-specific regulatory elements and a conserved core promoter encompassing the transcription start site. Core promoters in T. vaginalis appear to consist solely of a highly conserved initiator (Inr) element that is both a structural and a functional homologue of its metazoan counterpart. Using DNA affinity chromatography, we have isolated an Inr-binding protein from T. vaginalis. Cloning of the gene encoding the Inr binding protein identified a novel 39-kDa protein (IBP39). We show that IBP39 binds to both double and single Inr motifs found in T. vaginalis genes and that binding requires the conserved nucleotides necessary for Inr function in vivo. Analyses of the cloned IBP39 gene revealed no homology at the protein sequence level with identified proteins in other organisms or the presence of known DNA-binding domains. The relationship between IBP39 and Inr-binding proteins in metazoa presents interesting evolutionary questions. PMID:11604521

  13. Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity.

    PubMed

    Fan-Minogue, Hua; Bedwell, David M

    2008-01-01

    Recent studies of prokaryotic ribosomes have dramatically increased our knowledge of ribosomal RNA (rRNA) structure, functional centers, and their interactions with antibiotics. However, much less is known about how rRNA function differs between prokaryotic and eukaryotic ribosomes. The core decoding sites are identical in yeast and human 18S rRNAs, suggesting that insights obtained in studies with yeast rRNA mutants can provide information about ribosome function in both species. In this study, we examined the importance of key nucleotides of the 18S rRNA decoding site on ribosome function and aminoglycoside susceptibility in Saccharomyces cerevisiae cells expressing homogeneous populations of mutant ribosomes. We found that residues G577, A1755, and A1756 (corresponding to Escherichia coli residues G530, A1492, and A1493, respectively) are essential for cell viability. We also found that residue G1645 (A1408 in E. coli) and A1754 (G1491 in E. coli) both make significant and distinct contributions to aminoglycoside resistance. Furthermore, we found that mutations at these residues do not alter the basal level of translational accuracy, but influence both paromomycin-induced misreading of sense codons and readthrough of stop codons. This study represents the most comprehensive mutational analysis of the eukaryotic decoding site to date, and suggests that many fundamental features of decoding site function are conserved between prokaryotes and eukaryotes.

  14. Mutations in eukaryotic 18S ribosomal RNA affect translational fidelity and resistance to aminoglycoside antibiotics.

    PubMed

    Chernoff, Y O; Vincent, A; Liebman, S W

    1994-02-15

    Mutations have been created in the Saccharomyces cerevisiae 18S rRNA gene that correspond to those known to be involved in the control of translational fidelity or antibiotic resistance in prokaryotes. Yeast strains, in which essentially all chromosomal rDNA repeats are deleted and all cellular rRNAs are encoded by plasmid, have been constructed that contain only mutant 18S rRNA. In Escherichia coli, a C-->U substitution at position 912 of the small subunit rRNA causes streptomycin resistance. Eukaryotes normally carry U at the corresponding position and are naturally resistant to streptomycin. We show that a U-->C transition (rdn-4) at this position of the yeast 18S rRNA gene decreases resistance to streptomycin. The rdn-4 mutation also increases resistance to paromomycin and G-418, and inhibits nonsense suppression induced by paromomycin. The same phenotypes, as well as a slow growth phenotype, are also associated with rdn-2, whose prokaryotic counterpart, 517 G-->A, manifests itself as a suppressor rather than an antisuppressor. Neither rdn-2- nor rdn-4-related phenotypes could be detected in the presence of the normal level of wild-type rDNA repeats. Our data demonstrate that eukaryotic rRNA is involved in the control of translational fidelity, and indicate that rRNA features important for interactions with aminoglycosides have been conserved throughout evolution.

  15. Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6.

    PubMed

    Klinge, Sebastian; Voigts-Hoffmann, Felix; Leibundgut, Marc; Arpagaus, Sofia; Ban, Nenad

    2011-11-18

    Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.

  16. Eukaryotic elongation factor 2 controls TNF-α translation in LPS-induced hepatitis

    PubMed Central

    González-Terán, Bárbara; Cortés, José R.; Manieri, Elisa; Matesanz, Nuria; Verdugo, ρngeles; Rodríguez, María E.; González-Rodríguez, ρgueda; Valverde, ρngela; Martín, Pilar; Davis, Roger J.; Sabio, Guadalupe

    2012-01-01

    Bacterial LPS (endotoxin) has been implicated in the pathogenesis of acute liver disease through its induction of the proinflammatory cytokine TNF-α. TNF-α is a key determinant of the outcome in a well-established mouse model of acute liver failure during septic shock. One possible mechanism for regulating TNF-α expression is through the control of protein elongation during translation, which would allow rapid cell adaptation to physiological changes. However, the regulation of translational elongation is poorly understood. We found that expression of p38γ/δ MAPK proteins is required for the elongation of nascent TNF-α protein in macrophages. The MKK3/6-p38γ/δ pathway mediated an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) kinase, which in turn promoted eEF2 activation (dephosphorylation) and subsequent TNF-α elongation. These results identify a new signaling pathway that regulates TNF-α production in LPS-induced liver damage and suggest potential cell-specific therapeutic targets for liver diseases in which TNF-α production is involved. PMID:23202732

  17. Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation

    PubMed Central

    Chambers, Joseph E; Dalton, Lucy E; Clarke, Hanna J; Malzer, Elke; Dominicus, Caia S; Patel, Vruti; Moorhead, Greg; Ron, David; Marciniak, Stefan J

    2015-01-01

    Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR. DOI: http://dx.doi.org/10.7554/eLife.04872.001 PMID:25774599

  18. G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases

    PubMed Central

    Chen, Ruming; Rato, Cláudia; Yan, Yahui; Crespillo-Casado, Ana; Clarke, Hanna J; Harding, Heather P; Marciniak, Stefan J; Read, Randy J; Ron, David

    2015-01-01

    Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrate-selective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex. DOI: http://dx.doi.org/10.7554/eLife.04871.001 PMID:25774600

  19. Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation.

    PubMed

    Chambers, Joseph E; Dalton, Lucy E; Clarke, Hanna J; Malzer, Elke; Dominicus, Caia S; Patel, Vruti; Moorhead, Greg; Ron, David; Marciniak, Stefan J

    2015-01-01

    Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR.

  20. PTENα, a PTEN isoform translated through alternative initiation, regulates mitochondrial function and energy metabolism.

    PubMed

    Liang, Hui; He, Shiming; Yang, Jingyi; Jia, Xinying; Wang, Pan; Chen, Xi; Zhang, Zhong; Zou, Xiajuan; McNutt, Michael A; Shen, Wen Hong; Yin, Yuxin

    2014-05-01

    PTEN is one of the most frequently mutated genes in human cancer. It is known that PTEN has a wide range of biological functions beyond tumor suppression. Here, we report that PTENα, an N-terminally extended form of PTEN, functions in mitochondrial metabolism. Translation of PTENα is initiated from a CUG codon upstream of and in-frame with the coding region of canonical PTEN. Eukaryotic translation initiation factor 2A (eIF2A) controls PTENα translation, which requires a CUG-centered palindromic motif. We show that PTENα induces cytochrome c oxidase activity and ATP production in mitochondria. TALEN-mediated somatic deletion of PTENα impairs mitochondrial respiratory chain function. PTENα interacts with canonical PTEN to increase PINK1 protein levels and promote energy production. Our studies demonstrate the importance of eIF2A-mediated alternative translation for generation of protein diversity in eukaryotic systems and provide insights into the mechanism by which the PTEN family is involved in multiple cellular processes.

  1. Eukaryotic initiation factor 5B: a new player for the anti-hepatitis C virus effect of ribavirin?

    PubMed

    Galmozzi, E; Aghemo, A; Colombo, M

    2012-10-01

    The addition of the broad-spectrum antiviral agent ribavirin (RBV), a synthetic guanosine analog, to interferon-alpha (IFNα) monotherapy has been a major breakthrough in the treatment of patients with hepatitis C virus (HCV), as it greatly improved treatment response rates. Although several mechanisms of action have been proposed for RBV's antiviral activity, each with some experimental evidence, the precise mechanism by which it acts synergistically with IFNα has remained elusive. A cornerstone of the antiviral IFNα response is phosphorylation of the α subunit of eukaryotic initiation factor (eIF)2. This limits the availability of eIF2⋅GTP⋅Met-tRNA(i)(Met) ternary complexes, reduces formation of the 43S preinitiation complexes, ultimately blocking viral (and most cellular) mRNA translation. However recent studies indicated that translation driven by the HCV internal ribosome entry site (IRES) is insensitive to eIF2α phosphorylation. Particularly, in addition to the general eIF2-dependent pathway of translation, the HCV IRES makes use of a bacterial-like, eIF2-independent pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. Together, these observations support a model in which cellular stresses that induce eIF2α phosphorylation (e.g. treatment with IFNα) cause HCV IRES-directed translation to switch from an eIF2-dependent mode to an eIF5B-dependent mode, defining a tactic used by HCV to evade the INFα response. Eukaryotic eIF5B is a ribosome-dependent GTPase that is responsible for 80S complex formation in translation initiation but shows much lower affinities for GTP than to other GTPases, thus suggesting that it may mis-incorporate the RBV triphosphate (RTP) in place of GTP even at the RBV concentrations achieved in clinical use. Consequently, we theorize that RTP bound to eIF5B lowering its affinity for ribosome, blocks the 80S complex formation on HCV IRES inhibiting the eIF5B-dependent translation used by HCV to

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

    PubMed Central

    Joncourt, Raphael; Eberle, Andrea B.; Rufener, Simone C.; Mühlemann, Oliver

    2014-01-01

    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. PMID:25148142

  3. Leishmania donovani eukaryotic initiation factor 5A: molecular characterization, localization and homology modelling studies.

    PubMed

    Singh, Sushma; Raju, K; Jatekar, Deepika; Dinesh, Neeradi; Paul, M Stanley; Sobhia, M E

    2014-08-01

    Eukaryotic translation initiation factor 5A (eIF5A) is a small acidic protein highly conserved from archaea to mammals. eIF5A is the only protein which undergoes a unique lysine residue modification to hypusine. Hypusinylation is important for the function of eIF5A which is reported to be essential for cell viability. eIF5A promotes formation of the first peptide bond at the onset of protein synthesis. However, its function in Leishmania donovani is unclear. The present study focuses on the characterization and localization of L. donovani eIF5A protein. The eIF5A gene contains an ORF of 501×bp encoding 166 amino acid residues with a predicted molecular mass and isoelectric point of 17.8 kDa and 4.83 respectively. A phylogenetic tree analysis revealed its close proximity to trypanosomes however it is distantly located from Trichomonas vaginalis and Plasmodium falciparum. The L. donovani eIF5A was expressed as a 6× His tagged protein whose identity was confirmed by western blot and MALDI. Biophysical investigation by CD revealed the predominant presence of 49% β sheet structure which correlated well with secondary structure prediction. To gain insight into the role of eIF5A in L. donovani, we investigated the subcellular distribution of eIF5A. A GFP-fusion of L. donovani eIF5A was found to be localized in cytoplasm as confirmed by subcellular fractionation. Our studies indicated that eIF5A is primarily localized to cytoplasm and is undetectable in nuclear fraction. The homology model of eIF5A of L. donovani was built and the resulting model showed acceptable Ramachandran statistics. The model is reliable and can be used to study eIF5A binding with its effector molecules.

  4. Predicting Translation Initiation Rates for Designing Synthetic Biology

    PubMed Central

    Reeve, Benjamin; Hargest, Thomas; Gilbert, Charlie; Ellis, Tom

    2013-01-01

    In synthetic biology, precise control over protein expression is required in order to construct functional biological systems. A core principle of the synthetic biology approach is a model-guided design and based on the biological understanding of the process, models of prokaryotic protein production have been described. Translation initiation rate is a rate-limiting step in protein production from mRNA and is dependent on the sequence of the 5′-untranslated region and the start of the coding sequence. Translation rate calculators are programs that estimate protein translation rates based on the sequence of these regions of an mRNA, and as protein expression is proportional to the rate of translation initiation, such calculators have been shown to give good approximations of protein expression levels. In this review, three currently available translation rate calculators developed for synthetic biology are considered, with limitations and possible future progress discussed. PMID:25152877

  5. Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3.

    PubMed

    Zhou, Min; Sandercock, Alan M; Fraser, Christopher S; Ridlova, Gabriela; Stephens, Elaine; Schenauer, Matthew R; Yokoi-Fong, Theresa; Barsky, Daniel; Leary, Julie A; Hershey, John W; Doudna, Jennifer A; Robinson, Carol V

    2008-11-25

    The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.

  6. TARGETING THE eIF4F TRANSLATION INITIATION COMPLEX: A CRITICAL NEXUS FOR CANCER DEVELOPMENT

    PubMed Central

    Pelletier, Jerry; Graff, Jeremy; Ruggero, Davide; Sonenberg, Nahum

    2014-01-01

    Elevated protein synthesis is an important feature of many cancer cells and often arises as a consequence of increased signaling flux channeled to eukaryotic initiation factor (eIF) 4F, the key regulator of the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical models of cancer, eIF4F deregulation results in changes in translational efficiency of specific mRNA classes. Importantly, many of these mRNAs code for proteins that potently regulate critical cellular processes such as cell growth and proliferation, enhanced cell survival, and cell migration that ultimately impinge on several hallmarks of cancer, including increased angiogenesis, deregulated growth control, enhanced cellular survival, epithelial-to-mesenchymal transition, invasion and metastasis. By being positioned as the molecular nexus downstream of key oncogenic signaling pathways (e.g. Ras, PI3K/AKT/TOR, and Myc), eIF4F serves as a direct link between important steps in cancer development and translation initiation. Identification of mRNAs particularly responsive to elevated eIF4F activity that typifies tumorigenesis underscores the critical role of eIF4F in cancer and raises the exciting possibility of developing new-in-class small molecules targeting translation initiation as anti-neoplastic agents. PMID:25593033

  7. Influence of Translation Initiation on Organellar Protein Targeting in Arabidopsis

    SciTech Connect

    Sally A. Mackenzie

    2011-04-18

    A primary focus of the Mackenzie laboratory is the elucidation of processes and machinery for mitochondrial genome maintenance and transmission in higher plants. We have found that numerous organellar DNA maintenance components in plants appear to be dual targeted to mitochondria and plastids. Of particular interest was the observation that some twin (tandemly arrayed) dual targeting presequences appeared to utilize non-AUG alternative translation initiation, allowing for multiple translation starts at a single gene. Two aspects of this phenomenon were of particular interest: (1) Alternative translation initiation might provide a mechanism to regulate protein targeting temporally and spatially, a possibility that had not been demonstrated previously, and (2) alternative translation initiation might occur in genes involved in nuclear-controlled mitochondrial genome recombination, thought to be exclusively mitochondrial in their function. During the course of this research, we pursued three aims, with an emphasis on two specific genes of interest: POLgamma2, an organellar DNA polymerase, and MSH1, a MutS homolog thought to participate in mitochondrial, but not plastid, genome recombination surveillance. Our aims were to (1) Identify additional genes within Arabidopsis and other genomes that employ non-AUG alternative translation initiation, (2) Locate sequences upstream to the annotated AUG that confer alternative non-AUG translation initiation activity, and (3) Identify cis and trans factors that influence start site selection in genes with non-AUG starts. Toward these ends, we have shown that non-AUG initiation occurs in a number of genes, likely influencing targeting behavior of the protein. We have also shown that start site selection is strongly influenced by Kozak consensus sequence environment, indicating that alternative translation initiation in plants occurs by relaxation of ribosome scanning.

  8. PTENα is a PTEN isoform Translated through Alternative Initiation and Regulates Mitochondrial Function

    PubMed Central

    Liang, Hui; He, Shiming; Yang, Jingyi; Jia, Xinying; Wang, Pan; Chen, Xi; Zhang, Zhong; Zou, Xiajuan; McNutt, Michael A.; Shen, Wen Hong; Yin, Yuxin

    2014-01-01

    Summary PTEN is one of the most frequently mutated genes in human cancer. It is known that PTEN has a wide range of biological functions beyond tumor suppression. Here we report that PTENα, an N-terminally extended form of PTEN, functions in metabolism. Translation of PTENα is initiated from a CUG codon upstream of and in-frame with the coding region of canonical PTEN. Eukaryotic translation initiation factor 2A (eIF2A) controls PTENα translation and a CUG-centered palindromic motif is required in this process. PTENα induces cytochrome c oxidase activity and ATP production in mitochondria. TALEN-mediated somatic deletion of PTENα impairs mitochondrial respiratory chain function. We show that PTENα interacts with canonical PTEN to increase PINK1 and promote energy production. These data provide insights into the mechanism by which the PTEN family is involved in multiple cellular processes. Our studies suggest that mammalian cells can use alternate translation initiation mechanisms to generate protein isoforms. PMID:24768297

  9. An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation

    PubMed Central

    Toribio, René; Díaz-López, Irene; Boskovic, Jasminka; Ventoso, Iván

    2016-01-01

    During translation initiation, eukaryotic initiation factor 2 (eIF2) delivers the Met-tRNA to the 40S ribosomal subunit to locate the initiation codon (AUGi) of mRNA during the scanning process. Stress-induced eIF2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pathway in mammals. However, some viral mRNAs can initiate translation in the presence of phosphorylated eIF2 via stable RNA stem-loop structures (DLP; Downstream LooP) located in their coding sequence (CDS), which promote 43S preinitiation complex stalling on the initiation codon. We show here that during the scanning process, DLPs of Alphavirus mRNA become trapped in ES6S region (680–914 nt) of 18S rRNA that are projected from the solvent side of 40S subunit. This trapping can lock the progress of the 40S subunit on the mRNA in a way that places the upstream initiator AUGi on the P site of 40S subunit, obviating the participation of eIF2. Notably, the DLP structure is released from 18S rRNA upon 60S ribosomal subunit joining, suggesting conformational changes in ES6Ss during the initiation process. These novel findings illustrate how viral mRNA is threaded into the 40S subunit during the scanning process, exploiting the topology of the 40S subunit solvent side to enhance its translation in vertebrate hosts. PMID:26984530

  10. The importance of inter- and intramolecular base pairing for translation reinitiation on a eukaryotic bicistronic mRNA.

    PubMed

    Luttermann, Christine; Meyers, Gregor

    2009-02-01

    Calicivirus structure proteins are expressed from a subgenomic mRNA with two overlapping cistrons. The first ORF of this RNA codes for the viral major capsid protein VP1, and the second for the minor capsid protein VP2. Translation of VP2 is mediated by a termination/reinitiation mechanism, which depends on an upstream sequence element of approximately 70 nucleotides denoted "termination upstream ribosomal binding site" (TURBS). Two short sequence motifs within the TURBS were found to be essential for reinitiation. By a whole set of single site mutations and reciprocal base exchanges we demonstrate here for the first time conclusive evidence for the necessity of mRNA/18S rRNA hybridization for translation reinitiation in an eukaryotic system. Moreover, we show that motif 2 exhibits intramolecular hybridization with a complementary region upstream of motif 1, thus forming a secondary structure that positions post-termination ribosomes in an optimal distance to the VP2 start codon. Analysis of the essential elements of the TURBS led to a better understanding of the requirements for translation termination/reinitiation in eukaryotes.

  11. Translationally coupled initiation of protein synthesis in Bacillus subtilis.

    PubMed Central

    Sprengel, R; Reiss, B; Schaller, H

    1985-01-01

    The neomycin phosphotransferase gene (neo) from Transposon Tn5 is active in Gram-negative bacteria but silent in B. subtilis since it lacks an appropriate ribosome binding site for Gram-positive bacteria. Neo translation could be reactivated by coupling its initiation to the translational termination of the highly expressed beta-lactamase gene (penP) from B. licheniformis. This initiation occurred at the authentic neo start codon. Its efficiency was independent of the nucleotide sequence 5 to the neo gene, but strongly affected by the distance between the termination and initiation codon. It was the highest if both codons overlapped in the sequence ATGA. In B. licheniformis, a translationally coupled neo gene was inducible expressed as the penP gene demonstrating the potential of the technique to monitor the activity of expression units for which no direct assays exists. Images PMID:3923434

  12. TISdb: a database for alternative translation initiation in mammalian cells.

    PubMed

    Wan, Ji; Qian, Shu-Bing

    2014-01-01

    Proper selection of the translation initiation site (TIS) on mRNAs is crucial for the production of desired protein products. Recent studies using ribosome profiling technology uncovered a surprising variety of potential TIS sites in addition to the annotated start codon. The prevailing alternative translation reshapes the landscape of the proteome in terms of diversity and complexity. To identify the hidden coding potential of the transcriptome in mammalian cells, we developed global translation initiation sequencing (GTI-Seq) that maps genome-wide TIS positions at nearly a single nucleotide resolution. To facilitate studies of alternative translation, we created a database of alternative TIS sites identified from human and mouse cell lines based on multiple GTI-Seq replicates. The TISdb, available at http://tisdb.human.cornell.edu, includes 6991 TIS sites from 4961 human genes and 9973 TIS sites from 5668 mouse genes. The TISdb website provides a simple browser interface for query of high-confidence TIS sites and their associated open reading frames. The output of search results provides a user-friendly visualization of TIS information in the context of transcript isoforms. Together, the information in the database provides an easy reference for alternative translation in mammalian cells and will support future investigation of novel translational products.

  13. Plant Cap-binding Complexes Eukaryotic Initiation Factors eIF4F and eIFISO4F

    PubMed Central

    Mayberry, Laura K.; Allen, M. Leah; Nitka, Kelley R.; Campbell, Lara; Murphy, Patricia A.; Browning, Karen S.

    2011-01-01

    The initiation of translation in eukaryotes requires a suite of eIFs that include the cap-binding complex, eIF4F. eIF4F is comprised of the subunits eIF4G and eIF4E and often the helicase, eIF4A. The eIF4G subunit serves as an assembly point for other initiation factors, whereas eIF4E binds to the 7-methyl guanosine cap of mRNA. Plants have an isozyme form of eIF4F (eIFiso4F) with comparable subunits, eIFiso4E and eIFiso4G. Plant eIF4A is very loosely associated with the plant cap-binding complexes. The specificity of interaction of the individual subunits of the two complexes was previously unknown. To address this issue, mixed complexes (eIF4E-eIFiso4G or eIFiso4E-eIF4G) were expressed and purified from Escherichia coli for biochemical analysis. The activity of the mixed complexes in in vitro translation assays correlated with the large subunit of the respective correct complex. These results suggest that the eIF4G or eIFiso4G subunits influence translational efficiency more than the cap-binding subunits. The translation assays also showed varying responses of the mRNA templates to eIF4F or eIFiso4F, suggesting that some level of mRNA discrimination is possible. The dissociation constants for the correct complexes have KD values in the subnanomolar range, whereas the mixed complexes were found to have KD values in the ∼10 nm range. Displacement assays showed that the correct binding partner readily displaces the incorrect binding partner in a manner consistent with the difference in KD values. These results show molecular specificity for the formation of plant eIF4F and eIFiso4F complexes and suggest a role in mRNA discrimination during initiation of translation. PMID:21965660

  14. Unique role for translation initiation factor 3 in the light color regulation of photosynthetic gene expression.

    PubMed

    Gutu, Andrian; Nesbit, April D; Alverson, Andrew J; Palmer, Jeffrey D; Kehoe, David M

    2013-10-01

    Light-harvesting antennae are critical for collecting energy from sunlight and providing it to photosynthetic reaction centers. Their abundance and composition are tightly regulated to maintain efficient photosynthesis in changing light conditions. Many cyanobacteria alter their light-harvesting antennae in response to changes in ambient light-color conditions through the process of chromatic acclimation. The control of green light induction (Cgi) pathway is a light-color-sensing system that controls the expression of photosynthetic genes during chromatic acclimation, and while some evidence suggests that it operates via transcription attenuation, the components of this pathway have not been identified. We provide evidence that translation initiation factor 3 (IF3), an essential component of the prokaryotic translation initiation machinery that binds the 30S subunit and blocks premature association with the 50S subunit, is part of the control of green light induction pathway. Light regulation of gene expression has not been previously described for any translation initiation factor. Surprisingly, deletion of the IF3-encoding gene infCa was not lethal in the filamentous cyanobacterium Fremyella diplosiphon, and its genome was found to contain a second, redundant, highly divergent infC gene which, when deleted, had no effect on photosynthetic gene expression. Either gene could complement an Escherichia coli infC mutant and thus both encode bona fide IF3s. Analysis of prokaryotic and eukaryotic genome databases established that multiple infC genes are present in the genomes of diverse groups of bacteria and land plants, most of which do not undergo chromatic acclimation. This suggests that IF3 may have repeatedly evolved important roles in the regulation of gene expression in both prokaryotes and eukaryotes.

  15. Modifying the 5'-Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells.

    PubMed

    Holstein, Josephin M; Anhäuser, Lea; Rentmeister, Andrea

    2016-08-26

    The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox. PMID:27511141

  16. How MCM loading and spreading specify eukaryotic DNA replication initiation sites

    PubMed Central

    Hyrien, Olivier

    2016-01-01

    DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

  17. How MCM loading and spreading specify eukaryotic DNA replication initiation sites

    PubMed Central

    Hyrien, Olivier

    2016-01-01

    DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes. PMID:27635237

  18. How MCM loading and spreading specify eukaryotic DNA replication initiation sites.

    PubMed

    Hyrien, Olivier

    2016-01-01

    DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes. PMID:27635237

  19. Crystallization and preliminary X-ray analysis of eukaryotic initiation factor 4E from Pisum sativum

    PubMed Central

    Ashby, Jamie A.; Stevenson, Clare E. M.; Maule, Andrew J.; Lawson, David M.

    2009-01-01

    Crystals of an N-terminally truncated 20 kDa fragment of Pisum sativum eIF4E (ΔN-eIF4E) were grown by vapour diffusion. X-ray data were recorded to a resolution of 2.2 Å from a single crystal in-house. Indexing was consistent with primitive monoclinic symmetry and solvent-content estimations suggested that between four and nine copies of the eIF4E fragment were possible per crystallographic asymmetric unit. eIF4E is an essential component of the eukaryotic translation machinery and recent studies have shown that point mutations of plant eIF4Es can confer resistance to potyvirus infection. PMID:19652353

  20. Operons in eukaryotes.

    PubMed

    Blumenthal, Thomas

    2004-11-01

    It was thought that polycistronic transcription is a characteristic of bacteria and archaea, where many of the genes are clustered in operons composed of two to more than ten genes. By contrast, the genes of eukaryotes are generally considered to be monocistronic, each with its own promoter at the 5' end and a transcription terminator at the 3' end; however, it has recently become clear that not all eukaryotic genes are transcribed monocistronically. Numerous instances of polycistronic transcription in eukaryotes, from protists to chordates, have been reported. These can be divided into two broad types. Dicistronic transcription units specify a messenger RNA (mRNA) encoding two separate genes that is transported to the cytoplasm and translated in that form. Presumably, internal ribosome entry sites (IRES), or some form of translational re-initiation following the stop codon, are responsible for allowing translation of the downstream gene. In the other type, the initial transcript is processed by 3' end cleavage and trans-splicing to create monocistronic mRNAs that are transported to the cytoplasm and translated. Like bacterial operons, eukaryotic operons often result in co-expression of functionally related proteins.

  1. Analysis of a ubiquitous promoter element in a primitive eukaryote: early evolution of the initiator element.

    PubMed

    Liston, D R; Johnson, P J

    1999-03-01

    Typical metazoan core promoter elements, such as TATA boxes and Inr motifs, have yet to be identified in early-evolving eukaryotes, underscoring the extensive divergence of these organisms. Towards the identification of core promoters in protists, we have studied transcription of protein-encoding genes in one of the earliest-diverging lineages of Eukaryota, that represented by the parasitic protist Trichomonas vaginalis. A highly conserved element, comprised of a motif similar to a metazoan initiator (Inr) element, surrounds the start site of transcription in all examined T. vaginalis genes. In contrast, a metazoan-like TATA element appears to be absent in trichomonad promoters. We demonstrate that the conserved motif found in T. vaginalis protein-encoding genes is an Inr promoter element. This trichomonad Inr is essential for transcription, responsible for accurate start site selection, and interchangeable between genes, demonstrating its role as a core promoter element. The sequence requirements of the trichomonad Inr are similar to metazoan Inrs and can be replaced by a mammalian Inr. These studies show that the Inr is a ubiquitous, core promoter element for protein-encoding genes in an early-evolving eukaryote. Functional and structural similarities between this protist Inr and the metazoan Inr strongly indicate that the Inr promoter element evolved early in eukaryotic evolution.

  2. Supporting knowledge translation through collaborative translational research initiatives: 'bridging' versus 'blurring' boundary-spanning approaches in the UK CLAHRC initiative.

    PubMed

    Evans, Sarah; Scarbrough, Harry

    2014-04-01

    Recent policy initiatives in the UK and internationally have sought to promote knowledge translation between the 'producers' and 'users' of research. Within this paper we explore how boundary-spanning interventions used within such initiatives can support knowledge translation between diverse groups. Using qualitative data from a 3-year research study conducted from January 2010 to December 2012 of two case-sites drawn from the CLAHRC initiative in the UK, we distinguish two different approaches to supporting knowledge translation; a 'bridging' approach that involves designated roles, discrete events and activities to span the boundaries between communities, and a 'blurring' approach that de-emphasises the boundaries between groups, enabling a more continuous process of knowledge translation as part of day-to-day work-practices. In this paper, we identify and differentiate these boundary-spanning approaches and describe how they emerged from the context defined by the wider CLAHRC networks. This highlights the need to develop a more contextualised analysis of the boundary-spanning that underpins knowledge translation processes, relating this to the distinctive features of a particular case. PMID:24561773

  3. Supporting knowledge translation through collaborative translational research initiatives: 'bridging' versus 'blurring' boundary-spanning approaches in the UK CLAHRC initiative.

    PubMed

    Evans, Sarah; Scarbrough, Harry

    2014-04-01

    Recent policy initiatives in the UK and internationally have sought to promote knowledge translation between the 'producers' and 'users' of research. Within this paper we explore how boundary-spanning interventions used within such initiatives can support knowledge translation between diverse groups. Using qualitative data from a 3-year research study conducted from January 2010 to December 2012 of two case-sites drawn from the CLAHRC initiative in the UK, we distinguish two different approaches to supporting knowledge translation; a 'bridging' approach that involves designated roles, discrete events and activities to span the boundaries between communities, and a 'blurring' approach that de-emphasises the boundaries between groups, enabling a more continuous process of knowledge translation as part of day-to-day work-practices. In this paper, we identify and differentiate these boundary-spanning approaches and describe how they emerged from the context defined by the wider CLAHRC networks. This highlights the need to develop a more contextualised analysis of the boundary-spanning that underpins knowledge translation processes, relating this to the distinctive features of a particular case.

  4. Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A

    PubMed Central

    Hoque, Mainul; Hanauske-Abel, Hartmut M; Palumbo, Paul; Saxena, Deepti; D'Alliessi Gandolfi, Darlene; Park, Myung Hee; Pe'ery, Tsafi; Mathews, Michael B

    2009-01-01

    Background Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture. Results Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression. Conclusion At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs. PMID:19825182

  5. A translation system reconstituted with human factors proves that processing of encephalomyocarditis virus proteins 2A and 2B occurs in the elongation phase of translation without eukaryotic release factors.

    PubMed

    Machida, Kodai; Mikami, Satoshi; Masutani, Mamiko; Mishima, Kurumi; Kobayashi, Tominari; Imataka, Hiroaki

    2014-11-14

    The genomic RNA of encephalomyocarditis virus (EMCV) encodes a single polyprotein, and the primary scission of the polyprotein occurs between nonstructural proteins 2A and 2B by an unknown mechanism. To gain insight into the mechanism of 2A-2B processing, we first translated the 2A-2B region in vitro with eukaryotic and prokaryotic translation systems. The 2A-2B processing occurred only in the eukaryotic systems, not in the prokaryotic systems, and the unprocessed 2A-2B protein synthesized by a prokaryotic system remained uncleaved when incubated with a eukaryotic cell extract. These results suggest that 2A-2B processing is a eukaryote-specific, co-translational event. To define the translation factors required for 2A-2B processing, we constituted a protein synthesis system with eukaryotic elongation factors 1 and 2, eukaryotic release factors 1 and 3 (eRF1 and eRF3), aminoacyl-tRNA synthetases, tRNAs, ribosome subunits, and a plasmid template that included the hepatitis C virus internal ribosome entry site. We successfully reproduced 2A-2B processing in the reconstituted system even without eRFs. Our results indicate that this unusual event occurs in the elongation phase of translation.

  6. An Isoform of Eukaryotic Initiation Factor 4E from Chrysanthemum morifolium Interacts with Chrysanthemum Virus B Coat Protein

    PubMed Central

    Chen, Sumei; Sun, Zuxia; Guan, Zhiyong; Fang, Weimin; Teng, Nianjun; Chen, Fadi

    2013-01-01

    Background Eukaryotic translation initiation factor 4E (eIF4E) plays an important role in plant virus infection as well as the regulation of gene translation. Methodology/Principal Findings Here, we describe the isolation of a cDNA encoding CmeIF(iso)4E (GenBank accession no. JQ904592), an isoform of eIF4E from chrysanthemum, using RACE PCR. We used the CmeIF(iso)4E cDNA for expression profiling and to analyze the interaction between CmeIF(iso)4E and the Chrysanthemum virus B coat protein (CVBCP). Multiple sequence alignment and phylogenetic tree analysis showed that the sequence similarity of CmeIF(iso)4E with other reported plant eIF(iso)4E sequences varied between 69.12% and 89.18%, indicating that CmeIF(iso)4E belongs to the eIF(iso)4E subfamily of the eIF4E family. CmeIF(iso)4E was present in all chrysanthemum organs, but was particularly abundant in the roots and flowers. Confocal microscopy showed that a transiently transfected CmeIF(iso)4E-GFP fusion protein distributed throughout the whole cell in onion epidermis cells. A yeast two hybrid assay showed CVBCP interacted with CmeIF(iso)4E but not with CmeIF4E. BiFC assay further demonstrated the interaction between CmeIF(iso)4E and CVBCP. Luminescence assay showed that CVBCP increased the RLU of Luc-CVB, suggesting CVBCP might participate in the translation of viral proteins. Conclusions/Significance These results inferred that CmeIF(iso)4E as the cap-binding subunit eIF(iso)4F may be involved in Chrysanthemum Virus B infection in chrysanthemum through its interaction with CVBCP in spatial. PMID:23505421

  7. Molecular dissection of the eukaryotic initiation factor 4E (eIF4E) export-competent RNP.

    PubMed

    Topisirovic, Ivan; Siddiqui, Nadeem; Lapointe, Vincent Leroux; Trost, Matthias; Thibault, Pierre; Bangeranye, Catherine; Piñol-Roma, Serafin; Borden, Katherine L B

    2009-04-22

    The eukaryotic translation initiation factor 4E (eIF4E) controls gene expression through its effects on mRNA export and cap-dependent translation, both of which contribute to its oncogenic potential. In contrast to its translation function, the mRNA export function of eIF4E is poorly understood. Using an RNP isolation/mass spectrometry approach, we identified candidate cofactors of eIF4E mRNA export including LRPPRC. This protein associates with mRNAs containing the eIF4E-sensitivity element (4E-SE), and its overexpression alters the nuclear export of several eIF4E-sensitive mRNAs. LRPPRC-mediated alteration of eIF4E's mRNA export function requires the integrity of its eIF4E-binding site and it coincides with the subcellular re-distribution of eIF4E. The eIF4E export RNP is distinct in composition from the bulk mRNA export pathway, in that eIF4E- and eIF4E-sensitive mRNAs do not associate with general mRNA export factors such as TAP/NXF1 or REF/Aly. Our data indicate that mRNA export pathways have evolved for specific mRNAs enabling the differential regulation of biochemical pathways by modulating the expression of groups of genes at the level of their export.

  8. Interaction of the eukaryotic initiation factor 4E with 4E-BP2 at a dynamic bipartite interface.

    PubMed

    Lukhele, Sabelo; Bah, Alaji; Lin, Hong; Sonenberg, Nahum; Forman-Kay, Julie D

    2013-12-01

    Cap-dependent translation initiation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with eIF4E binding proteins (4E-BPs). Whereas the binding of 4E-BP peptides containing the eIF4E-binding ⁵⁴YXXXXLΦ⁶⁰ motif has been studied, atomic-level characterization of the interaction of eIF4E with full-length 4E-BPs has been lacking. Here, we use isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to characterize the dynamic, structural and binding properties of 4E-BP2. Although disordered, 4E-BP2 contains significant fluctuating secondary structure and binds eIF4E at an extensive bipartite interface including the canonical ⁵⁴YXXXXLΦ⁶⁰ and ⁷⁸IPGVT⁸² sites. Each of the two binding elements individually has submicromolar affinity and exchange on and off of the eIF4E surface within the context of the overall nanomolar complex. This dynamic interaction facilitates exposure of regulatory phosphorylation sites within the complex. The 4E-BP2 interface on eIF4E overlaps yet is more extensive than the eIF4G:eIF4E interface, suggesting that these key interactions may be differentially targeted for therapeutics.

  9. Directional transition from initiation to elongation in bacterial translation

    PubMed Central

    Goyal, Akanksha; Belardinelli, Riccardo; Maracci, Cristina; Milón, Pohl; Rodnina, Marina V.

    2015-01-01

    The transition of the 30S initiation complex (IC) to the translating 70S ribosome after 50S subunit joining provides an important checkpoint for mRNA selection during translation in bacteria. Here, we study the timing and control of reactions that occur during 70S IC formation by rapid kinetic techniques, using a toolbox of fluorescence-labeled translation components. We present a kinetic model based on global fitting of time courses obtained with eight different reporters at increasing concentrations of 50S subunits. IF1 and IF3 together affect the kinetics of subunit joining, but do not alter the elemental rates of subsequent steps of 70S IC maturation. After 50S subunit joining, IF2-dependent reactions take place independent of the presence of IF1 or IF3. GTP hydrolysis triggers the efficient dissociation of fMet-tRNAfMet from IF2 and promotes the dissociation of IF2 and IF1 from the 70S IC, but does not affect IF3. The presence of non-hydrolyzable GTP analogs shifts the equilibrium towards a stable 70S–mRNA–IF1–IF2–fMet-tRNAfMet complex. Our kinetic analysis reveals the molecular choreography of the late stages in translation initiation. PMID:26338773

  10. Intrauterine growth restriction inhibits expression of eukaryotic elongation factor 2 kinase, a regulator of protein translation.

    PubMed

    McKnight, Robert A; Yost, Christian C; Zinkhan, Erin K; Fu, Qi; Callaway, Christopher W; Fung, Camille M

    2016-08-01

    Nutrient deprivation suppresses protein synthesis by blocking peptide elongation. Transcriptional upregulation and activation of eukaryotic elongation factor 2 kinase (eEF2K) blocks peptide elongation by phosphorylating eukaryotic elongation factor 2. Previous studies examining placentas from intrauterine growth restricted (IUGR) newborn infants show decreased eEF2K expression and activity despite chronic nutrient deprivation. However, the effect of IUGR on hepatic eEF2K expression in the fetus is unknown. We, therefore, examined the transcriptional regulation of hepatic eEF2K gene expression in a Sprague-Dawley rat model of IUGR. We found decreased hepatic eEF2K mRNA and protein levels in IUGR offspring at birth compared with control, consistent with previous placental observations. Furthermore, the CpG island within the eEF2K promoter demonstrated increased methylation at a critical USF 1/2 transcription factor binding site. In vitro methylation of this binding site caused near complete loss of eEF2K promoter activity, designating this promoter as methylation sensitive. The eEF2K promotor in IUGR offspring also lost the protective histone covalent modifications associated with unmethylated CGIs. In addition, the +1 nucleosome was displaced 3' and RNA polymerase loading was reduced at the IUGR eEF2K promoter. Our findings provide evidence to explain why IUGR-induced chronic nutrient deprivation does not result in the upregulation of eEF2K gene transcription. PMID:27317589

  11. Mechanism of chromosomal DNA replication initiation and replication fork stabilization in eukaryotes.

    PubMed

    Wu, LiHong; Liu, Yang; Kong, DaoChun

    2014-05-01

    Chromosomal DNA replication is one of the central biological events occurring inside cells. Due to its large size, the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called DNA origins so that the replication could be completed in a limited time. Further, eukaryotic DNA replication is sophisticatedly regulated, and this regulation guarantees that each origin fires once per S phase and each segment of DNA gets duplication also once per cell cycle. The first step of replication initiation is the assembly of pre-replication complex (pre-RC). Since 1973, four proteins, Cdc6/Cdc18, MCM, ORC and Cdt1, have been extensively studied and proved to be pre-RC components. Recently, a novel pre-RC component called Sap1/Girdin was identified. Sap1/Girdin is required for loading Cdc18/Cdc6 to origins for pre-RC assembly in the fission yeast and human cells, respectively. At the transition of G1 to S phase, pre-RC is activated by the two kinases, cyclindependent kinase (CDK) and Dbf4-dependent kinase (DDK), and subsequently, RPA, primase-polα, PCNA, topoisomerase, Cdc45, polδ, and polɛ are recruited to DNA origins for creating two bi-directional replication forks and initiating DNA replication. As replication forks move along chromatin DNA, they frequently stall due to the presence of a great number of replication barriers on chromatin DNA, such as secondary DNA structures, protein/DNA complexes, DNA lesions, gene transcription. Stalled forks must require checkpoint regulation for their stabilization. Otherwise, stalled forks will collapse, which results in incomplete DNA replication and genomic instability. This short review gives a concise introduction regarding the current understanding of replication initiation and replication fork stabilization.

  12. Translation and developmental regulation of RNA encoded by the eukaryotic transposable element copia.

    PubMed

    Flavell, A J; Ruby, S W; Toole, J J; Roberts, B E; Rubin, G M

    1980-12-01

    copia-specific RNA was isolated from Drosophila melanogaster tissue culture cells by hybridization of cytoplasmic polyadenylylated RNA to copia DNA immobilized on cellulose. The purified RNA was translated in reticulocyte lysates. One major polypeptide of approximately 51,000 daltons was synthesized in addition to several others between 18,000 and 38,000 daltons. The 51,000-dalton polypeptide and several of the others are encoded by mRNAs of about 2000 nucleotides. The approximate locations on the copia element of the coding sequences for the 51,000-dalton polypeptide and several other proteins were determined by hybrid-arrested translation with copia restriction fragments. The relative abundance of copia-specific RNA was determined at various stages of the Drosophila life cycle. The level of copia-specific RNA is modulated during development of the organism, with the highest level occurring during the larval stages.

  13. An in vivo control map for the eukaryotic mRNA translation machinery.

    PubMed

    Firczuk, Helena; Kannambath, Shichina; Pahle, Jürgen; Claydon, Amy; Beynon, Robert; Duncan, John; Westerhoff, Hans; Mendes, Pedro; McCarthy, John Eg

    2013-01-01

    Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNA(i) to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5'untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5'UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.

  14. Eukaryotic Translation Elongation Factor 1A Induces Anoikis by Triggering Cell Detachment*

    PubMed Central

    Itagaki, Keisuke; Naito, Toshihiko; Iwakiri, Ryota; Haga, Makoto; Miura, Shougo; Saito, Yohei; Owaki, Toshiyuki; Kamiya, Sadahiro; Iyoda, Takuya; Yajima, Hirofumi; Iwashita, Shintaro; Ejiri, Shin-Ichiro; Fukai, Fumio

    2012-01-01

    Anoikis, apoptosis because of loss of cell anchorage, is crucial for tissue homeostasis. Fibronectin not only provides a scaffold for cell anchorage but also harbors a cryptic antiadhesive site capable of inducing β1-integrin inactivation. In this study, this cryptic antiadhesive site is implicated in spontaneous induction of anoikis. Nontransformed fibroblasts (NIH3T3) adhering to a fibronectin substratum underwent anoikis during serum starvation culture. This anoikis was caused by proteolytic exposure of the cryptic antiadhesive site in fibronectin by matrix metalloproteinase. Eukaryotic elongation factor 1A (eEF1A) was identified as a membrane receptor for the exposed antiadhesive site. Serum starvation raised the membrane residence of eEF1A, and siRNA-based disruption of this increase rendered cells anoikis-resistant. By contrast, cells became more susceptible to anoikis in parallel with increased membrane residence of eEF1A by enforced expression. These results demonstrate that eEF1A acts as a membrane receptor for the cryptic antiadhesive site of fibronectin, which contributes to cell regulation, including anoikis, through negative regulation of cell anchorage. PMID:22399298

  15. Viral Genome-Linked Protein (VPg) Is Essential for Translation Initiation of Rabbit Hemorrhagic Disease Virus (RHDV)

    PubMed Central

    Zhu, Jie; Wang, Binbin; Miao, Qiuhong; Tan, Yonggui; Li, Chuanfeng; Chen, Zongyan; Guo, Huimin; Liu, Guangqing

    2015-01-01

    Rabbit hemorrhagic disease virus (RHDV), the causative agent of rabbit hemorrhagic disease, is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, limiting the study of the pathogenesis of RHDV. In addition, the mechanisms underlying RHDV translation and replication are largely unknown compared with other caliciviridae viruses. The RHDV replicon recently constructed in our laboratory provides an appropriate model to study the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon, we demonstrated that the viral genome-linked protein (VPg) is essential for RHDV translation in RK-13 cells for the first time. In addition, we showed that VPg interacts with eukaryotic initiation factor 4E (eIF4E) in vivo and in vitro and that eIF4E silencing inhibits RHDV translation, suggesting the interaction between VPg and eIF4E is involved in RHDV translation. Our results support the hypothesis that VPg serves as a novel cap substitute during the initiation of RHDV translation. PMID:26599265

  16. Viral Genome-Linked Protein (VPg) Is Essential for Translation Initiation of Rabbit Hemorrhagic Disease Virus (RHDV).

    PubMed

    Zhu, Jie; Wang, Binbin; Miao, Qiuhong; Tan, Yonggui; Li, Chuanfeng; Chen, Zongyan; Guo, Huimin; Liu, Guangqing

    2015-01-01

    Rabbit hemorrhagic disease virus (RHDV), the causative agent of rabbit hemorrhagic disease, is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, limiting the study of the pathogenesis of RHDV. In addition, the mechanisms underlying RHDV translation and replication are largely unknown compared with other caliciviridae viruses. The RHDV replicon recently constructed in our laboratory provides an appropriate model to study the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon, we demonstrated that the viral genome-linked protein (VPg) is essential for RHDV translation in RK-13 cells for the first time. In addition, we showed that VPg interacts with eukaryotic initiation factor 4E (eIF4E) in vivo and in vitro and that eIF4E silencing inhibits RHDV translation, suggesting the interaction between VPg and eIF4E is involved in RHDV translation. Our results support the hypothesis that VPg serves as a novel cap substitute during the initiation of RHDV translation. PMID:26599265

  17. Discovery of the first known small-molecule inhibitors of heme-regulated eukaryotic initiation factor 2alpha (HRI) kinase.

    PubMed

    Rosen, Mark D; Woods, Craig R; Goldberg, Steven D; Hack, Michael D; Bounds, A Dawn; Yang, Young; Wagaman, Pamela C; Phuong, Victor K; Ameriks, Angela P; Barrett, Terrance D; Kanelakis, Kimon C; Chuang, Jui Chang; Chang, Jui; Shankley, Nigel P; Rabinowitz, Michael H

    2009-12-01

    A series of indeno[1,2-c]pyrazoles were discovered to be the first known inhibitors of heme-regulated eukaryotic initiation factor 2alpha (HRI) kinase. The synthesis, structure-activity relationship profile, and in-vitro pharmacological characterization of this inaugural series of HRI kinase inhibitors are detailed.

  18. Distinct eRF3 Requirements Suggest Alternate eRF1 Conformations Mediate Peptide Release During Eukaryotic Translation Termination

    PubMed Central

    Fan-Minogue, Hua; Du, Ming; Pisarev, Andrey V.; Kallmeyer, Adam K.; Salas-Marco, Joe; Keeling, Kim M.; Thompson, Sunnie R.; Pestova, Tatyana V.; Bedwell, David M.

    2008-01-01

    Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, while variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination. PMID:18538658

  19. Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon

    SciTech Connect

    Feng, Yue; Gunter, L.E.; Organ, E.L.; Cavener, D.R. )

    1991-04-01

    The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the [minus]3 position did not affect relative translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region [minus]1 to [minus]9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.

  20. Process of Hypertrophic Scar Formation: Expression of Eukaryotic Initiation Factor 6

    PubMed Central

    Yang, Qing-Qing; Yang, Si-Si; Tan, Jiang-Lin; Luo, Gao-Xing; He, Wei-Feng; Wu, Jun

    2015-01-01

    Background: Hypertrophic scar is one of the most common complications and often causes the disfigurement or deformity in burn or trauma patients. Therapeutic methods on hypertrophic scar treatment have limitations due to the poor understanding of mechanisms of hypertrophic scar formation. To throw light on the molecular mechanism of hypertrophic scar formation will definitely improve the outcome of the treatment. This study aimed to illustrate the negative role of eukaryotic initiation factor 6 (eIF6) in the process of human hypertrophic scar formation, and provide a possible indicator of hypertrophic scar treatment and a potential target molecule for hypertrophic scar. Methods: In the present study, we investigated the protein expression of eIF6 in the human hypertrophic scar of different periods by immunohistochemistry and Western blot analysis. Results: In the hypertrophic scar tissue, eIF6 expression was significantly decreased and absent in the basal layer of epidermis in the early period, and increased slowly and began to appear in the basal layer of epidermis by the scar formation time. Conclusions: This study confirmed that eIF6 expression was significantly related to the development of hypertrophic scar, and the eIF6 may be a target molecule for hypertrophic scar control or could be an indicator of the outcomes for other treatment modalities. PMID:26481747

  1. Gene and translation initiation site prediction in metagenomic sequences

    SciTech Connect

    Hyatt, Philip Douglas; LoCascio, Philip F; Hauser, Loren John; Uberbacher, Edward C

    2012-01-01

    Gene prediction in metagenomic sequences remains a difficult problem. Current sequencing technologies do not achieve sufficient coverage to assemble the individual genomes in a typical sample; consequently, sequencing runs produce a large number of short sequences whose exact origin is unknown. Since these sequences are usually smaller than the average length of a gene, algorithms must make predictions based on very little data. We present MetaProdigal, a metagenomic version of the gene prediction program Prodigal, that can identify genes in short, anonymous coding sequences with a high degree of accuracy. The novel value of the method consists of enhanced translation initiation site identification, ability to identify sequences that use alternate genetic codes and confidence values for each gene call. We compare the results of MetaProdigal with other methods and conclude with a discussion of future improvements.

  2. Regulation of cap-dependent translation initiation in the early stage porcine parthenotes.

    PubMed

    Susor, Andrej; Jelínková, Lucie; Karabínová, Pavla; Torner, Helmut; Tomek, Wolfgang; Kovárová, Hana; Kubelka, Michal

    2008-12-01

    The binding of mRNAs to ribosomes is mediated by the protein complex eIF4F in conjunction with eIF4B (eukaryotic initiation factor 4F and 4B). EIF4F is a three subunit complex consisting of eIF4A (RNA helicase), eIF4E (mRNA cap binding protein), and eIF4G (bridging protein). The crucial role is played by eIF4E, which directly binds the 5'-cap structure of the mRNA and facilitates the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. EIF4E binding to mRNA and to other initiation factors is regulated on several levels, including its phosphorylation on Ser-209, and association with its regulatory protein 4E-binding protein (4E-BP1). In this study we document that both the translation initiation factor eIF4E and its regulator 4E-BP1 become dephosphorylated in the early stage porcine zygotes already 8 hr post-activation. Similarly, the activities of ERK1/2 MAP and Mnk1 kinases, which are both involved in eIF4E phosphorylation, gradually decrease during this period with the timing similar to that of eIF4E dephosphorylation. The formation of an active eIF4F complex is also diminished after 9-15 hr post-activation, although substantial amounts of this complex have been detected also 24 hr post-activation (2-cell stage). The overall protein synthesis in the parthenotes decreases gradually from 12 hr post-activation reaching a minimum after 48 hr (4-cell stage). Although the translation is gradually decreasing during early preimplantation development, the eIF4F complex, which is temporarily formed, might be a premise for the translation of a small subset of mRNAs at this period of development.

  3. N-terminal Proteomics and Ribosome Profiling Provide a Comprehensive View of the Alternative Translation Initiation Landscape in Mice and Men*

    PubMed Central

    Van Damme, Petra; Gawron, Daria; Van Criekinge, Wim; Menschaert, Gerben

    2014-01-01

    Usage of presumed 5′UTR or downstream in-frame AUG codons, next to non-AUG codons as translation start codons contributes to the diversity of a proteome as protein isoforms harboring different N-terminal extensions or truncations can serve different functions. Recent ribosome profiling data revealed a highly underestimated occurrence of database nonannotated, and thus alternative translation initiation sites (aTIS), at the mRNA level. N-terminomics data in addition showed that in higher eukaryotes around 20% of all identified protein N termini point to such aTIS, to incorrect assignments of the translation start codon, translation initiation at near-cognate start codons, or to alternative splicing. We here report on more than 1700 unique alternative protein N termini identified at the proteome level in human and murine cellular proteomes. Customized databases, created using the translation initiation mapping obtained from ribosome profiling data, additionally demonstrate the use of initiator methionine decoded near-cognate start codons besides the existence of N-terminal extended protein variants at the level of the proteome. Various newly identified aTIS were confirmed by mutagenesis, and meta-analyses demonstrated that aTIS reside in strong Kozak-like motifs and are conserved among eukaryotes, hinting to a possible biological impact. Finally, TargetP analysis predicted that the usage of aTIS often results in altered subcellular localization patterns, providing a mechanism for functional diversification. PMID:24623590

  4. Activation of GCN2 kinase by ribosome stalling links translation elongation with translation initiation

    PubMed Central

    Ishimura, Ryuta; Nagy, Gabor; Dotu, Ivan; Chuang, Jeffrey H; Ackerman, Susan L

    2016-01-01

    Ribosome stalling during translation has recently been shown to cause neurodegeneration, yet the signaling pathways triggered by stalled elongation complexes are unknown. To investigate these pathways we analyzed the brain of C57BL/6J-Gtpbp2nmf205-/- mice in which neuronal elongation complexes are stalled at AGA codons due to deficiencies in a tRNAArgUCU tRNA and GTPBP2, a mammalian ribosome rescue factor. Increased levels of phosphorylation of eIF2α (Ser51) were detected prior to neurodegeneration in these mice and transcriptome analysis demonstrated activation of ATF4, a key transcription factor in the integrated stress response (ISR) pathway. Genetic experiments showed that this pathway was activated by the eIF2α kinase, GCN2, in an apparent deacylated tRNA-independent fashion. Further we found that the ISR attenuates neurodegeneration in C57BL/6J-Gtpbp2nmf205-/- mice, underscoring the importance of cellular and stress context on the outcome of activation of this pathway. These results demonstrate the critical interplay between translation elongation and initiation in regulating neuron survival during cellular stress. DOI: http://dx.doi.org/10.7554/eLife.14295.001 PMID:27085088

  5. Activation of GCN2 kinase by ribosome stalling links translation elongation with translation initiation.

    PubMed

    Ishimura, Ryuta; Nagy, Gabor; Dotu, Ivan; Chuang, Jeffrey H; Ackerman, Susan L

    2016-01-01

    Ribosome stalling during translation has recently been shown to cause neurodegeneration, yet the signaling pathways triggered by stalled elongation complexes are unknown. To investigate these pathways we analyzed the brain of C57BL/6J-Gtpbp2(nmf205)(-/-) mice in which neuronal elongation complexes are stalled at AGA codons due to deficiencies in a tRNA(Arg)UCU tRNA and GTPBP2, a mammalian ribosome rescue factor. Increased levels of phosphorylation of eIF2α (Ser51) were detected prior to neurodegeneration in these mice and transcriptome analysis demonstrated activation of ATF4, a key transcription factor in the integrated stress response (ISR) pathway. Genetic experiments showed that this pathway was activated by the eIF2α kinase, GCN2, in an apparent deacylated tRNA-independent fashion. Further we found that the ISR attenuates neurodegeneration in C57BL/6J-Gtpbp2(nmf205)(-/-) mice, underscoring the importance of cellular and stress context on the outcome of activation of this pathway. These results demonstrate the critical interplay between translation elongation and initiation in regulating neuron survival during cellular stress. PMID:27085088

  6. [Study of molecular mechanisms of translation initiation in mammals using in vitro reconstruction of initiation complexes].

    PubMed

    Shatskiĭ, I N

    2001-01-01

    Papers on the mechanisms of translation initiation in mammals studied by reconstruction of initiation complexes from individual components are reviewed. The author points to the constraints of this approach and to the pitfalls ignoring which one might come to erroneous conclusions and even artifacts. In addition, some methods employed in the field as well as some technical problems are discussed in the paper, together with the means of obviating them. The review could be a guidebook for newcomers into this quite labor-consuming field.

  7. Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution

    PubMed Central

    Armache, Jean-Paul; Jarasch, Alexander; Anger, Andreas M.; Villa, Elizabeth; Becker, Thomas; Bhushan, Shashi; Jossinet, Fabrice; Habeck, Michael; Dindar, Gülcin; Franckenberg, Sibylle; Marquez, Viter; Mielke, Thorsten; Thomm, Michael; Berninghausen, Otto; Beatrix, Birgitta; Söding, Johannes; Westhof, Eric; Wilson, Daniel N.; Beckmann, Roland

    2010-01-01

    Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES–ES and r-protein–ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome. PMID:20980660

  8. Mutations in Caenorhabditis elegans eIF2β Permit Translation Initiation From Non-AUG Start Codons

    PubMed Central

    Zhang , Yinhua; Maduzia , Lisa L.

    2010-01-01

    Recognition of the AUG start codon on mRNAs during translation initiation in eukaryotes occurs in a preinitiation complex that includes small ribosomal subunits and multiple translation initiation factors. The complexity of this process and the lack of appropriate tools have prevented its genetic study in multicellular organisms. Here we describe a genetic system in the nematode Caenorhabditis elegans to study how the AUG start codon is selected. We have generated a sensitive reporter assay that allows for the isolation of mutants with reduced fidelity to recognize the AUG start codon. Two mutants were identified to have dominant missense mutations in iftb-1, which encodes the β-subunit of eIF2 (eIF2β). Both mutations occur in a conserved region located outside of the C2–C2 zinc finger domain where yeast SUI3 mutations are localized in Saccharomyces cerevisiae eIF2β. C. elegans iftb-1, as well as mutant eIF2βs carrying the equivalent SUI3 mutations, are able to initiate translation at non-AUG codons that retain two potential base-pairing interactions with the anticodon of the initiator methionyl tRNA. These analyses further support the critical role of eIF2β in start codon selection, and two functional domains within eIF2β are likely involved, one defined by our C. elegans mutants and the other by the yeast SUI3 mutants. PMID:20215469

  9. Ribosomal Initiation Complex Assembly within the Wild-Strain of Coxsackievirus B3 and Live-Attenuated Sabin3-like IRESes during the Initiation of Translation

    PubMed Central

    Souii, Amira; M’hadheb-Gharbi, Manel Ben; Sargueil, Bruno; Brossard, Audrey; Chamond, Nathalie; Aouni, Mahjoub; Gharbi, Jawhar

    2013-01-01

    Coxsackievirus B3 (CVB3) is an enterovirus of the family of Picornaviridae. The Group B coxsackieviruses include six serotypes (B1 to B6) that cause a variety of human diseases, including myocarditis, meningitis, and diabetes. Among the group B, the B3 strain is mostly studied for its cardiovirulence and its ability to cause acute and persistent infections. Translation initiation of CVB3 RNA has been shown to be mediated by a highly ordered structure of the 5′-untranslated region (5′UTR), which harbors an internal ribosome entry site (IRES). Translation initiation is a complex process in which initiator tRNA, 40S and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of the mRNA. We have previously addressed the question of whether the attenuating mutations of domain V of the poliovirus IRES were specific for a given genomic context or whether they could be transposed and extrapolated to a genomic related virus, i.e., CVB3 wild-type strain. In this context, we have described that Sabin3-like mutation (U473→C) introduced in CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In this study, we analyzed the efficiency of formation of ribosomal initiation complexes 48S and 80S through 10%–30% and 10%–50% sucrose gradients using rabbit reticulocyte lysates (RRLs) and stage-specific translation inhibitors: 5′-Guanylyl-imidodiphosphate (GMP-PNP) and Cycloheximide (CHX), respectively. We demonstrated that the interaction of 48S and 80S ribosomal complexes within the mutant CVB3 RNA was abolished compared with the wild-type RNA by ribosome assembly analysis. Taken together, it is possible that the mutant RNA was unable to interact with some trans-acting factors critical for enhanced IRES function. PMID:23439549

  10. eIF1A/eIF5B interaction network and its functions in translation initiation complex assembly and remodeling

    PubMed Central

    Nag, Nabanita; Lin, Kai Ying; Edmonds, Katherine A.; Yu, Jielin; Nadkarni, Devika; Marintcheva, Boriana; Marintchev, Assen

    2016-01-01

    Eukaryotic translation initiation is a highly regulated process involving multiple steps, from 43S pre-initiation complex (PIC) assembly, to ribosomal subunit joining. Subunit joining is controlled by the G-protein eukaryotic translation initiation factor 5B (eIF5B). Another protein, eIF1A, is involved in virtually all steps, including subunit joining. The intrinsically disordered eIF1A C-terminal tail (eIF1A-CTT) binds to eIF5B Domain-4 (eIF5B-D4). The ribosomal complex undergoes conformational rearrangements at every step of translation initiation; however, the underlying molecular mechanisms are poorly understood. Here we report three novel interactions involving eIF5B and eIF1A: (i) a second binding interface between eIF5B and eIF1A; (ii) a dynamic intramolecular interaction in eIF1A between the folded domain and eIF1A-CTT; and (iii) an intramolecular interaction between eIF5B-D3 and -D4. The intramolecular interactions within eIF1A and eIF5B interfere with one or both eIF5B/eIF1A contact interfaces, but are disrupted on the ribosome at different stages of translation initiation. Therefore, our results indicate that the interactions between eIF1A and eIF5B are being continuously rearranged during translation initiation. We present a model how the dynamic eIF1A/eIF5B interaction network can promote remodeling of the translation initiation complexes, and the roles in the process played by intrinsically disordered protein segments. PMID:27325746

  11. Eukaryotic initiation factor 3C silencing inhibits cell proliferation and promotes apoptosis in human glioma.

    PubMed

    Hao, Jinmin; Wang, Zhiming; Wang, Yaowu; Liang, Zhaohui; Zhang, Xin; Zhao, Zongmao; Jiao, Baohua

    2015-06-01

    Eukaryotic initiation factor 3, subunit c (eIF3c), an oncogene overexpressed in human cancers, plays an important role in cell tumorigenesis and proliferation. However, studies assessing its function in gliomas are scarce. The present study evaluated for the first time, the role of eIF3c in gliomas. Immunohistochemistry was carried out to assess eIF3c expression in 95 human glioma samples and normal brain tissues. Then, the eIF3c mRNA levels were detected in tumor and normal brain specimens by quantitative RT-PCR. In addition, eIF3c mRNA levels were assessed in four glioma cell lines (U87, U251, A172 and U373) by semi-quantitative RT-PCR. The RNA interference (RNAi) technology was employed to knock down the eIF3c gene in the U251 cells. Western blot analysis, BrdU assay and flow cytometry were used to measure eIF3c protein levels, cell proliferation, cell apoptosis and cell cycle, respectively. The eIF3c protein was overexpressed in the human glioma specimens. In agreement, the eIF3c mRNA expression levels were significantly higher in the human glioma tissues compared with the normal brain samples (P<0.0001). In addition, eIF3c mRNA was detected in all the glioma cell lines. Silencing the eIF3c gene in the U251 cells by RNAi significantly suppressed cell proliferation (P<0.01) and increased apoptosis (P<0.01). Finally, a stark decrease was observed in the G1 phase cell number (P<0.01), while the S and G2 phase cells were significantly increased (P<0.01) after eIF3c knockdown. These findings suggest that eIF3c is overexpressed in human gliomas and essential for their proliferation and survival. Therefore, inhibiting eIF3c expression may constitute an effective therapy for human glioma.

  12. Mapping codon usage of the translation initiation region in porcine reproductive and respiratory syndrome virus genome

    PubMed Central

    2011-01-01

    Background Porcine reproductive and respitatory syndrome virus (PRRSV) is a recently emerged pathogen and severely affects swine populations worldwide. The replication of PRRSV is tightly controlled by viral gene expression and the codon usage of translation initiation region within each gene could potentially regulate the translation rate. Therefore, a better understanding of the codon usage pattern of the initiation translation region would shed light on the regulation of PRRSV gene expression. Results In this study, the codon usage in the translation initiation region and in the whole coding sequence was compared in PRRSV ORF1a and ORFs2-7. To investigate the potential role of codon usage in affecting the translation initiation rate, we established a codon usage model for PRRSV translation initiation region. We observed that some non-preferential codons are preferentially used in the translation initiation region in particular ORFs. Although some positions vary with codons, they intend to use codons with negative CUB. Furthermore, our model of codon usage showed that the conserved pattern of CUB is not directly consensus with the conserved sequence, but shaped under the translation selection. Conclusions The non-variation pattern with negative CUB in the PRRSV translation initiation region scanned by ribosomes is considered the rate-limiting step in the translation process. PMID:22014033

  13. A novel interaction of Cap-binding protein complexes eukaryotic initiation factor (eIF) 4F and eIF(iso)4F with a region in the 3'-untranslated region of satellite tobacco necrosis virus.

    PubMed

    Gazo, Brandy M; Murphy, Patricia; Gatchel, Jennifer R; Browning, Karen S

    2004-04-01

    Satellite tobacco necrosis virus (STNV) RNA is naturally uncapped at its 5' end and lacks polyadenylation at its 3' end. Despite lacking these two hallmarks of eukaryotic mRNAs, STNV-1 RNA is translated very efficiently. A approximately 130-nucleotide translational enhancer (TED), located 3' to the termination codon, is necessary for efficient cap-independent translation of STNV-1 RNA. The STNV-1 TED RNA fragment binds to the eukaryotic cap-binding complexes, initiation factor (eIF) 4F and eIF(iso)4F, as measured by nitrocellulose binding and fluorescence titration. STNV-1 TED is a potent inhibitor of in vitro translation when added in trans. This inhibition is reversed by the addition of eIF4F or eIF(iso)4F, and the subunits of eIF4F and eIF(iso)4F cross-link to STNV-1 TED, providing additional evidence that these factors interact directly with STNV-1 TED. Deletion mutagenesis of the STNV-1 TED indicates that a minimal region of approximately 100 nucleotides is necessary to promote cap-independent translation primarily through interaction with the cap binding subunits (eIF4E or eIF(iso)4E) of eIF4F or eIF(iso)4F.

  14. Mitotic phosphorylation of eukaryotic initiation factor 4G1 (eIF4G1) at Ser1232 by Cdk1:cyclin B inhibits eIF4A helicase complex binding with RNA.

    PubMed

    Dobrikov, Mikhail I; Shveygert, Mayya; Brown, Michael C; Gromeier, Matthias

    2014-02-01

    During mitosis, global translation is suppressed, while synthesis of proteins with vital mitotic roles must go on. Prior evidence suggests that the mitotic translation shift involves control of initiation. Yet, no signals specifically targeting translation initiation factors during mitosis have been identified. We used phosphoproteomics to investigate the central translation initiation scaffold and "ribosome adaptor," eukaryotic initiation factor 4G1 (eIF4G1) in interphase or nocodazole-arrested mitotic cells. This approach and kinase inhibition assays, in vitro phosphorylation with recombinant kinase, and kinase depletion-reconstitution experiments revealed that Ser1232 in eIF4G1 is phosphorylated by cyclin-dependent kinase 1 (Cdk1):cyclin B during mitosis. Ser1232 is located in an unstructured region of the C-terminal portion of eIF4G1 that coordinates assembly of the eIF4G/-4A/-4B helicase complex and binding of the mitogen-activated protein kinase (MAPK) signal-integrating kinase, Mnk. Intense phosphorylation of Ser1232 in mitosis strongly enhanced the interactions of eIF4A with HEAT domain 2 of eIF4G and decreased association of eIF4G/-4A with RNA. Our findings implicate phosphorylation of eIF4G1(Ser1232) by Cdk1:cyclin B and its inhibitory effects on eIF4A helicase activity in the mitotic translation initiation shift.

  15. Prediction of translation initiation sites in human mRNA sequences with AUG start codon in weak Kozak context: A neural network approach.

    PubMed

    Tikole, Suhas; Sankararamakrishnan, Ramasubbu

    2008-05-16

    Translation of eukaryotic mRNAs is often regulated by nucleotides around the start codon. A purine at position -3 and a guanine at position +4 contribute significantly to enhance the translation efficiency. Algorithms to predict the translation initiation site often fail to predict the start site if the sequence context is not present. We have developed a neural network method to predict the initiation site of mRNA sequences that lack the preferred nucleotides at the positions -3 and +4 surrounding the translation initiation site. Neural networks of various architectures comprising different number of hidden layers were designed and tested for various sizes of windows of nucleotides surrounding translation initiation sites. We found that the neural network with two hidden layers showed a sensitivity of 83% and specificity of 73% indicating a vastly improved performance in successfully predicting the translation initiation site of mRNA sequences with weak Kozak context. WeakAUG server is freely available at http://bioinfo.iitk.ac.in/AUGPred/.

  16. eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation.

    PubMed

    Lee, Amy S; Kranzusch, Philip J; Doudna, Jennifer A; Cate, Jamie H D

    2016-08-01

    Eukaryotic mRNAs contain a 5′ cap structure that is crucial for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression despite inactivation of eIF4E. Here we describe a cap-dependent pathway of translation initiation in human cells that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eIF3 complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modelling of cap recognition by eIF3d. eIF3d makes specific contacts with the cap, as exemplified by cap analogue competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs such as the cell proliferation regulator c-Jun (also known as JUN). The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a mechanism of cap-dependent translation that is independent of eIF4E, and illustrate how modular RNA elements work together to direct specialized forms of translation initiation. PMID:27462815

  17. Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

    SciTech Connect

    Simonetti, Angelita; Fabbretti, Attilio; Hazemann, Isabelle; Jenner, Lasse; Gualerzi, Claudio O.; Klaholz, Bruno P.

    2013-06-01

    The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.

  18. Initiation on the divergent Type I cadicivirus IRES: factor requirements and interactions with the translation apparatus

    PubMed Central

    Asnani, Mukta; Pestova, Tatyana V.; Hellen, Christopher U.T.

    2016-01-01

    Cadicivirus (CDV) is unique amongst picornaviruses in having a dicistronic genome with internal ribosomal entry sites (IRESs) preceding both open reading frames. Here, we investigated initiation on the 5′-terminal IRES. We report that the 982-nt long 5′UTR comprises 12 domains (d1-d12), five of which (d8-d12, nts 341–950) constitute a divergent Type I IRES. It comprises central elements (the apex of d10, d11 and the following polypyrimidine tract) that are homologous to corresponding elements in canonical Type 1 IRESs, and non-canonical flanking domains (d8, d9 and d12). In vitro reconstitution revealed that as with canonical Type I IRESs, 48S complex formation requires eukaryotic initiation factors (eIFs) 1, 1A, 2, 3, 4A, 4B and 4G, and the poly(C) binding protein 2 (PCBP2), and starts with specific binding of eIF4G/eIF4A to d11. However, in contrast to canonical Type I IRESs, subsequent recruitment of 43S ribosomal complexes does not require direct interaction of their eIF3 constituent with the IRES-bound eIF4G. On the other hand, the CDV IRES forms a 40S/eIF3/IRES ternary complex, with multiple points of contact. These additional interactions with translational components could potentially stimulate recruitment of the 43S complex and alleviate the necessity for direct eIF4G/eIF3 interaction. PMID:26873921

  19. 70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

    PubMed Central

    Yamamoto, Hiroshi; Wittek, Daniela; Gupta, Romi; Qin, Bo; Ueda, Takuya; Krause, Roland; Yamamoto, Kaori; Albrecht, Renate; Pech, Markus; Nierhaus, Knud H.

    2016-01-01

    According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1–IF3). Here, we describe a novel type of initiation termed “70S-scanning initiation,” where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine–Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism. PMID:26888283

  20. Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex

    PubMed Central

    Erzberger, Jan P.; Stengel, Florian; Pellarin, Riccardo; Zhang, Suyang; Schaefer, Tanja; Aylett, Christopher H.S.; Cimermančič, Peter; Boehringer, Daniel; Sali, Andrej; Aebersold, Ruedi; Ban, Nenad

    2014-01-01

    Summary Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA. PMID:25171412

  1. Enhancement of translation initiation by A/T-rich sequences downstream of the initiation codon in Escherichia coli.

    PubMed

    Qing, Guoliang; Xia, Bing; Inouye, Masayori

    2003-01-01

    The region located downstream of the initiation codon constitutes part of the translation initiation signal, significantly affecting the level of protein expression in E. coli. In order to determine its influence on translation initiation, we inserted random 12-base sequences downstream of the initiation codon of the lacZ gene. A total of 119 random clones showing higher beta-galactosidase activities than the control lacZ gene were isolated and subsequently sequenced. Analysis of these clones revealed that their insertion sequences are strikingly rich in A and T, but poor in G, with no consensus sequences among them. Toeprinting experiments and polysome profile analysis confirmed that the A/T-rich sequences enhance translation at the level of initiation. Collectively, the present data demonstrate that A/T richness of the region following the initiation codon plays a significant role in E. coli gene expression. PMID:15153766

  2. Interferon-dependent engagement of eukaryotic initiation factor 4B via S6 kinase (S6K)- and ribosomal protein S6K-mediated signals.

    PubMed

    Kroczynska, Barbara; Kaur, Surinder; Katsoulidis, Efstratios; Majchrzak-Kita, Beata; Sassano, Antonella; Kozma, Sara C; Fish, Eleanor N; Platanias, Leonidas C

    2009-05-01

    Although the roles of Jak-Stat pathways in type I and II interferon (IFN)-dependent transcriptional regulation are well established, the precise mechanisms of mRNA translation for IFN-sensitive genes remain to be defined. We examined the effects of IFNs on the phosphorylation/activation of eukaryotic translation initiation factor 4B (eIF4B). Our data show that eIF4B is phosphorylated on Ser422 during treatment of sensitive cells with alpha IFN (IFN-alpha) or IFN-gamma. Such phosphorylation is regulated, in a cell type-specific manner, by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) and results in enhanced interaction of the protein with eIF3A (p170/eIF3A) and increased associated ATPase activity. Our data also demonstrate that IFN-inducible eIF4B activity and IFN-stimulated gene 15 protein (ISG15) or IFN-gamma-inducible chemokine CXCL-10 protein expression are diminished in S6k1/S6k2 double-knockout mouse embryonic fibroblasts. In addition, IFN-alpha-inducible ISG15 protein expression is blocked by eIF4B or eIF3A knockdown, establishing a requirement for these proteins in mRNA translation/protein expression by IFNs. Importantly, the generation of IFN-dependent growth inhibitory effects on primitive leukemic progenitors is dependent on activation of the S6K/eIF4B or RSK/eIF4B pathway. Taken together, our findings establish critical roles for S6K and RSK in the induction of IFN-dependent biological effects and define a key regulatory role for eIF4B as a common mediator and integrator of IFN-generated signals from these kinases. PMID:19289497

  3. Promotion of Viral IRES-Mediated Translation Initiation under Mild Hypothermia

    PubMed Central

    Licursi, Maria; Carmona-Martinez, Ricardo A.; Razavi, Seyd; Hirasawa, Kensuke

    2015-01-01

    Internal ribosome entry site (IRES)-mediated translation is an essential replication step for certain viruses. As IRES-mediated translation is regulated differently from cap-dependent translation under various cellular conditions, we sought to investigate whether temperature influences efficiency of viral IRES-mediated translation initiation by using bicistronic reporter constructs containing an IRES element of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), human rhinovirus (HRV) or poliovirus (PV). Under mild hypothermic conditions (30 and 35°C), we observed increases in the efficiency of translation initiation by HCV and HRV IRES elements compared to translation initiation at 37°C. The promotion of HRV IRES activity was observed as early as 2 hours after exposure to mild hypothermia. We also confirmed the promotion of translation initiation by HRV IRES under mild hypothermia in multiple cell lines. The expression levels and locations of polypyrimidine tract-binding protein (PTB) and upstream of N-Ras (unr), the IRES trans-acting factors (ITAFs) of HCV and HRV IRES elements, were not modulated by the temperature shift from 37°C to 30°C. Taken together, this study demonstrates that efficiency of translation initiation by some viral IRES elements is temperature dependent. PMID:25951166

  4. An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5' UTRs and its implications for eukaryotic gene translation regulation.

    PubMed

    Pánek, Josef; Kolár, Michal; Vohradský, Jirí; Shivaya Valásek, Leos

    2013-09-01

    There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA-rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5' untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5' UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5' UTRs of mRNAs.

  5. A Distinct Translation Initiation Mechanism Generates Cryptic Peptides for Immune Surveillance

    PubMed Central

    Starck, Shelley R.; Ow, Yongkai; Jiang, Vivian; Tokuyama, Maria; Rivera, Mark; Qi, Xin; Roberts, Richard W.; Shastri, Nilabh

    2008-01-01

    MHC class I molecules present a comprehensive mixture of peptides on the cell surface for immune surveillance. The peptides represent the intracellular protein milieu produced by translation of endogenous mRNAs. Unexpectedly, the peptides are encoded not only in conventional AUG initiated translational reading frames but also in alternative cryptic reading frames. Here, we analyzed how ribosomes recognize and use cryptic initiation codons in the mRNA. We find that translation initiation complexes assemble at non-AUG codons but differ from canonical AUG initiation in response to specific inhibitors acting within the peptidyl transferase and decoding centers of the ribosome. Thus, cryptic translation at non-AUG start codons can utilize a distinct initiation mechanism which could be differentially regulated to provide peptides for immune surveillance. PMID:18941630

  6. Supporting knowledge translation through collaborative translational research initiatives: ‘Bridging’ versus ‘blurring’ boundary-spanning approaches in the UK CLAHRC initiative

    PubMed Central

    Evans, Sarah; Scarbrough, Harry

    2014-01-01

    Recent policy initiatives in the UK and internationally have sought to promote knowledge translation between the ‘producers’ and ‘users’ of research. Within this paper we explore how boundary-spanning interventions used within such initiatives can support knowledge translation between diverse groups. Using qualitative data from a 3-year research study conducted from January 2010 to December 2012 of two case-sites drawn from the CLAHRC initiative in the UK, we distinguish two different approaches to supporting knowledge translation; a ‘bridging’ approach that involves designated roles, discrete events and activities to span the boundaries between communities, and a ‘blurring’ approach that de-emphasises the boundaries between groups, enabling a more continuous process of knowledge translation as part of day-to-day work-practices. In this paper, we identify and differentiate these boundary-spanning approaches and describe how they emerged from the context defined by the wider CLAHRC networks. This highlights the need to develop a more contextualised analysis of the boundary-spanning that underpins knowledge translation processes, relating this to the distinctive features of a particular case. PMID:24561773

  7. A departmental initiative for clinical and translational research.

    PubMed

    Colombo, Christopher J; Baer, Stephanie; Blake, Lindsay; Bollag, Wendy B; Colombo, Rhonda; Diamond, Matthew; George, Varghese; Huber, Lu; Merchen, Lee; Miles, Kathy; Yang, Frances; Nahman, N Stanley

    2016-06-01

    To encourage departmental research activities, the Department of Medicine of the Medical College of Georgia (MCG) introduced an internally funded Translational Research Program (TRP) in 2014. Patterned after the Vanderbilt Institute for Clinical and Translational Research, the program offers research studios for project guidance, research mentoring and the availability of limited financial support through research vouchers. Additional academic services include abstract reviewing, conducting research conferences, organizing departmental research programs for students, and offering courses in biostatistics. During the first 15 months of its existence, the TRP working group addressed 132 distinct activities. Research mentoring, publications, and the conduct of research studios or voucher approvals encompassed 49% of working group activities. Other academic services constituted the remaining 51%. Twenty-four per cent of TRP committee activities involved research mentoring of 32 investigators (25% faculty and 75% trainees). Mentored projects generated 17 abstracts, 2 manuscripts and $87,000 in funds. The TRP conducted 13 research studios; trainees presented 54%. The TRP reviewed 36 abstracts for local and state organizations. Monthly research conferences and statistical courses were conducted and well attended. Our experience thus far indicates that a departmental TRP may serve to facilitate the growth of patient-oriented research with minimal financial support. It requires active engagement of volunteer faculty and departmental leadership willing to balance research with the other demands of the academic mission. PMID:27073213

  8. Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

    PubMed Central

    Han, Yan; Gao, Xiangwei; Liu, Botao; Wan, Ji; Zhang, Xingqian; Qian, Shu-Bing

    2014-01-01

    The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation. PMID:24903108

  9. Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

    PubMed Central

    Tsotakos, Nikolaos; Silveyra, Patricia; Lin, Zhenwu; Thomas, Neal; Vaid, Mudit

    2014-01-01

    Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5′-untranslated regions (5′-UTR) due to differential splicing. The 5′-UTR variant ACD′ is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication. PMID:25326576

  10. Deep sequencing reveals global patterns of mRNA recruitment during translation initiation

    PubMed Central

    Gao, Rong; Yu, Kai; Nie, Jukui; Lian, Tengfei; Jin, Jianshi; Liljas, Anders; Su, Xiao-Dong

    2016-01-01

    In this work, we developed a method to systematically study the sequence preference of mRNAs during translation initiation. Traditionally, the dynamic process of translation initiation has been studied at the single molecule level with limited sequencing possibility. Using deep sequencing techniques, we identified the sequence preference at different stages of the initiation complexes. Our results provide a comprehensive and dynamic view of the initiation elements in the translation initiation region (TIR), including the S1 binding sequence, the Shine-Dalgarno (SD)/anti-SD interaction and the second codon, at the equilibrium of different initiation complexes. Moreover, our experiments reveal the conformational changes and regional dynamics throughout the dynamic process of mRNA recruitment. PMID:27460773

  11. Deep sequencing reveals global patterns of mRNA recruitment during translation initiation.

    PubMed

    Gao, Rong; Yu, Kai; Nie, Jukui; Lian, Tengfei; Jin, Jianshi; Liljas, Anders; Su, Xiao-Dong

    2016-07-27

    In this work, we developed a method to systematically study the sequence preference of mRNAs during translation initiation. Traditionally, the dynamic process of translation initiation has been studied at the single molecule level with limited sequencing possibility. Using deep sequencing techniques, we identified the sequence preference at different stages of the initiation complexes. Our results provide a comprehensive and dynamic view of the initiation elements in the translation initiation region (TIR), including the S1 binding sequence, the Shine-Dalgarno (SD)/anti-SD interaction and the second codon, at the equilibrium of different initiation complexes. Moreover, our experiments reveal the conformational changes and regional dynamics throughout the dynamic process of mRNA recruitment.

  12. An accurately preorganized IRES RNA structure enables eIF4G capture for initiation of viral translation.

    PubMed

    Imai, Shunsuke; Kumar, Parimal; Hellen, Christopher U T; D'Souza, Victoria M; Wagner, Gerhard

    2016-09-01

    Many viruses bypass canonical cap-dependent translation in host cells by using internal ribosomal entry sites (IRESs) in their transcripts; IRESs hijack initiation factors for the assembly of initiation complexes. However, it is currently unknown how IRES RNAs recognize initiation factors that have no endogenous RNA binding partners; in a prominent example, the IRES of encephalomyocarditis virus (EMCV) interacts with the HEAT-1 domain of eukaryotic initiation factor 4G (eIF4G). Here we report the solution structure of the J-K region of this IRES and show that its stems are precisely organized to position protein-recognition bulges. This multisite interaction mechanism operates on an all-or-nothing principle in which all domains are required. This preorganization is accomplished by an 'adjuster module': a pentaloop motif that acts as a dual-sided docking station for base-pair receptors. Because subtle changes in the orientation abrogate protein capture, our study highlights how a viral RNA acquires affinity for a target protein. PMID:27525590

  13. Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

    PubMed

    Mead, Emma J; Masterton, Rosalyn J; Feary, Marc; Obrezanova, Olga; Zhang, Lin; Young, Robert; Smales, C Mark

    2015-12-15

    Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity. PMID:26420881

  14. Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

    PubMed

    Mead, Emma J; Masterton, Rosalyn J; Feary, Marc; Obrezanova, Olga; Zhang, Lin; Young, Robert; Smales, C Mark

    2015-12-15

    Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity.

  15. Biochemical and functional analysis of a 9-nt RNA sequence that affects translation efficiency in eukaryotic cells.

    PubMed

    Chappell, Stephen A; Edelman, Gerald M; Mauro, Vincent P

    2004-06-29

    We previously identified an internal ribosome entry site (IRES) within the 5' leader of the mRNA encoding the Gtx homeodomain protein and showed that shorter nonoverlapping segments of this 5' leader could enhance the translation of a second cistron in a dicistronic mRNA. One of these segments was 9 nt in length, and when multiple copies of this IRES module were linked together, IRES activity was greatly enhanced. To further expand the potential uses of these synthetic constructs and facilitate analyses of the mechanism by which they affect translation, we show here that an IRES containing five linked copies of the 9-nt sequence can also enhance translation in the 5' leader of a monocistronic mRNA. Moreover, a search for interactions of the IRES module with cellular factors revealed specific binding to 40S ribosomal subunits but not to other cellular components. Based on the results of earlier studies suggesting that this sequence could bind to a complementary segment of 18S rRNA, we tested various sequences for possible links between the length of the complementary match, their binding to ribosomes, and their influence on translational efficiency. We found that the length of the complementary match was directly correlated with the ability of RNA probes to bind to ribosomes. In addition, translation was maximally enhanced ( approximately 8-fold) by a 7-nt segment of the 9-nt element; the enhancement declined progressively as the complementary stretches became progressively longer or shorter. The results suggest that the Gtx 9-nt sequence affects translation efficiency by a mechanism that involves base pairing to 18S rRNA.

  16. Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: an eIF5-dependent cell-free translation system.

    PubMed

    Maiti, T; Das, S; Maitra, U

    2000-02-22

    Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound GTP. In Saccharomyces cerevisiae, eIF5, a protein of 45346Da, is encoded by a single-copy essential gene, TIF5. In this paper, we have isolated a temperature-sensitive S. cerevisiae strain, TMY5-1, by replacing the wild-type chromosomal copy of TIF5 with one mutagenized in vitro. The mutant yeast cells rapidly cease protein synthesis when grown under non-permissive conditions, lose polyribosomes and accumulate free 80S ribosomes. Further characterization of mutant eIF5 showed that the mutant protein, expressed in Escherichia coli, is defective both in its interaction with eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S initiation complex and consequently in the formation of the 80S initiation complex. Additionally, the availability of a yeast strain containing temperature-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for translation of mRNAs in vitro. PMID:10689193

  17. Expression of eukaryotic polypeptides in chloroplasts

    DOEpatents

    Mayfield, Stephen P.

    2013-06-04

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  18. Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes.

    PubMed Central

    Pestova, T V; Shatsky, I N; Hellen, C U

    1996-01-01

    Eukaryotic translation is initiated following binding of ribosomes either to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are both mediated by eukaryotic initiation factor 4F (eIF4F), which consists of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G subunits. Here we present a functional analysis of eIF4F which defines the subunits and subunit domains necessary for its function in initiation mediated by the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified translation components and lacking eIF4A and -4F, IRES-mediated initiation did not require the cap-binding protein eIF4E but was absolutely dependent on eIF4A and the central third of eIF4G. This central domain of eIF4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation codon in an ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES. The central domain of eIF4G was itself UV cross-linked to the IRES and strongly stimulated UV cross-linking of eIF4A to the IRES in conjunction with either eIF4B or with the carboxy-terminal third of eIF4G. PMID:8943342

  19. Yeast eukaryotic initiation factor 4B (eIF4B) enhances complex assembly between eIF4A and eIF4G in vivo.

    PubMed

    Park, Eun-Hee; Walker, Sarah E; Zhou, Fujun; Lee, Joseph M; Rajagopal, Vaishnavi; Lorsch, Jon R; Hinnebusch, Alan G

    2013-01-25

    Translation initiation factor eIF4F (eukaryotic initiation factor 4F), composed of eIF4E, eIF4G, and eIF4A, binds to the m(7)G cap structure of mRNA and stimulates recruitment of the 43S preinitiation complex and subsequent scanning to the initiation codon. The HEAT domain of eIF4G stabilizes the active conformation of eIF4A required for its RNA helicase activity. Mammalian eIF4B also stimulates eIF4A activity, but this function appears to be lacking in yeast, making it unclear how yeast eIF4B (yeIF4B/Tif3) stimulates translation. We identified Ts(-) mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A·eIF4G complexes in vivo, and the rescue of specific mutant eIF4A·eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.

  20. Why are we where we are? Understanding replication origins and initiation sites in eukaryotes using ChIP-approaches.

    PubMed

    Schepers, Aloys; Papior, Peer

    2010-01-01

    DNA replication initiates from origins of replication following a strict sequential activation programme and a conserved temporal order of activation. The number of replication initiation sites varies between species, according to the complexity of the genomes, with an average spacing of 100,000 bp. In contrast to yeast genomes, the location and definition of origins in mammalian genomes has been elusive. Historically, mammalian replication initiation sites have been mapped in situ by systematically searching specific genomic loci for sites that preferentially initiated DNA replication, potential origins by start-site mapping and autonomously replicating sequence experiments, and potential ORC and pre-replicative complex (pre-RC) sites by chromatin immunoprecipitation (ChIP) using antibodies for pre-RC proteins. In the past decade, ChIP has become an important method for analyzing protein/DNA interactions. Classically, ChIP is combined with Southern blotting or PCR. Recently, whole genome-ChIP methods have been very successful in unicellular eukaryotes to understand molecular mechanisms coordinating replication initiation and its flexibility in response to environmental changes. However, in mammalian systems, ChIP with pre-RC antibodies has often been challenging and genome-wide studies are scarce. In this review, we will appraise the progress that has been made in understanding replication origin organization using immunoprecipitation of the ORC and Mcm2-7 complexes. A special focus will be on the advantages and disadvantages of genome-wide ChIP-technologies and their potential impact on understanding metazoan replicators. PMID:19904620

  1. Amphetamine elevates phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the rat forebrain via activating dopamine D1 and D2 receptors.

    PubMed

    Xue, Bing; Fitzgerald, Cole A; Jin, Dao-Zhong; Mao, Li-Min; Wang, John Q

    2016-09-01

    Psychostimulants have an impact on protein synthesis, although underlying molecular mechanisms are unclear. Eukaryotic initiation factor 2α-subunit (eIF2α) is a key player in initiation of protein translation and is regulated by phosphorylation. While this factor is sensitive to changing synaptic input and is critical for synaptic plasticity, its sensitivity to stimulants is poorly understood. Here we systematically characterized responses of eIF2α to a systemic administration of the stimulant amphetamine (AMPH) in dopamine responsive regions of adult rat brains. Intraperitoneal injection of AMPH at 5mg/kg increased eIF2α phosphorylation at serine 51 in the striatum. This increase was transient. In the medial prefrontal cortex (mPFC), AMPH induced a relatively delayed phosphorylation of the factor. Pretreatment with a dopamine D1 receptor antagonist SCH23390 blocked the AMPH-stimulated eIF2α phosphorylation in both the striatum and mPFC. Similarly, a dopamine D2 receptor antagonist eticlopride reduced the effect of AMPH in the two regions. Two antagonists alone did not alter basal eIF2α phosphorylation. AMPH and two antagonists did not change the amount of total eIF2α proteins in both regions. These results demonstrate the sensitivity of eIF2α to stimulant exposure. AMPH possesses the ability to stimulate eIF2α phosphorylation in striatal and mPFC neurons in vivo in a D1 and D2 receptor-dependent manner. PMID:27338925

  2. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A.

    PubMed Central

    Pause, A; Sonenberg, N

    1992-01-01

    eIF-4A is a translation initiation factor that exhibits bidirectional RNA unwinding activity in vitro in the presence of another translation initiation factor, eIF-4B and ATP. This activity is thought to be responsible for the melting of secondary structure in the 5' untranslated region of eukaryotic mRNAs to facilitate ribosome binding. eIF-4A is a member of a fast growing family of proteins termed the DEAD family. These proteins are believed to be RNA helicases, based on the demonstrated in vitro RNA helicase activity of two members (eIF-4A and p68) and their homology in eight amino acid regions. Several related biochemical activities were attributed to eIF-4A: (i) ATP binding, (ii) RNA-dependent ATPase and (iii) RNA helicase. To determine the contribution of the highly conserved regions to these activities, we performed site-directed mutagenesis. First we show that recombinant eIF-4A, together with recombinant eIF-4B, exhibit RNA helicase activity in vitro. Mutations in the ATPase A motif (AXXXXGKT) affect ATP binding, whereas mutations in the predicted ATPase B motif (DEAD) affect ATP hydrolysis. We report here that the DEAD region couples the ATPase with the RNA helicase activity. Furthermore, two other regions, whose functions were unknown, have also been characterized. We report that the first residue in the HRIGRXXR region is involved in ATP hydrolysis and that the SAT region is essential for RNA unwinding. Our results suggest that the highly conserved regions in the DEAD box family are critical for RNA helicase activity. Images PMID:1378397

  3. Targeting Eukaryotic Translation in Mesothelioma Cells with an eIF4E-Specific Antisense Oligonucleotide

    PubMed Central

    Jacobson, Blake A.; Thumma, Saritha C.; Jay-Dixon, Joseph; Patel, Manish R.; Dubear Kroening, K.; Kratzke, Marian G.; Etchison, Ryan G.; Konicek, Bruce W.; Graff, Jeremy R.; Kratzke, Robert A.

    2013-01-01

    Background Aberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO) is assessed as a therapy for mesothelioma. Methods Mesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and β-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment. Results eIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number. Conclusion 4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity. PMID:24260583

  4. Expression of Eukaryotic Initiation Factor 5A and Hypusine Forming Enzymes in Glioblastoma Patient Samples: Implications for New Targeted Therapies

    PubMed Central

    Preukschas, Michael; Hagel, Christian; Schulte, Alexander; Weber, Kristoffer; Lamszus, Katrin; Sievert, Henning; Pällmann, Nora; Bokemeyer, Carsten; Hauber, Joachim; Braig, Melanie; Balabanov, Stefan

    2012-01-01

    Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. Despite a broad range of new and more specific treatment strategies, therapy of glioblastomas remains challenging and tumors relapse in all cases. Recent work demonstrated that the posttranslational hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is a crucial regulator of cell proliferation, differentiation and an important factor in tumor formation, progression and maintenance. Here we report that eIF-5A as well as the hypusine-forming enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are highly overexpressed in glioblastoma patient samples. Importantly, targeting eIF-5A and its hypusine modification with GC7, a specific DHS-inhibitor, showed a strong antiproliferative effect in glioblastoma cell lines in vitro, while normal human astrocytes were not affected. Furthermore, we identified p53 dependent premature senescence, a permanent cell cycle arrest, as the primary outcome in U87-MG cells after treatment with GC7. Strikingly, combined treatment with clinically relevant alkylating agents and GC7 had an additive antiproliferative effect in glioblastoma cell lines. In addition, stable knockdown of eIF-5A and DHS by short hairpin RNA (shRNA) could mimic the antiproliferative effects of GC7. These findings suggest that pharmacological inhibition of eIF-5A may represent a novel concept to treat glioblastomas and may help to substantially improve the clinical course of this tumor entity. PMID:22927971

  5. Sld3-MCM Interaction Facilitated by Dbf4-Dependent Kinase Defines an Essential Step in Eukaryotic DNA Replication Initiation

    PubMed Central

    Fang, Dingqiang; Cao, Qinhong; Lou, Huiqiang

    2016-01-01

    Sld3/Treslin is an evolutionarily conserved protein essential for activation of DNA helicase Mcm2-7 and replication initiation in all eukaryotes. Nevertheless, it remains elusive how Sld3 is recruited to origins. Here, we have identified the direct physical association of Sld3 with Mcm2 and Mcm6 subunits in vitro, which is significantly enhanced by DDK in vivo. The Sld3-binding domain (SBD) is mapped to the N-termini of Mcm2 and Mcm6, both of them are essential for cell viability and enriched with the DDK phosphorylation sites. Glutamic acid substitution of four conserved positively charged residues of Sld3 (sld3-4E), near the Cdc45-binding region, interrupts its interaction with Mcm2/6 and causes cell death. By using a temperature-inducible degron (td), we show that deletion of Mcm6 SBD (mcm6ΔN122) abolishes not only Sld3 enrichment at early origins in G1 phase, but also subsequent recruitment of GINS and RPA during S phase. These findings elucidate the in vivo molecular details of the DDK-dependent Sld3-MCM association, which plays a crucial role in MCM helicase activation and origin unwinding. PMID:27375603

  6. The soy isoflavone equol may increase cancer malignancy via up-regulation of eukaryotic protein synthesis initiation factor eIF4G.

    PubMed

    de la Parra, Columba; Otero-Franqui, Elisa; Martinez-Montemayor, Michelle; Dharmawardhane, Suranganie

    2012-12-01

    Dietary soy is thought to be cancer-preventive; however, the beneficial effects of soy on established breast cancer is controversial. We recently demonstrated that dietary daidzein or combined soy isoflavones (genistein, daidzein, and glycitein) increased primary mammary tumor growth and metastasis. Cancer-promoting molecules, including eukaryotic protein synthesis initiation factors (eIF) eIF4G and eIF4E, were up-regulated in mammary tumors from mice that received dietary daidzein. Herein, we show that increased eIF expression in tumor extracts of mice after daidzein diets is associated with protein expression of mRNAs with internal ribosome entry sites (IRES) that are sensitive to eIF4E and eIF4G levels. Results with metastatic cancer cell lines show that some of the effects of daidzein in vivo can be recapitulated by the daidzein metabolite equol. In vitro, equol, but not daidzein, up-regulated eIF4G without affecting eIF4E or its regulator, 4E-binding protein (4E-BP), levels. Equol also increased metastatic cancer cell viability. Equol specifically increased the protein expression of IRES containing cell survival and proliferation-promoting molecules and up-regulated gene and protein expression of the transcription factor c-Myc. Moreover, equol increased the polysomal association of mRNAs for p 120 catenin and eIF4G. The elevated eIF4G in response to equol was not associated with eIF4E or 4E-binding protein in 5' cap co-capture assays or co-immunoprecipitations. In dual luciferase assays, IRES-dependent protein synthesis was increased by equol. Therefore, up-regulation of eIF4G by equol may result in increased translation of pro-cancer mRNAs with IRESs and, thus, promote cancer malignancy.

  7. Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells.

    PubMed Central

    Senthil, D; Faulkner, J L; Choudhury, G G; Abboud, H E; Kasinath, B S

    2001-01-01

    Interaction between angiotensin II, which binds a G-protein-coupled receptor, and insulin, a ligand for receptor tyrosine kinase, was examined in renal proximal tubular epithelial cells. Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) which follows the release of the factor from a heterodimeric complex by phosphorylation of its binding protein, 4E-BP1. Angiotensin II (1 nM) or insulin (1 nM) individually stimulated 4E-BP1 phosphorylation. However, pre-incubation with angiotensin II abrogated insulin-induced phosphorylation of 4E-BP1, resulting in persistent binding to eIF4E. Although angiotensin II and insulin individually activated phosphoinositide 3-kinase and extracellular signal-regulated kinase (ERK)-1/-2-type mitogen-activated protein (MAP) kinase, pre-incubation with angiotensin II abolished insulin-induced stimulation of these kinases, suggesting more proximal events in insulin signalling may be intercepted. Pretreatment with angiotensin II markedly inhibited insulin-stimulated tyrosine phosphorylation of insulin-receptor beta-chain and insulin-receptor substrate 1. Losartan prevented angiotensin II inhibition of insulin-induced ERK-1/-2-type MAP kinase activation and 4E-BP1 phosphorylation, suggesting mediation of the effect of angiotensin II by its type 1 receptor. Insulin-stimulated de novo protein synthesis was also abolished by pre-incubation with angiotensin II. These data show that angiotensin II inhibits 4E-BP1 phosphorylation and stimulation of protein synthesis induced by insulin by interfering with proximal events in insulin signalling. Our data provide a mechanistic basis for insulin insensitivity induced by angiotensin II. PMID:11695995

  8. Phosphoinositide 3-Kinases Upregulate System xc− via Eukaryotic Initiation Factor 2α and Activating Transcription Factor 4 – A Pathway Active in Glioblastomas and Epilepsy

    PubMed Central

    Baxter, Paul; Kassubek, Rebecca; Albrecht, Philipp; Van Liefferinge, Joeri; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Karpel-Massler, Georg; Meakin, Paul J.; Hayes, John D.; Aronica, Eleonora; Smolders, Ilse; Ludolph, Albert C.; Methner, Axel; Conrad, Marcus; Massie, Ann; Hardingham, Giles E.

    2014-01-01

    Abstract Aims: Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc− imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc− and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. Results: PI3Ks induce system xc− through glycogen synthase kinase 3β (GSK-3β) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2α phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. Innovation: Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3β, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc−. Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. Conclusion: PI3K-regulated system xc− activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate. Antioxid. Redox Signal. 20: 2907–2922. PMID:24219064

  9. NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII.

    PubMed

    Aumayr, Martina; Fedosyuk, Sofiya; Ruzicska, Katharina; Sousa-Blin, Carla; Kontaxis, Georg; Skern, Tim

    2015-12-01

    Messenger RNA is recruited to the eukaryotic ribosome by a complex including the eukaryotic initiation factor (eIF) 4E (the cap-binding protein), the scaffold protein eIF4G and the RNA helicase eIF4A. To shut-off host-cell protein synthesis, eIF4G is cleaved during picornaviral infection by a virally encoded proteinase; the structural basis of this reaction and its stimulation by eIF4E is unclear. We have structurally and biochemically investigated the interaction of purified foot-and-mouth disease virus (FMDV) leader proteinase (Lb(pro)), human rhinovirus 2 (HRV2) 2A proteinase (2A(pro)) and coxsackievirus B4 (CVB4) 2A(pro) with purified eIF4GII, eIF4E and the eIF4GII/eIF4E complex. Using nuclear magnetic resonance (NMR), we completed (13)C/(15) N sequential backbone assignment of human eIF4GII residues 551-745 and examined their binding to murine eIF4E. eIF4GII551-745 is intrinsically unstructured and remains so when bound to eIF4E. NMR and biophysical techniques for determining stoichiometry and binding constants revealed that the papain-like Lb(pro) only forms a stable complex with eIF4GII(551-745) in the presence of eIF4E, with KD values in the low nanomolar range; Lb(pro) contacts both eIF4GII and eIF4E. Furthermore, the unrelated chymotrypsin-like 2A(pro) from HRV2 and CVB4 also build a stable complex with eIF4GII/eIF4E, but with K(D) values in the low micromolar range. The HRV2 enzyme also forms a stable complex with eIF4E; however, none of the proteinases tested complex stably with eIF4GII alone. Thus, these three picornaviral proteinases have independently evolved to establish distinct triangular heterotrimeric protein complexes that may actively target ribosomes involved in mRNA recruitment to ensure efficient host cell shut-off.

  10. The 5′ Untranslated Region of the Human T-Cell Lymphotropic Virus Type 1 mRNA Enables Cap-Independent Translation Initiation

    PubMed Central

    Olivares, Eduardo; Landry, Dori M.; Cáceres, C. Joaquín; Pino, Karla; Rossi, Federico; Navarrete, Camilo; Huidobro-Toro, Juan Pablo; Thompson, Sunnie R.

    2014-01-01

    ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5′-untranslated region (5′UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5′UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon. IMPORTANCE The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene

  11. ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes.

    PubMed

    Ellederová, Zdenka; Cais, Ondrej; Susor, Andrej; Uhlírová, Katka; Kovárová, Hana; Jelínková, Lucie; Tomek, Wolfgang; Kubelka, Michal

    2008-02-01

    Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E undergoes regulated phosphorylation on Ser-209 and this phosphorylation is believed to be important for its binding to mRNA and to other initiation factors. The findings showing that the translation initiation factor eIF4E becomes gradually phosphorylated during in vitro maturation (IVM) of pig oocytes with a maximum in metaphase II (M II) stage oocytes have been documented by us recently (Ellederova et al., 2006). The aim of this work was to study in details the metabolic pathways involved in this process. Using inhibitors of cyclin-dependent kinases, Butyrolactone I (BL I) and protein phosphatases, okadaic acid (OA) we show that ERK1/2 MAP kinase pathway is involved in this phosphorylation. We also demonstrate that activation and phosphorylation of ERK1/2 MAP kinase and eIF4E is associated with the activating phosphorylation of Mnk1 kinase, one of the two main kinases phosphorylating eIF4E in somatic cells.

  12. Rewarding Excellent Teaching: The Translation of a Policy Initiative in the United Kingdom

    ERIC Educational Resources Information Center

    Turner, Rebecca; Gosling, David

    2012-01-01

    The need to provide more significant rewards for "teaching excellence" in order to provide parity of status with research in higher education has often been asserted. This paper examines ways in which the idea of rewarding excellent teaching has been understood and translated within a large teaching and learning initiative that was overtly based…

  13. Comparative analysis of contextual bias around the translation initiation sites in plant genomes.

    PubMed

    Gupta, Paras; Rangan, Latha; Ramesh, T Venkata; Gupta, Mudit

    2016-09-01

    Nucleotide distribution around translation initiation site (TIS) is thought to play an important role in determining translation efficiency. Kozak in vertebrates and later Joshi et al. in plants identified context sequence having a key role in translation efficiency, but a great variation regarding this context sequence has been observed among different taxa. The present study aims to refine the context sequence around initiation codon in plants and addresses the sampling error problem by using complete genomes of 7 monocots and 7 dicots separately. Besides positions -3 and +4, significant conservation at -2 and +5 positions was also found and nucleotide bias at the latter two positions was shown to directly influence translation efficiency in the taxon studied. About 1.8% (monocots) and 2.4% (dicots) of the total sequences fit the context sequence from positions -3 to +5, which might be indicative of lower number of housekeeping genes in the transcriptome. A three base periodicity was observed in 5' UTR and CDS of monocots and only in CDS of dicots as confirmed against random occurrence and annotation errors. Deterministic enrichment of GCNAUGGC in monocots, AANAUGGC in dicots and GCNAUGGC in plants around TIS was also established (where AUG denotes the start codon), which can serve as an arbiter of putative TIS with efficient translation in plants. PMID:27316311

  14. Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1.

    PubMed

    Eckhardt, Katrin; Troger, Juliane; Reissmann, Jana; Katschinski, Dörthe M; Wagner, Klaus F; Stengel, Petra; Paasch, Uwe; Hunziker, Peter; Borter, Emanuela; Barth, Sandra; Schlafli, Philipp; Spielmann, Patrick; Stiehl, Daniel P; Camenisch, Gieri; Wenger, Roland H

    2007-01-01

    PASKIN links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in pancreatic beta-cells. Using newly generated monoclonal antibodies, PASKIN was localized in the nuclei of human testis germ cells and in the midpiece of human sperm tails. A speckle-like nuclear pattern was observed for endogenous PASKIN in HeLa cells in addition to its cytoplasmic localization. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN and to the C-terminus of eEF1A1 using mammalian two-hybrid and GST pull-down assays. Kinase assays, mass spectrometry and site-directed mutagenesis revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation mainly but not exclusively at Thr432. Wild-type but not kinase-inactive PASKIN increased the in vitro translation of a reporter cRNA. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins also showed a remarkably similar localization in the midpiece of the sperm tail. These data suggest regulation of eEF1A1 by PASKIN-dependent phosphorylation in somatic as well as in sperm cells. PMID:17595531

  15. Positive mRNA Translational Control in Germ Cells by Initiation Factor Selectivity

    PubMed Central

    Friday, Andrew J.; Keiper, Brett D.

    2015-01-01

    Ultimately, the production of new proteins in undetermined cells pushes them to new fates. Other proteins hold a stem cell in a mode of self-renewal. In germ cells, these decision-making proteins are produced largely from translational control of preexisting mRNAs. To date, all of the regulation has been attributed to RNA binding proteins (RBPs) that repress mRNAs in many models of germ cell development (Drosophila, mouse, C. elegans, and Xenopus). In this review, we focus on the selective, positive function of translation initiation factors eIF4E and eIF4G, which recruit mRNAs to ribosomes upon derepression. Evidence now shows that the two events are not separate but rather are coordinated through composite complexes of repressors and germ cell isoforms of eIF4 factors. Strikingly, the initiation factor isoforms are themselves mRNA selective. The mRNP complexes of translation factors and RBPs are built on specific populations of mRNAs to prime them for subsequent translation initiation. Simple rearrangement of the partners causes a dormant mRNP to become synthetically active in germ cells when and where they are required to support gametogenesis. PMID:26357652

  16. The E3 Ubiquitin Ligase ARIH1 Protects against Genotoxic Stress by Initiating a 4EHP-Mediated mRNA Translation Arrest

    PubMed Central

    von Stechow, Louise; Typas, Dimitris; Carreras Puigvert, Jordi; Oort, Laurens; Siddappa, Ramakrishnaiah; Pines, Alex; Vrieling, Harry; van de Water, Bob

    2015-01-01

    DNA damage response signaling is crucial for genome maintenance in all organisms and is corrupted in cancer. In an RNA interference (RNAi) screen for (de)ubiquitinases and sumoylases modulating the apoptotic response of embryonic stem (ES) cells to DNA damage, we identified the E3 ubiquitin ligase/ISGylase, ariadne homologue 1 (ARIH1). Silencing ARIH1 sensitized ES and cancer cells to genotoxic compounds and ionizing radiation, irrespective of their p53 or caspase-3 status. Expression of wild-type but not ubiquitinase-defective ARIH1 constructs prevented sensitization caused by ARIH1 knockdown. ARIH1 protein abundance increased after DNA damage through attenuation of proteasomal degradation that required ATM signaling. Accumulated ARIH1 associated with 4EHP, and in turn, this competitive inhibitor of the eukaryotic translation initiation factor 4E (eIF4E) underwent increased nondegradative ubiquitination upon DNA damage. Genotoxic stress led to an enrichment of ARIH1 in perinuclear, ribosome-containing regions and triggered 4EHP association with the mRNA 5′ cap as well as mRNA translation arrest in an ARIH1-dependent manner. Finally, restoration of DNA damage-induced translation arrest in ARIH1-depleted cells by means of an eIF2 inhibitor was sufficient to reinstate resistance to genotoxic stress. These findings identify ARIH1 as a potent mediator of DNA damage-induced translation arrest that protects stem and cancer cells against genotoxic stress. PMID:25624349

  17. Human Translation Initiation Factor eIF4G1 Possesses a Low-Affinity ATP Binding Site Facing the ATP-Binding Cleft of eIF4A in the eIF4G/eIF4A Complex

    PubMed Central

    2015-01-01

    Eukaryotic translation initiation factor 4G (eIF4G) plays a crucial role in translation initiation, serving as a scaffolding protein binding several other initiation factors, other proteins, and RNA. Binding of eIF4G to the ATP-dependent RNA helicase eukaryotic translation initiation factor 4A (eIF4A) enhances the activity of eIF4A in solution and in crowded environments. Previously, this activity enhancement was solely attributed to eIF4G, conferring a closed, active conformation upon eIF4A. Here we show that eIF4G contains a low-affinity binding site at the entrance to the ATP-binding cleft on eIF4A, suggesting that regulation of the local ATP concentration may be an additional reason for the enhancement in activity. PMID:25255371

  18. MicroRNA-33a-5p Modulates Japanese Encephalitis Virus Replication by Targeting Eukaryotic Translation Elongation Factor 1A1

    PubMed Central

    Chen, Zheng; Ye, Jing; Ashraf, Usama; Li, Yunchuan; Wei, Siqi; Wan, Shengfeng; Zohaib, Ali; Song, Yunfeng; Chen, Huanchun

    2016-01-01

    ABSTRACT Japanese encephalitis virus (JEV) is a typical mosquito-borne flavivirus responsible for acute encephalitis and meningitis in humans. However, the molecular mechanism for JEV pathogenesis is still unclear. MicroRNAs (miRNAs) are small noncoding RNAs that act as gene regulators. They are directly or indirectly involved in many cellular functions owing to their ability to target mRNAs for degradation or translational repression. However, how cellular miRNAs are regulated and their functions during JEV infection are largely unknown. In the present study, we found that JEV infection downregulated the expression of endogenous cellular miR-33a-5p. Notably, artificially transfecting with miR-33a-5p mimics led to a significant decrease in viral replication, suggesting that miR-33a-5p acts as a negative regulator of JEV replication. A dual-luciferase reporter assay identified eukaryotic translation elongation factor 1A1 (EEF1A1) as one of the miR-33a-5p target genes. Our study further demonstrated that EEF1A1 can interact with the JEV proteins NS3 and NS5 in replicase complex. Through this interaction, EEF1A1 can stabilize the components of viral replicase complex and thus facilitates viral replication during JEV infection. Taken together, these results suggest that miR-33a-5p is downregulated during JEV infection, which contributes to viral replication by increasing the intracellular level of EEF1A1, an interaction partner of JEV NS3 and NS5. This study provides a better understanding of the molecular mechanisms of JEV pathogenesis. IMPORTANCE MiRNAs are critical regulators of gene expression that utilize sequence complementarity to bind to and modulate the stability or translation efficiency of target mRNAs. Accumulating data suggest that miRNAs regulate a wide variety of molecular mechanisms in the host cells during viral infections. JEV, a neurotropic flavivirus, is one of the major causes of acute encephalitis in humans worldwide. The roles of cellular mi

  19. IRE1α-Dependent Decay of CReP/Ppp1r15b mRNA Increases Eukaryotic Initiation Factor 2α Phosphorylation and Suppresses Protein Synthesis

    PubMed Central

    So, Jae-Seon; Cho, Sungyun; Min, Sang-Hyun; Kimball, Scot R.

    2015-01-01

    The unfolded protein response (UPR) regulates endoplasmic reticulum (ER) homeostasis and protects cells from ER stress. IRE1α is a central regulator of the UPR that activates the transcription factor XBP1s through an unconventional splicing mechanism using its endoribonuclease activity. IRE1α also cleaves certain mRNAs containing XBP1-like secondary structures to promote the degradation of these mRNAs, a process known as regulated IRE1α-dependent decay (RIDD). We show here that the mRNA of CReP/Ppp1r15b, a regulatory subunit of eukaryotic translation initiation factor 2α (eIF2α) phosphatase, is a RIDD substrate. eIF2α plays a central role in the integrated stress response by mediating the translational attenuation to decrease the stress level in the cell. CReP expression was markedly suppressed in XBP1-deficient mice livers due to hyperactivated IRE1α. Decreased CReP expression caused the induction of eIF2α phosphorylation and the attenuation of protein synthesis in XBP1-deficient livers. ER stress also suppressed CReP expression in an IRE1α-dependent manner, which increased eIF2α phosphorylation and consequently attenuated protein synthesis. Taken together, the results of our study reveal a novel function of IRE1α in the regulation of eIF2α phosphorylation and the translational control. PMID:26031337

  20. Translation initiation rate determines the impact of ribosome stalling on bacterial protein synthesis.

    PubMed

    Hersch, Steven J; Elgamal, Sara; Katz, Assaf; Ibba, Michael; Navarre, William Wiley

    2014-10-10

    Ribosome stalling during translation can be caused by a number of characterized mechanisms. However, the impact of elongation stalls on protein levels is variable, and the reasons for this are often unclear. To investigate this relationship, we examined the bacterial translation elongation factor P (EF-P), which plays a critical role in rescuing ribosomes stalled at specific amino acid sequences including polyproline motifs. In previous proteomic analyses of both Salmonella and Escherichia coli efp mutants, it was evident that not all proteins containing a polyproline motif were dependent on EF-P for efficient expression in vivo. The α- and β-subunits of ATP synthase, AtpA and AtpD, are translated from the same mRNA transcript, and both contain a PPG motif; however, proteomic analysis revealed that AtpD levels are strongly dependent on EF-P, whereas AtpA levels are independent of EF-P. Using these model proteins, we systematically determined that EF-P dependence is strongly influenced by elements in the 5'-untranslated region of the mRNA. By mutating either the Shine-Dalgarno sequence or the start codon, we find that EF-P dependence correlates directly with the rate of translation initiation where strongly expressed proteins show the greatest dependence on EF-P. Our findings demonstrate that polyproline-induced stalls exert a net effect on protein levels only if they limit translation significantly more than initiation. This model can be generalized to explain why sequences that induce pauses in translation elongation to, for example, facilitate folding do not necessarily exact a penalty on the overall production of the protein. PMID:25148683

  1. Involvement of the eukaryotic initiation factor 6 and kermit2/gipc2 in Xenopus laevis pronephros formation.

    PubMed

    Tussellino, Margherita; De Marco, Nadia; Campanella, Chiara; Carotenuto, Rosa

    2012-01-01

    The translation initiation factor Eif6 has been implicated as a regulator of ribosome assembly, selective mRNA translation and apoptosis. Many of these activities depend upon the phosphorylation of eif6 Serine 235 by protein kinase C (PKC). Eif6-60S is probably part of the RNA-induced silencing complex (RISC). eif6 over-expression in Xenopus embryos causes aberrant eye development. kermit2/gipc2 morphants have an eye phenotype similar to that of the eif6 overexpressors. Eye formation is regulated by insulin growth factor (IGF) signalling. eif6 interacts with the IGF receptor (IGFR) and kermit2/gipc2, which also binds to igfr. eif6 over-expression in Xenopus causes also the formation of antero-ventral oedema, suggesting a malfunction of the excretory system. Here we evaluated the pronephros phenotype. The oedema grows into the nephrocoel, expanding its boundary and is accompanied by a strong reduction of the pronephros. The three main components of the pronephros are severely impaired in eif6 over-expressors, while are not affected in eif6 morphants. Conversely, gipc2 depletion induces the oedema phenotype and reduction of the pronephros, while gipc2 overexpression does not. p110*, a constitutively active p110 subunit of the PI3 kinase partially recovers the oedema phenotype. We also determined that PKC-dependent phosphorylation of Ser235 in eif6 is not required to produce defective pronephroi. These results indicate that the levels of eif6 are highly regulated during development and instrumental for proper morphogenesis of the pronephros. Moreover, it appears that for proper pronephros development the gipc2 level should be kept within or over the physiological range and that the oedema phenotype is partly due to the inhibition of IGF signalling. PMID:22689378

  2. A new database (GCD) on genome composition for eukaryote and prokaryote genome sequences and their initial analyses.

    PubMed

    Kryukov, Kirill; Sumiyama, Kenta; Ikeo, Kazuho; Gojobori, Takashi; Saitou, Naruya

    2012-01-01

    Eukaryote genomes contain many noncoding regions, and they are quite complex. To understand these complexities, we constructed a database, Genome Composition Database, for the whole genome composition statistics for 101 eukaryote genome data, as well as more than 1,000 prokaryote genomes. Frequencies of all possible one to ten oligonucleotides were counted for each genome, and these observed values were compared with expected values computed under observed oligonucleotide frequencies of length 1-4. Deviations from expected values were much larger for eukaryotes than prokaryotes, except for fungal genomes. Mammalian genomes showed the largest deviation among animals. The results of comparison are available online at http://esper.lab.nig.ac.jp/genome-composition-database/.

  3. Translation Initiation is Controlled by RNA Folding Kinetics via a Ribosome Drafting Mechanism.

    PubMed

    Espah Borujeni, Amin; Salis, Howard M

    2016-06-01

    RNA folding plays an important role in controlling protein synthesis as well as other cellular processes. Existing models have focused on how RNA folding energetics control translation initiation rate under equilibrium conditions but have largely ignored the effects of nonequilibrium RNA folding. We introduce a new mechanism, called "ribosome drafting", that explains how a mRNA's folding kinetics and the ribosome's binding rate collectively control its translation initiation rate. During cycles of translation, ribosome drafting emerges whenever successive ribosomes bind to a mRNA faster than the mRNA can refold, maintaining it in a nonequilibrium state with an acceleration of protein synthesis. Using computational design, time-correlated single photon counting, and expression measurements, we demonstrate that slow-folding and fast-folding RNA structures with equivalent folding energetics can vary protein synthesis rates by 1000-fold. We determine the necessary conditions for ribosome drafting by characterizing mRNAs with rationally designed ribosome binding rates, folding kinetics, and folding energetics, confirming the predictions of a nonequilibrium Markov model of translation. Our results have widespread implications, illustrating how competitive folding and assembly kinetics can shape the gene expression machinery's sequence-structure-function relationship inside cells. PMID:27199273

  4. Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation.

    PubMed

    Hussain, Tanweer; Llácer, Jose L; Wimberly, Brian T; Kieft, Jeffrey S; Ramakrishnan, V

    2016-09-22

    In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1-3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA(fMet)) into the P site for start codon recognition. PMID:27662086

  5. Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex.

    PubMed

    Gilbert, Robert J C; Gordiyenko, Yulya; von der Haar, Tobias; Sonnen, Andreas F-P; Hofmann, Gregor; Nardelli, Maria; Stuart, David I; McCarthy, John E G

    2007-04-01

    In the process of protein synthesis, the small (40S) subunit of the eukaryotic ribosome is recruited to the capped 5' end of the mRNA, from which point it scans along the 5' untranslated region in search of a start codon. However, the 40S subunit alone is not capable of functional association with cellular mRNA species; it has to be prepared for the recruitment and scanning steps by interactions with a group of eukaryotic initiation factors (eIFs). In budding yeast, an important subset of these factors (1, 2, 3, and 5) can form a multifactor complex (MFC). Here, we describe cryo-EM reconstructions of the 40S subunit, of the MFC, and of 40S complexes with MFC factors plus eIF1A. These studies reveal the positioning of the core MFC on the 40S subunit, and show how eIF-binding induces mobility in the head and platform and reconfigures the head-platform-body relationship. This is expected to increase the accessibility of the mRNA channel, thus enabling the 40S subunit to convert to a recruitment-competent state.

  6. Enterovirus-induced miR-141 contributes to shutoff of host protein translation by targeting the translation initiation factor eIF4E.

    PubMed

    Ho, Bing-Ching; Yu, Sung-Liang; Chen, Jeremy J W; Chang, Sui-Yuan; Yan, Bo-Shiun; Hong, Qi-Sheng; Singh, Sher; Kao, Chuan-Liang; Chen, Hsuan-Yu; Su, Kang-Yi; Li, Ker-Chau; Cheng, Chiou-Ling; Cheng, Hao-Wei; Lee, Jen-Yi; Lee, Chun-Nan; Yang, Pan-Chyr

    2011-01-20

    Viruses rely on the host translation machinery to complete their life cycles. Picornaviruses use an internal ribosome entry site to initiate cap-independent protein translation and in parallel host cap-dependent translation is shut off. This process is thought to occur primarily via cleavage of host translation initiation factors eIF4GI and eIF4GII by viral proteases. Here we describe another mechanism whereby miR-141 induced upon enterovirus infection targets the cap-dependent translation initiation factor, eIF4E, for shutoff of host protein synthesis. Knockdown of miR-141 reduces viral propagation, and silencing of eIF4E can completely reverse the inhibitory effect of the miR-141 antagomiR on viral propagation. Ectopic expression of miR-141 promotes the switch from cap-dependent to cap-independent translation. Moreover, we identified a transcription factor, EGR1, which is partly responsible for miR-141 induction in response to enterovirus infection. Our results suggest that upregulation of miR-141 upon enterovirus infection can facilitate viral propagation by expediting the translational switch.

  7. Effect of phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 on the function of reversing factor in the initiation of protein synthesis.

    PubMed Central

    Matts, R L; Levin, D H; London, I M

    1983-01-01

    The reticulocyte reversing factor (RF) isolated as a complex with eukaryotic initiation factor 2 (eIF-2) acts catalytically in restoring protein synthesis in reticulocyte lysates inhibited by heme deficiency. In reconstituted in vitro assay mixtures containing Mg2+ (0.25-0.5 mM), RF catalyzes the formation of the binary complex (eIF-2-GDP) but this effect is inhibited when eIF-2 is phosphorylated by the heme-regulated kinase for the alpha-subunit of eIF-2 (HRI). More significantly, RF catalyzes the rapid dissociation of (eIF-2-GDP), which permits the exchange of GTP for GDP and, in the presence of Met-tRNAf, promotes the formation of the ternary complex (eIF-2-Met-tRNAf X GTP). However, phosphorylation of the binary complex by HRI prevents its dissociation by RF and, as a consequence, ternary complex formation is inhibited. Our results indicate that phosphorylated binary complex [eIF-2(alpha P).GDP] interacts with RF to form a [RF . eIF-2(alpha P)] that is not readily dissociable. This binding of RF renders it unavailable to catalyze the dissociation of unphosphorylated binary complex, thereby blocking the recycling of eIF-2. Since RF is present in lysates at a limited concentration relative to that of eIF-2, the sequestering of RF in this manner could account for the observation that the phosphorylation of a small proportion of eIF-2 in heme-deficient lysates is sufficient to inhibit protein synthesis. Images PMID:6573671

  8. MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2.

    PubMed

    Zhu, Qiuzhen; Zhang, Yuefan; Liu, Yulan; Cheng, Hao; Wang, Jing; Zhang, Yue; Rui, Yaocheng; Li, Tiejun

    2016-01-01

    Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury. PMID:26918757

  9. MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2

    PubMed Central

    Liu, Yulan; Cheng, Hao; Wang, Jing; Zhang, Yue; Rui, Yaocheng; Li, Tiejun

    2016-01-01

    Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury. PMID:26918757

  10. Expanding the eukaryotic genetic code

    DOEpatents

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2010-09-14

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  11. Expanding the eukaryotic genetic code

    DOEpatents

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2009-10-27

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  12. Expanding the eukaryotic genetic code

    SciTech Connect

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2009-11-17

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  13. Expanding the eukaryotic genetic code

    SciTech Connect

    Chin, Jason W; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G

    2015-02-03

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  14. Expanding the eukaryotic genetic code

    DOEpatents

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2012-02-14

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  15. Expanding the eukaryotic genetic code

    SciTech Connect

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2009-12-01

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  16. Expanding the eukaryotic genetic code

    SciTech Connect

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2013-01-22

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  17. Expanding the eukaryotic genetic code

    DOEpatents

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2012-05-08

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  18. The phosphorylation state of translation initiation factors is regulated developmentally and following heat shock in wheat.

    PubMed

    Gallie, D R; Le, H; Caldwell, C; Tanguay, R L; Hoang, N X; Browning, K S

    1997-01-10

    Several translation initiation factors in mammals and yeast are regulated by phosphorylation. The phosphorylation state of these factors is subject to alteration during development, environmental stress (heat shock, starvation, or heme deprivation), or viral infection. The phosphorylation state and the effect of changes in phosphorylation of the translation initiation factors of higher plants have not been previously investigated. We have determined the isoelectric states for the wheat translation initiation factors eIF-4A, eIF-4B, eIF-4F, eIF-iso4F, and eIF-2 and the poly(A)-binding protein in the seed, during germination, and following heat shock of wheat seedlings using two-dimensional gel electrophoresis and Western analysis. We found that the developmentally induced changes in isoelectric state observed during germination or the stress-induced changes were consistent with changes in phosphorylation. Treatment of the phosphorylated forms of the factors with phosphatases confirmed that the nature of the modification was due to phosphorylation. The isoelectric states of eIF-4B, eIF-4F (eIF-4E, p26), eIF-iso4F (eIF-iso4E, p28), and eIF-2alpha (p42) were altered during germination, suggesting that phosphorylation of these factors is developmentally regulated and correlates with the resumption of protein synthesis that occurs during germination. The phosphorylation of eIF-2beta (p38) or poly(A)-binding protein did not change either during germination or following a thermal stress. Only the phosphorylation state of two factors, eIF-4A and eIF-4B, changed following a heat shock, suggesting that plants may differ significantly from animals in the way in which their translational machinery is modified in response to a thermal stress. PMID:8995401

  19. The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies

    SciTech Connect

    Salsman, Jayme; Pinder, Jordan; Tse, Brenda; Corkery, Dale; Dellaire, Graham

    2013-10-15

    The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein–protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoform I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs. - Highlights: • The PML-I C-terminus, encoded by exon 9, interacts with translation factor eIF3K. • We identify a novel eIF3K isoform that excludes exon 2 (eIF3K-2). • eIF3K-2 preferentially associates with PML bodies enriched in PML-I vs. PML-IV. • Alternative splicing of eIF3K regulates association with PML bodies.

  20. Divergent LIN28-mRNA associations result in translational suppression upon the initiation of differentiation.

    PubMed

    Tan, Shen Mynn; Altschuler, Gabriel; Zhao, Tian Yun; Ang, Haw Siang; Yang, Henry; Lim, Bing; Vardy, Leah; Hide, Winston; Thomson, Andrew M; Lareu, Ricky R

    2014-07-01

    LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.

  1. Coding theory based models for protein translation initiation in prokaryotic organisms.

    SciTech Connect

    May, Elebeoba Eni; Bitzer, Donald L. (North Carolina State University, Raleigh, NC); Rosnick, David I. (North Carolina State University, Raleigh, NC); Vouk, Mladen A.

    2003-03-01

    Our research explores the feasibility of using communication theory, error control (EC) coding theory specifically, for quantitatively modeling the protein translation initiation mechanism. The messenger RNA (mRNA) of Escherichia coli K-12 is modeled as a noisy (errored), encoded signal and the ribosome as a minimum Hamming distance decoder, where the 16S ribosomal RNA (rRNA) serves as a template for generating a set of valid codewords (the codebook). We tested the E. coli based coding models on 5' untranslated leader sequences of prokaryotic organisms of varying taxonomical relation to E. coli including: Salmonella typhimurium LT2, Bacillus subtilis, and Staphylococcus aureus Mu50. The model identified regions on the 5' untranslated leader where the minimum Hamming distance values of translated mRNA sub-sequences and non-translated genomic sequences differ the most. These regions correspond to the Shine-Dalgarno domain and the non-random domain. Applying the EC coding-based models to B. subtilis, and S. aureus Mu50 yielded results similar to those for E. coli K-12. Contrary to our expectations, the behavior of S. typhimurium LT2, the more taxonomically related to E. coli, resembled that of the non-translated sequence group.

  2. [Translation and cultural adaptation of the Global Appraisal of Individual Needs - Initial].

    PubMed

    Claro, Heloísa Garcia; de Oliveira, Márcia Aparecida Ferreira; Paglione, Heloisa Barbosa; Pinho, Paula Hayasi; Pereira, Maria Odete; de Vargas, Divane

    2012-10-01

    The objective of this study was to perform the translation and cultural adaptation of the Global Appraisal of Individual Needs - Initial instrument, and calculate its content validity index. This is a methodological study designed for the cultural adaptation of the instrument. The instrument was translated into Portuguese in two versions that originated the synthesis of the translations, which were then submitted to the evaluation of four judges, experts in the field of alcohol and other drugs. After the suggested changes were made, the instrument was back-translated and resubmitted to the judges and authors of the original instrument, resulting in the final version of the instrument, Avaliação Global das Necessidades Individuais - Inicial. The content validity index of the instrument was 0.91, considered valid according to the literature. The instrument Avaliação Global das Necessidades Individuais - Inicial was culturally adapted to the Portuguese language spoken in Brazil; however, it was not submitted to tests with the target population, which suggests further studies should be performed to test its reliability and validity.

  3. Inhibition of Influenza Virus Replication by DNA Aptamers Targeting a Cellular Component of Translation Initiation

    PubMed Central

    Rodriguez, Paloma; Pérez-Morgado, M Isabel; Gonzalez, Víctor M; Martín, M Elena; Nieto, Amelia

    2016-01-01

    The genetic diversity of the influenza virus hinders the use of broad spectrum antiviral drugs and favors the appearance of resistant strains. Single-stranded DNA aptamers represent an innovative approach with potential application as antiviral compounds. The mRNAs of influenza virus possess a 5′cap structure and a 3′poly(A) tail that makes them structurally indistinguishable from cellular mRNAs. However, selective translation of viral mRNAs occurs in infected cells through a discriminatory mechanism, whereby viral polymerase and NS1 interact with components of the translation initiation complex, such as the eIF4GI and PABP1 proteins. We have studied the potential of two specific aptamers that recognize PABP1 (ApPABP7 and ApPABP11) to act as anti-influenza drugs. Both aptamers reduce viral genome expression and the production of infective influenza virus particles. The interaction of viral polymerase with the eIF4GI translation initiation factor is hindered by transfection of infected cells with both PABP1 aptamers, and ApPABP11 also inhibits the association of NS1 with PABP1 and eIF4GI. These results indicate that aptamers targeting the host factors that interact with viral proteins may potentially have a broad therapeutic spectrum, reducing the appearance of escape mutants and resistant subtypes. PMID:27070300

  4. Alternative translation initiation site in the DA strain of Theiler's murine encephalomyelitis virus.

    PubMed Central

    Kong, W P; Roos, R P

    1991-01-01

    Polyprotein processing studies of Theiler's murine encephalomyelitis virus (TMEV), a group of mouse picornaviruses, demonstrated synthesis of a protein we have called l during in vitro translations from the RNA of DA, a demyelinating strain of TMEV, but not GDVII, an acute neurovirulent strain. We have proposed that l is synthesized from an alternative initiation site in the DA leader (L) coding area out of phase with the polyprotein reading frame (R. P. Roos, W.-P. Kong, B. L. Semler, J. Virol. 63:5344-5353, 1989). We now provide support for this proposal from experiments involving in vitro translation of three separate mutations of an infectious DA cDNA clone: DA"l"-1, which contains a base mismatch at the putative initiation codon of l, DAL-1, which contains a base mismatch at the presumed authentic initiation site of L at the beginning of the polyprotein; and DAL:NheI, which contains nucleotides coding for a four-amino-acid insertion in the L coding area with a termination codon in the l reading frame. Our results demonstrate that the DA strain uses an alternative initiation site and reading frame to in vitro synthesize l. l may have a role in the biological activity of the virus. Images PMID:2033677

  5. SBI-0640756 Attenuates the Growth of Clinically Unresponsive Melanomas by Disrupting the eIF4F Translation Initiation Complex.

    PubMed

    Feng, Yongmei; Pinkerton, Anthony B; Hulea, Laura; Zhang, Tongwu; Davies, Michael A; Grotegut, Stefan; Cheli, Yann; Yin, Hongwei; Lau, Eric; Kim, Hyungsoo; De, Surya K; Barile, Elisa; Pellecchia, Maurizio; Bosenberg, Marcus; Li, Jian-Liang; James, Brian; Hassig, Christian A; Brown, Kevin M; Topisirovic, Ivan; Ronai, Ze'ev A

    2015-12-15

    Disrupting the eukaryotic translation initiation factor 4F (eIF4F) complex offers an appealing strategy to potentiate the effectiveness of existing cancer therapies and to overcome resistance to drugs such as BRAF inhibitors (BRAFi). Here, we identified and characterized the small molecule SBI-0640756 (SBI-756), a first-in-class inhibitor that targets eIF4G1 and disrupts the eIF4F complex. SBI-756 impaired the eIF4F complex assembly independently of mTOR and attenuated growth of BRAF-resistant and BRAF-independent melanomas. SBI-756 also suppressed AKT and NF-κB signaling, but small-molecule derivatives were identified that only marginally affected these pathways while still inhibiting eIF4F complex formation and melanoma growth, illustrating the potential for further structural and functional manipulation of SBI-756 as a drug lead. In the gene expression signature patterns elicited by SBI-756, DNA damage, and cell-cycle regulatory factors were prominent, with mutations in melanoma cells affecting these pathways conferring drug resistance. SBI-756 inhibited the growth of NRAS, BRAF, and NF1-mutant melanomas in vitro and delayed the onset and reduced the incidence of Nras/Ink4a melanomas in vivo. Furthermore, combining SBI-756 and a BRAFi attenuated the formation of BRAFi-resistant human tumors. Taken together, our findings show how SBI-756 abrogates the growth of BRAF-independent and BRAFi-resistant melanomas, offering a preclinical rationale to evaluate its antitumor effects in other cancers. PMID:26603897

  6. Translation initiation of the replication initiator repB gene of promiscuous plasmid pMV158 is led by an extended non-SD sequence.

    PubMed

    López-Aguilar, Celeste; Ruiz-Masó, José A; Rubio-Lepe, Tania Samir; Sanz, Marta; del Solar, Gloria

    2013-07-01

    RepB is the pMV158-encoded protein that initiates rolling-circle replication of this promiscuous plasmid. Availability of RepB is rate-limiting for the plasmid replication process, and therefore the repB gene encoding the protein is subjected to strict control. Two trans-acting plasmid elements, CopG and the antisense RNAII, are involved in controlling the synthesis of the initiator at the transcriptional and translational level, respectively. In addition to this dual control of repB expression that senses and corrects fluctuations in plasmid copy number, proper availability of RepB also relies on the adequate functionality of the transcription and translation initiation regulatory signals. Translation of repB has been postulated to depend on an atypical ribosome binding site that precedes its start codon, although such a hypothesis has never been proved. To define sequences involved in translation of repB, several mutations in the translation initiation region of the repB mRNA have been characterized by using an Escherichia coli in vitro expression system wherein the synthesis of RepB was detected and quantified. We showed that translation of repB is not coupled to that of copG and depends only on its own initiation signals. The atypical ribosome binding site, as it was defined, is not involved in translation initiation. However, the sequence just upstream of the repB start codon, encompassing the proximal box of the atypical ribosome binding site and the four bases immediately downstream of it, is indeed important for efficient translation of repB. The high degree of conservation of this sequence among the rep genes of plasmids of the same pMV158 family supports its relevancy as a translation initiation signal in mRNAs without a recognizable Shine-Dalgarno sequence.

  7. L-serine deficiency caused by genetic Phgdh deletion leads to robust induction of 4E-BP1 and subsequent repression of translation initiation in the developing central nervous system.

    PubMed

    Sayano, Tomoko; Kawakami, Yuriko; Kusada, Wataru; Suzuki, Takeshi; Kawano, Yuki; Watanabe, Akihiro; Takashima, Kana; Arimoto, Yashiho; Esaki, Kayoko; Wada, Akira; Yoshizawa, Fumiaki; Watanabe, Masahiko; Okamoto, Masahiro; Hirabayashi, Yoshio; Furuya, Shigeki

    2013-03-01

    Targeted disruption in mice of the gene encoding D-3-phosphoglycerate dehydrogenase (Phgdh) results in embryonic lethality associated with a striking reduction in free L-serine and growth retardation including severe brain malformation. We previously observed a severe impairment in neurogenesis of the central nervous system of Phgdh knockout (KO) embryos and a reduction in the protein content of their brains. Although these findings suggest that L-serine deficiency links attenuation of mRNA translation to severe developmental malformation of the central nervous system, the underlying key molecular event remains unexplored. Here we demonstrate that mRNA of Eif4ebp1 encoding eukaryotic initiation factor 4 binding protein 1 and its protein, 4E-BP1, are markedly induced in the central nervous system of Phgdh KO embryos, whereas a modest induction is observed in the liver. The increase in 4E-BP1 was associated with a decrease in the cap initiation complex in the brain, as shown by lower levels of eukaryotic translation initiation factor 4G bound to eukaryotic translation initiation factor 4E (eIF4E) and increased eIF4E interaction with 4E-BP1 based on 7-methyl-GTP chromatography. eIF4E protein and polysomes were also diminished in Phgdh KO embryos. Induction of Eif4ebp1 mRNA and of 4E-BP1 was reproduced in mouse embryonic fibroblasts established from Phgdh KO embryos under the condition of L-serine deprivation. Induction of Eif4ebp1 mRNA was suppressed only when L-serine was supplemented in the culture medium, indicating that reduced L-serine availability regulates the induction of Eif4ebp1/4E-BP1. These data suggest that elevated levels of 4E-BP1 may be involved in a mechanism to arrest brain development in Phgdh KO embryos. PMID:23350942

  8. Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism.

    PubMed Central

    Boiziau, C; Kurfurst, R; Cazenave, C; Roig, V; Thuong, N T; Toulmé, J J

    1991-01-01

    We have used alpha-oligomers as antisense oligonucleotides complementary to three different sequences of the rabbit beta-globin mRNA: a region adjacent to the cap site, a region spanning the AUG initiation codon or a sequence in the coding region. These alpha-oligonucleotides were synthesized either with a free 5' OH group or linked to an acridine derivative. The effect of these oligonucleotides on mRNA translation was investigated in cell-free extracts and in Xenopus oocytes. In rabbit reticulocyte lysate and in wheat germ extracts oligomers targeted to the cap site and the initiation codon reduced beta-globin synthesis in a dose-dependent manner, whereas the target mRNA remained intact. The anti-cap alpha-oligomer was even more efficient that its beta-counterpart in rabbit reticulocyte lysate. In contrast, only the alpha-oligomer, linked to the acridine derivative, complementary to the cap region displayed significant antisense properties in Xenopus oocytes. Therefore initiation of translation can be arrested by oligonucleotide/RNA hybrids which are not substrates for RNase-H. Images PMID:1850511

  9. Initiation of Translation by Cricket Paralysis Virus IRES Requires Its Translocation in the Ribosome

    PubMed Central

    Fernández, Israel S.; Bai, Xiao-Chen; Murshudov, Garib; Scheres, Sjors H.W.; Ramakrishnan, V.

    2014-01-01

    Summary The cricket paralysis virus internal ribosome entry site (CrPV-IRES) is a folded structure in a viral mRNA that allows initiation of translation in the absence of any host initiation factors. By using recent advances in single-particle electron cryomicroscopy, we have solved the structure of CrPV-IRES bound to the ribosome of the yeast Kluyveromyces lactis in both the canonical and rotated states at overall resolutions of 3.7 and 3.8 Å, respectively. In both states, the pseudoknot PKI of the CrPV-IRES mimics a tRNA/mRNA interaction in the decoding center of the A site of the 40S ribosomal subunit. The structure and accompanying factor-binding data show that CrPV-IRES binding mimics a pretranslocation rather than initiation state of the ribosome. Translocation of the IRES by elongation factor 2 (eEF2) is required to bring the first codon of the mRNA into the A site and to allow the start of translation. PMID:24792965

  10. Identification of Plasmodium falciparum Translation Initiation eIF2β Subunit: Direct Interaction with Protein Phosphatase Type 1

    PubMed Central

    Tellier, Géraldine; Lenne, Astrid; Cailliau-Maggio, Katia; Cabezas-Cruz, Alejandro; Valdés, James J.; Martoriati, Alain; Aliouat, El M.; Gosset, Pierre; Delaire, Baptiste; Fréville, Aline; Pierrot, Christine; Khalife, Jamal

    2016-01-01

    Protein phosphatase 1 (PP1c) is one of the main phosphatases whose function is shaped by many regulators to confer a specific location and a selective function for this enzyme. Here, we report that eukaryotic initiation factor 2β of Plasmodium falciparum (PfeIF2β) is an interactor of PfPP1c. Sequence analysis of PfeIF2β revealed a deletion of 111 amino acids when compared to its human counterpart and the presence of two potential binding motifs to PfPP1 (29FGEKKK34, 103KVAW106). As expected, we showed that PfeIF2β binds PfeIF2γ and PfeIF5, confirming its canonical interaction with partners of the translation complex. Studies of the PfeIF2β-PfPP1 interaction using wild-type, single and double mutated versions of PfeIF2β revealed that both binding motifs are critical. We next showed that PfeIF2β is able to induce Germinal Vesicle Break Down (GVBD) when expressed in Xenopus oocytes, an indicator of its capacity to regulate PP1. Only combined mutations of both binding motifs abolished the interaction with PP1 and the induction of GVBD. In P. falciparum, although the locus is accessible for genetic manipulation, PfeIF2β seems to play an essential role in intraerythrocytic cycle as no viable knockout parasites were detectable. Interestingly, as for PfPP1, the subcellular fractionation of P. falciparum localized PfeIF2β in cytoplasm and nuclear extracts, suggesting a potential effect on PfPP1 in both compartments and raising the question of a non-canonical function of PfeIf2β in the nucleus. Hence, the role played by PfeIF2β in blood stage parasites could occur at multiple levels involving the binding to proteins of the translational complex and to PfPP1. PMID:27303372

  11. Identification of Plasmodium falciparum Translation Initiation eIF2β Subunit: Direct Interaction with Protein Phosphatase Type 1.

    PubMed

    Tellier, Géraldine; Lenne, Astrid; Cailliau-Maggio, Katia; Cabezas-Cruz, Alejandro; Valdés, James J; Martoriati, Alain; Aliouat, El M; Gosset, Pierre; Delaire, Baptiste; Fréville, Aline; Pierrot, Christine; Khalife, Jamal

    2016-01-01

    Protein phosphatase 1 (PP1c) is one of the main phosphatases whose function is shaped by many regulators to confer a specific location and a selective function for this enzyme. Here, we report that eukaryotic initiation factor 2β of Plasmodium falciparum (PfeIF2β) is an interactor of PfPP1c. Sequence analysis of PfeIF2β revealed a deletion of 111 amino acids when compared to its human counterpart and the presence of two potential binding motifs to PfPP1 ((29)FGEKKK(34), (103)KVAW(106)). As expected, we showed that PfeIF2β binds PfeIF2γ and PfeIF5, confirming its canonical interaction with partners of the translation complex. Studies of the PfeIF2β-PfPP1 interaction using wild-type, single and double mutated versions of PfeIF2β revealed that both binding motifs are critical. We next showed that PfeIF2β is able to induce Germinal Vesicle Break Down (GVBD) when expressed in Xenopus oocytes, an indicator of its capacity to regulate PP1. Only combined mutations of both binding motifs abolished the interaction with PP1 and the induction of GVBD. In P. falciparum, although the locus is accessible for genetic manipulation, PfeIF2β seems to play an essential role in intraerythrocytic cycle as no viable knockout parasites were detectable. Interestingly, as for PfPP1, the subcellular fractionation of P. falciparum localized PfeIF2β in cytoplasm and nuclear extracts, suggesting a potential effect on PfPP1 in both compartments and raising the question of a non-canonical function of PfeIf2β in the nucleus. Hence, the role played by PfeIF2β in blood stage parasites could occur at multiple levels involving the binding to proteins of the translational complex and to PfPP1. PMID:27303372

  12. Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p).

    PubMed

    Giraldo, R; Diaz-Orejas, R

    2001-04-24

    The proteins responsible for the initiation of DNA replication are thought to be essentially unrelated in bacteria and archaea/eukaryotes. Here we show that RepA, the initiator from the Pseudomonas plasmid pPS10, and the C-terminal domain of ScOrc4p, a subunit of Saccharomyces cerevisiae (Sc) origin recognition complex (ORC), share sequence similarities. Based on biochemical and spectroscopic evidence, these similarities include common structural elements, such as a winged-helix domain and a leucine-zipper dimerization motif. We have also found that ScOrc4p, as previously described for RepA-type initiators, interacts with chaperones of the Hsp70 family both in vitro and in vivo, most probably to regulate the assembly of active ORC. In evolutionary terms, our results are compatible with the recruitment of the same protein module for initiation of DNA replication by the ancestors of present-day Gram-negative bacteria plasmids, archaea, and eukaryotes. PMID:11296251

  13. Stage-specific assembly events of the 6-MDa small-subunit processome initiate eukaryotic ribosome biogenesis.

    PubMed

    Chaker-Margot, Malik; Hunziker, Mirjam; Barandun, Jonas; Dill, Brian D; Klinge, Sebastian

    2015-11-01

    Eukaryotic ribosome biogenesis involves a plethora of ribosome-assembly factors, and their temporal order of association with preribosomal RNA is largely unknown. By using Saccharomyces cerevisiae as a model organism, we developed a system that recapitulates and arrests ribosome assembly at early stages, thus providing in vivo snapshots of nascent preribosomal particles. Here we report the stage-specific order in which 70 ribosome-assembly factors associate with preribosomal RNA domains, thereby forming the 6-MDa small-subunit processome. PMID:26479197

  14. mTORC1 mediated translational elongation limits intestinal tumour initiation and growth

    PubMed Central

    Faller, William J.; Ridgway, Rachel A.; Jamieson, Thomas; Karim, Saadia A.; Jones, Carolyn; Radulescu, Sorina; Huels, David J.; Myant, Kevin B.; Dudek, Kate M.; Casey, Helen A.; Scopelliti, Alessandro; Cordero, Julia B.; Vidal, Marcos; Pende, Mario; Ryazanov, Alexey G.; Sonenberg, Nahum; Meyuhas, Oded; Hall, Michael N.; Bushell, Martin; Willis, Anne E.; Sansom, Owen J.

    2014-01-01

    Inactivation of APC is a strongly predisposing event in the development of colorectal cancer1,2, prompting us to search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth3-5 and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP16,7. This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP18, would be ineffective in limiting cancer progression in APC deficient lesions. Here we show that mTORC1 activity is absolutely required for the proliferation of APC deficient (but not wild type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC deficient cells show the expected increases in protein synthesis, our studies reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1 mediated inhibition of eEF2 kinase is required for the proliferation of APC deficient cells. Importantly, treatment of established APC deficient adenomas with rapamycin (which can target eEF2 through the mTORC1 – S6K – eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together our data suggest that inhibition of translation elongation using existing, clinically approved drugs such as the Rapalogs, would provide clear therapeutic benefit for patients at high-risk of developing colorectal cancer. PMID:25383520

  15. Entrainment to periodic initiation and transition rates in a computational model for gene translation.

    PubMed

    Margaliot, Michael; Sontag, Eduardo D; Tuller, Tamir

    2014-01-01

    Periodic oscillations play an important role in many biomedical systems. Proper functioning of biological systems that respond to periodic signals requires the ability to synchronize with the periodic excitation. For example, the sleep/wake cycle is a manifestation of an internal timing system that synchronizes to the solar day. In the terminology of systems theory, the biological system must entrain or phase-lock to the periodic excitation. Entrainment is also important in synthetic biology. For example, connecting several artificial biological systems that entrain to a common clock may lead to a well-functioning modular system. The cell-cycle is a periodic program that regulates DNA synthesis and cell division. Recent biological studies suggest that cell-cycle related genes entrain to this periodic program at the gene translation level, leading to periodically-varying protein levels of these genes. The ribosome flow model (RFM) is a deterministic model obtained via a mean-field approximation of a stochastic model from statistical physics that has been used to model numerous processes including ribosome flow along the mRNA. Here we analyze the RFM under the assumption that the initiation and/or transition rates vary periodically with a common period T. We show that the ribosome distribution profile in the RFM entrains to this periodic excitation. In particular, the protein synthesis pattern converges to a unique periodic solution with period T. To the best of our knowledge, this is the first proof of entrainment in a mathematical model for translation that encapsulates aspects such as initiation and termination rates, ribosomal movement and interactions, and non-homogeneous elongation speeds along the mRNA. Our results support the conjecture that periodic oscillations in tRNA levels and other factors related to the translation process can induce periodic oscillations in protein levels, and may suggest a new approach for re-engineering genetic systems to obtain a

  16. Synthetic 15 - 19-meric oligoribonucleotides as models for translation initiation of the phage fr replicase gene

    SciTech Connect

    Kumpin`sh, V.Kh.; Litsis, N.G.; Renhof, R.F.

    1995-12-01

    Automatic solid-phase synthesis of 15 - 19-meric oligoribonucleotides was carried out using 5{prime}-O-dimethoxytrityl-2{prime}-O-(2-tetrahydropyranyl)- [or 2{prime}-O-(2-tetrahydrofuranyl)-] N-acylribonucleoside-3{prime}-O- (methyl-N,N-diisopropyl) phosphoramidite synthons and 1-H-tetrazole as an activator. Comparative analysis of the template activity of the oligoribonucleotides synthesized, which are models of the translation initiation region of the replicase gene of MS2 and fr phages, showed that the minimal active fragment of RNA is a 16-mer containing the initiation AUG codon of the gene, a short spacer, a Shine-Dalgarno domain, and the 5{prime}-terminal AUGA sequence with a functionally important termination AUG codon. 31 refs., 1 fig., 1 tab.

  17. Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2

    PubMed Central

    Lu, Phoebe D; Jousse, Céline; Marciniak, Stefan J; Zhang, Yuhong; Novoa, Isabel; Scheuner, Donalyn; Kaufman, Randal J; Ron, David; Harding, Heather P

    2004-01-01

    Transient phosphorylation of the α-subunit of translation initiation factor 2 (eIF2α) represses translation and activates select gene expression under diverse stressful conditions. Defects in the eIF2α phosphorylation-dependent integrated stress response impair resistance to accumulation of malfolded proteins in the endoplasmic reticulum (ER stress), to oxidative stress and to nutrient deprivations. To study the hypothesized protective role of eIF2α phosphorylation in isolation of parallel stress signaling pathways, we fused the kinase domain of pancreatic endoplasmic reticulum kinase (PERK), an ER stress-inducible eIF2α kinase that is normally activated by dimerization, to a protein module that binds a small dimerizer molecule. The activity of this artificial eIF2α kinase, Fv2E-PERK, is subordinate to the dimerizer and is uncoupled from upstream stress signaling. Fv2E-PERK activation enhanced the expression of numerous stress-induced genes and protected cells from the lethal effects of oxidants, peroxynitrite donors and ER stress. Our findings indicate that eIF2α phosphorylation can initiate signaling in a cytoprotective gene expression pathway independently of other parallel stress-induced signals and that activation of this pathway can single-handedly promote a stress-resistant preconditioned state. PMID:14713949

  18. Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation

    PubMed Central

    Andreev, Dmitri E.; Hirnet, Juliane; Terenin, Ilya M.; Dmitriev, Sergey E.; Niepmann, Michael; Shatsky, Ivan N.

    2012-01-01

    Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5′ end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNAGly anticodon stem–loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus–host cell interaction are discussed. PMID:22373920

  19. De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum.

    PubMed

    Jagannathan, Sujatha; Reid, David W; Cox, Amanda H; Nicchitta, Christopher V

    2014-10-01

    The specialized protein synthesis functions of the cytosol and endoplasmic reticulum compartments are conferred by the signal recognition particle (SRP) pathway, which directs the cotranslational trafficking of signal sequence-encoding mRNAs from the cytosol to the endoplasmic reticulum (ER). Although subcellular mRNA distributions largely mirror the binary pattern predicted by the SRP pathway model, studies in mammalian cells, yeast, and Drosophila have also demonstrated that cytosolic protein-encoding mRNAs are broadly represented on ER-bound ribosomes. A mechanism for such noncanonical mRNA localization remains, however, to be identified. Here, we examine the hypothesis that de novo translation initiation on ER-bound ribosomes serves as a mechanism for localizing cytosolic protein-encoding mRNAs to the ER. As a test of this hypothesis, we performed single molecule RNA fluorescence in situ hybridization studies of subcellular mRNA distributions and report that a substantial fraction of mRNAs encoding the cytosolic protein GAPDH resides in close proximity to the ER. Consistent with these data, analyses of subcellular mRNA and ribosome distributions in multiple cell lines demonstrated that cytosolic protein mRNA-ribosome distributions were strongly correlated, whereas signal sequence-encoding mRNA-ribosome distributions were divergent. Ribosome footprinting studies of ER-bound polysomes revealed a substantial initiation codon read density enrichment for cytosolic protein-encoding mRNAs. We also demonstrate that eukaryotic initiation factor 2α is bound to the ER via a salt-sensitive, ribosome-independent mechanism. Combined, these data support ER-localized translation initiation as a mechanism for mRNA recruitment to the ER.

  20. The conserved GTPase LepA contributes mainly to translation initiation in Escherichia coli

    PubMed Central

    Balakrishnan, Rohan; Oman, Kenji; Shoji, Shinichiro; Bundschuh, Ralf; Fredrick, Kurt

    2014-01-01

    LepA is a paralog of EF-G found in all bacteria. Deletion of lepA confers no obvious growth defect in Escherichia coli, and the physiological role of LepA remains unknown. Here, we identify nine strains (ΔdksA, ΔmolR1, ΔrsgA, ΔtatB, ΔtonB, ΔtolR, ΔubiF, ΔubiG or ΔubiH) in which ΔlepA confers a synthetic growth phenotype. These strains are compromised for gene regulation, ribosome assembly, transport and/or respiration, indicating that LepA contributes to these functions in some way. We also use ribosome profiling to deduce the effects of LepA on translation. We find that loss of LepA alters the average ribosome density (ARD) for hundreds of mRNA coding regions in the cell, substantially reducing ARD in many cases. By contrast, only subtle and codon-specific changes in ribosome distribution along mRNA are seen. These data suggest that LepA contributes mainly to the initiation phase of translation. Consistent with this interpretation, the effect of LepA on ARD is related to the sequence of the Shine–Dalgarno region. Global perturbation of gene expression in the ΔlepA mutant likely explains most of its phenotypes. PMID:25378333

  1. Translation initiation factor (iso) 4E interacts with BTF3, the beta subunit of the nascent polypeptide-associated complex.

    PubMed

    Freire, Miguel Angel

    2005-01-31

    A two-hybrid screen with the translation initiation factor, eIF(iso)4E from Arabidopsis, identified a clone encoding a lipoxygenase type 2 [Freire, M.A., et al., 2000. Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein. Plant Molecular Biology 44, 129-140], and three cDNA clones encoding the homologue of the mammalian BTF3 factor, the beta subunit of the nascent polypeptide-associated complex (NAC). Here we report on the interaction between the translation initiation factor eIF(iso)4E and AtBTF3. AtBTF3 protein is able to interact with the wheat initiation factors eIF4E and eIF(iso)4E. AtBTF3 contains a sequence related to the prototypic motif found on most of the 4E-binding proteins, and competes with the translation initiation factor eIF(iso)4G for eIF4(iso)4E binding, in a two hybrid interference assay. These findings provide a molecular link between the translation initiation mechanism and the emergence of the nascent polypeptide chains.

  2. Deadenylation and its regulation in eukaryotic cells.

    PubMed

    Zhang, Xiaokan; Kleiman, Frida E; Devany, Emral

    2014-01-01

    Messenger RNA deadenylation is a process that allows rapid regulation of gene expression in response to different cellular conditions. The change of the mRNA poly(A) tail length by the activation of deadenylation might regulate gene expression by affecting mRNA stability, mRNA transport, or translation initiation. Activation of deadenylation processes are highly regulated and associated with different cellular conditions such as cancer, development, mRNA surveillance, DNA damage response, and cell differentiation. In the last few years, new technologies for studying deadenylation have been developed. Here we overview concepts related to deadenylation and its regulation in eukaryotic cells. We also describe some of the most commonly used protocols to study deadenylation in eukaryotic cells.

  3. PreTIS: A Tool to Predict Non-canonical 5’ UTR Translational Initiation Sites in Human and Mouse

    PubMed Central

    Reuter, Kerstin; Helms, Volkhard

    2016-01-01

    Translation of mRNA sequences into proteins typically starts at an AUG triplet. In rare cases, translation may also start at alternative non–AUG codons located in the annotated 5’ UTR which leads to an increased regulatory complexity. Since ribosome profiling detects translational start sites at the nucleotide level, the properties of these start sites can then be used for the statistical evaluation of functional open reading frames. We developed a linear regression approach to predict in–frame and out–of–frame translational start sites within the 5’ UTR from mRNA sequence information together with their translation initiation confidence. Predicted start codons comprise AUG as well as near–cognate codons. The underlying datasets are based on published translational start sites for human HEK293 and mouse embryonic stem cells that were derived by the original authors from ribosome profiling data. The average prediction accuracy of true vs. false start sites for HEK293 cells was 80%. When applied to mouse mRNA sequences, the same model predicted translation initiation sites observed in mouse ES cells with an accuracy of 76%. Moreover, we illustrate the effect of in silico mutations in the flanking sequence context of a start site on the predicted initiation confidence. Our new webservice PreTIS visualizes alternative start sites and their respective ORFs and predicts their ability to initiate translation. Solely, the mRNA sequence is required as input. PreTIS is accessible at http://service.bioinformatik.uni-saarland.de/pretis. PMID:27768687

  4. UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly.

    PubMed

    Hunziker, Mirjam; Barandun, Jonas; Petfalski, Elisabeth; Tan, Dongyan; Delan-Forino, Clémentine; Molloy, Kelly R; Kim, Kelly H; Dunn-Davies, Hywel; Shi, Yi; Chaker-Margot, Malik; Chait, Brian T; Walz, Thomas; Tollervey, David; Klinge, Sebastian

    2016-06-29

    Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes-UtpA and UtpB-interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5' end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5' ETS and U3 snoRNA as well as the 3' boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.

  5. UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly

    NASA Astrophysics Data System (ADS)

    Hunziker, Mirjam; Barandun, Jonas; Petfalski, Elisabeth; Tan, Dongyan; Delan-Forino, Clémentine; Molloy, Kelly R.; Kim, Kelly H.; Dunn-Davies, Hywel; Shi, Yi; Chaker-Margot, Malik; Chait, Brian T.; Walz, Thomas; Tollervey, David; Klinge, Sebastian

    2016-06-01

    Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes--UtpA and UtpB--interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5' end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5' ETS and U3 snoRNA as well as the 3' boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.

  6. UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly

    PubMed Central

    Hunziker, Mirjam; Barandun, Jonas; Petfalski, Elisabeth; Tan, Dongyan; Delan-Forino, Clémentine; Molloy, Kelly R.; Kim, Kelly H.; Dunn-Davies, Hywel; Shi, Yi; Chaker-Margot, Malik; Chait, Brian T.; Walz, Thomas; Tollervey, David; Klinge, Sebastian

    2016-01-01

    Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes—UtpA and UtpB—interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA–protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5′ end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5′ ETS and U3 snoRNA as well as the 3′ boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly. PMID:27354316

  7. Eukaryotic origins.

    PubMed

    Lake, James A

    2015-09-26

    The origin of the eukaryotes is a fundamental scientific question that for over 30 years has generated a spirited debate between the competing Archaea (or three domains) tree and the eocyte tree. As eukaryotes ourselves, humans have a personal interest in our origins. Eukaryotes contain their defining organelle, the nucleus, after which they are named. They have a complex evolutionary history, over time acquiring multiple organelles, including mitochondria, chloroplasts, smooth and rough endoplasmic reticula, and other organelles all of which may hint at their origins. It is the evolutionary history of the nucleus and their other organelles that have intrigued molecular evolutionists, myself included, for the past 30 years and which continues to hold our interest as increasingly compelling evidence favours the eocyte tree. As with any orthodoxy, it takes time to embrace new concepts and techniques.

  8. Eukaryotic origins

    PubMed Central

    Lake, James A.

    2015-01-01

    The origin of the eukaryotes is a fundamental scientific question that for over 30 years has generated a spirited debate between the competing Archaea (or three domains) tree and the eocyte tree. As eukaryotes ourselves, humans have a personal interest in our origins. Eukaryotes contain their defining organelle, the nucleus, after which they are named. They have a complex evolutionary history, over time acquiring multiple organelles, including mitochondria, chloroplasts, smooth and rough endoplasmic reticula, and other organelles all of which may hint at their origins. It is the evolutionary history of the nucleus and their other organelles that have intrigued molecular evolutionists, myself included, for the past 30 years and which continues to hold our interest as increasingly compelling evidence favours the eocyte tree. As with any orthodoxy, it takes time to embrace new concepts and techniques. PMID:26323753

  9. The European Network for Translational Research in Atrial Fibrillation (EUTRAF): objectives and initial results.

    PubMed

    Schotten, Ulrich; Hatem, Stephane; Ravens, Ursula; Jaïs, Pierre; Müller, Frank-Ulrich; Goette, Andres; Rohr, Stephan; Antoons, Gudrun; Pieske, Burkert; Scherr, Daniel; Oto, Ali; Casadei, Barbara; Verheule, Sander; Cartlidge, David; Steinmeyer, Klaus; Götsche, Thorsten; Dobrev, Dobromir; Kockskämper, Jens; Lendeckel, Uwe; Fabritz, Larissa; Kirchhof, Paulus; Camm, A John

    2015-10-01

    Atrial fibrillation (AF) is the most common sustained arrhythmia in the general population. As an age-related arrhythmia AF is becoming a huge socio-economic burden for European healthcare systems. Despite significant progress in our understanding of the pathophysiology of AF, therapeutic strategies for AF have not changed substantially and the major challenges in the management of AF are still unmet. This lack of progress may be related to the multifactorial pathogenesis of atrial remodelling and AF that hampers the identification of causative pathophysiological alterations in individual patients. Also, again new mechanisms have been identified and the relative contribution of these mechanisms still has to be established. In November 2010, the European Union launched the large collaborative project EUTRAF (European Network of Translational Research in Atrial Fibrillation) to address these challenges. The main aims of EUTRAF are to study the main mechanisms of initiation and perpetuation of AF, to identify the molecular alterations underlying atrial remodelling, to develop markers allowing to monitor this processes, and suggest strategies to treat AF based on insights in newly defined disease mechanisms. This article reports on the objectives, the structure, and initial results of this network. PMID:26364316

  10. eIF4B, eIF4G and RNA regulate eIF4A activity in translation initiation by modulating the eIF4A conformational cycle.

    PubMed

    Harms, Ulf; Andreou, Alexandra Zoi; Gubaev, Airat; Klostermeier, Dagmar

    2014-07-01

    Eukaryotic translation initiation factor eIF4A is a DEAD-box helicase that resolves secondary structure elements in the 5'-UTR of mRNAs during ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. DEAD-box proteins alternate between open and closed conformations during RNA unwinding. The transition to the closed conformation is linked to duplex destabilization. eIF4A is a special DEAD-box protein that can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by the translation initiation factor eIF4G and a closed state in the presence of eIF4G and eIF4B. We show here that eIF4A alone does not measurably sample the closed conformation. The translation initiation factors eIF4B and eIF4G accelerate the eIF4A conformational cycle. eIF4G increases the rate of closing more than the opening rate, and eIF4B selectively increases the closing rate. Strikingly, the rate constants and the effect of eIF4B are different for different RNAs, and are related to the presence of single-stranded regions. Modulating the kinetics of the eIF4A conformational cycle is thus central for the multi-layered regulation of its activity, and for its role as a regulatory hub in translation initiation. PMID:24848014

  11. Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms.

    PubMed

    Rathore, Om Singh; Faustino, Alexandra; Prudêncio, Pedro; Van Damme, Petra; Cox, Cymon J; Martinho, Rui Gonçalo

    2016-02-10

    Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes.

  12. Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms

    PubMed Central

    Rathore, Om Singh; Faustino, Alexandra; Prudêncio, Pedro; Van Damme, Petra; Cox, Cymon J.; Martinho, Rui Gonçalo

    2016-01-01

    Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes. PMID:26861501

  13. Human eukaryotic initiation factor 4G (eIF4G) protein binds to eIF3c, -d, and -e to promote mRNA recruitment to the ribosome.

    PubMed

    Villa, Nancy; Do, Angelie; Hershey, John W B; Fraser, Christopher S

    2013-11-15

    Recruitment of mRNA to the 40S ribosomal subunit requires the coordinated interaction of a large number of translation initiation factors. In mammals, the direct interaction between eukaryotic initiation factor 4G (eIF4G) and eIF3 is thought to act as the molecular bridge between the mRNA cap-binding complex and the 40S subunit. A discrete ∼90 amino acid domain in eIF4G is responsible for binding to eIF3, but the identity of the eIF3 subunit(s) involved is less clear. The eIF3e subunit has been shown to directly bind eIF4G, but the potential role of other eIF3 subunits in stabilizing this interaction has not been investigated. It is also not clear if the eIF4A helicase plays a role in stabilizing the interaction between eIF4G and eIF3. Here, we have used a fluorescence anisotropy assay to demonstrate that eIF4G binds to eIF3 independently of eIF4A binding to the middle region of eIF4G. By using a site-specific cross-linking approach, we unexpectedly show that the eIF4G-binding surface in eIF3 is comprised of the -c, -d and -e subunits. Screening multiple cross-linker positions reveals that eIF4G contains two distinct eIF3-binding subdomains within the previously identified eIF3-binding domain. Finally, by employing an eIF4G-dependent translation assay, we establish that both of these subdomains are required for efficient mRNA recruitment to the ribosome and stimulate translation. Our study reveals unexpected complexity to the eIF3-eIF4G interaction that provides new insight into the regulation of mRNA recruitment to the human ribosome.

  14. Crosslinking of eukaryotic initiation factor eIF3 to the 40S ribosomal subunit from rabbit reticulocytes.

    PubMed

    Tolan, D R; Hershey, J W; Traut, R T

    1983-07-01

    Complexes of purified 40S ribosomal subunits and initiation factor 3 from rabbit reticulocytes were crosslinked using the reversible protein crosslinking reagent, 2-iminothiolane, under conditions shown previously to lead to the formation of dimers between 40S proteins but not higher multimers. The activity of both the 40S subunits and initiation factor 3 was maintained. Protein crosslinked to the factor was purified by sucrose density gradient centrifugation following nuclease digestion of the ribosomal subunit: alternatively, the total protein was extracted from 40S: factor complexes. The protein obtained by either method was analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Ribosomal proteins were found in multimeric complexes of high molecular weight due to their crosslinking to components of eIF3. Identification of the ribosomal proteins appearing below the diagonal was accomplished by elution, radioiodination, two-dimensional polyacrylamide/urea gel electrophoresis, and radioautography. Proteins S2, S3, S3a, S4, S5, S6, S8, S9, S11, S12, S14, S15, S16, S19, S24, S25, and S26 were identified. Because many of the proteins in this group form crosslinked dimers with each other, it was impossible to distinguish proteins directly crosslinked to eIF3 from those crosslinked indirectly through one bridging protein. The results nonetheless imply that the 40S ribosomal proteins identified are at or near the binding site for initiation factor 3.

  15. Aim-less translation: loss of Saccharomyces cerevisiae mitochondrial translation initiation factor mIF3/Aim23 leads to unbalanced protein synthesis

    PubMed Central

    Kuzmenko, Anton; Derbikova, Ksenia; Salvatori, Roger; Tankov, Stoyan; Atkinson, Gemma C.; Tenson, Tanel; Ott, Martin; Kamenski, Piotr; Hauryliuk, Vasili

    2016-01-01

    The mitochondrial genome almost exclusively encodes a handful of transmembrane constituents of the oxidative phosphorylation (OXPHOS) system. Coordinated expression of these genes ensures the correct stoichiometry of the system’s components. Translation initiation in mitochondria is assisted by two general initiation factors mIF2 and mIF3, orthologues of which in bacteria are indispensible for protein synthesis and viability. mIF3 was thought to be absent in Saccharomyces cerevisiae until we recently identified mitochondrial protein Aim23 as the missing orthologue. Here we show that, surprisingly, loss of mIF3/Aim23 in S. cerevisiae does not indiscriminately abrogate mitochondrial translation but rather causes an imbalance in protein production: the rate of synthesis of the Atp9 subunit of F1F0 ATP synthase (complex V) is increased, while expression of Cox1, Cox2 and Cox3 subunits of cytochrome c oxidase (complex IV) is repressed. Our results provide one more example of deviation of mitochondrial translation from its bacterial origins. PMID:26728900

  16. Translational networks in healthcare? Evidence on the design and initiation of organizational networks for knowledge mobilization.

    PubMed

    Fitzgerald, Louise; Harvey, Gill

    2015-08-01

    International attention has focussed on the variations between research evidence and practice in healthcare. This prompted the creation of formalized translational networks consisting of academic-service partnerships. The English Collaborations for Leadership in Applied Health Research and Care (CLAHRCs) are one example of a translational network. Using longitudinal, archival case study data from one CLAHRC over a 3-year period (2008-11), this article explores the relationship between organizational form and the function(s) of a translational network. The article focuses on the research gaps on the effective structures and appropriate governance to support a translational network. Data analysis suggested that the policy of setting up translational networks is insufficient of itself to produce positive translational activity. The data indicate that to leverage the benefits of the whole network, attention must be paid to devising a structure which integrates research production and use and facilitates lateral cross-disciplinary and cross-organizational communication. Equally, appropriate governance arrangements are necessary, particularly in large, multi-stakeholder networks, where shared governance may be questionable. Inappropriate network structure and governance inhibits the potential of the translational network. Finally, the case provides insights into the movement of knowledge within and between network organizations. The data demonstrate that knowledge mobilization extends beyond knowledge translation; knowledge mobilization includes the negotiated utilization of knowledge - a balanced power form of collaboration. Whilst much translational effort is externally focused on the health system, our findings highlight the essential need for the internal negotiation and mobilization of knowledge within academia.

  17. Translation initiation factor eIF3 promotes programmed stop codon readthrough

    PubMed Central

    Beznosková, Petra; Wagner, Susan; Jansen, Myrte Esmeralda; von der Haar, Tobias; Valášek, Leoš Shivaya

    2015-01-01

    Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans. PMID:25925566

  18. Solution structure of the C1-subdomain of Bacillus stearothermophilus translation initiation factor IF2

    PubMed Central

    Wienk, Hans; Tomaselli, Simona; Bernard, Cédric; Spurio, Roberto; Picone, Delia; Gualerzi, Claudio O.; Boelens, Rolf

    2005-01-01

    IF2 is one of three bacterial translation initiation factors that are conserved through all kingdoms of life. It binds the 30S and 50S ribosomal subunits, as well as fMet-tRNAfMet. After these interactions, fMet-tRNAfMet is oriented to the ribosomal P-site where the first amino acid of the nascent polypeptide, formylmethionine, is presented. The C-terminal domain of Bacillus stearothermophilus IF2, which is responsible for recognition and binding of fMet-tRNAfMet, contains two structured modules. Previously, the solution structure of the most C-terminal module, IF2-C2, has been elucidated by NMR spectroscopy and direct interactions between this subdomain and fMet-tRNAfMet were reported. In the present NMR study we have obtained the spectral assignment of the other module of the C-terminal domain (IF2-C1) and determined its solution structure and backbone dynamics. The IF2-C1 core forms a flattened fold consisting of a central four-stranded parallel β-sheet flanked by three α-helices. Although its overall organization resembles that of subdomain III of the archaeal IF2-homolog eIF5B whose crystal structure had previously been reported, some differences of potential functional significance are evident. PMID:16081655

  19. A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation.

    PubMed

    Leen, Eoin N; Sorgeloos, Frédéric; Correia, Samantha; Chaudhry, Yasmin; Cannac, Fabien; Pastore, Chiara; Xu, Yingqi; Graham, Stephen C; Matthews, Stephen J; Goodfellow, Ian G; Curry, Stephen

    2016-01-01

    Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.

  20. A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation

    PubMed Central

    Leen, Eoin N.; Sorgeloos, Frédéric; Correia, Samantha; Chaudhry, Yasmin; Cannac, Fabien; Pastore, Chiara; Xu, Yingqi; Graham, Stephen C.; Matthews, Stephen J.; Goodfellow, Ian G.; Curry, Stephen

    2016-01-01

    Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy. PMID:26734730

  1. Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae.

    PubMed

    Roque, Sylvain; Cerciat, Marie; Gaugué, Isabelle; Mora, Liliana; Floch, Aurélie G; de Zamaroczy, Miklos; Heurgué-Hamard, Valérie; Kervestin, Stephanie

    2015-01-01

    Eukaryotic release factor 3 (eRF3) is implicated in translation termination and also interacts with the poly(A)-binding protein (PABP, Pab1 in yeast), a major player in mRNA metabolism. Despite conservation of this interaction, its precise function remains elusive. First, we showed experimentally that yeast eRF3 does not contain any obvious consensus PAM2 (PABP-interacting motif 2). Thus, in yeast this association is different from the well described interaction between the metazoan factors. To gain insight into the exact function of this interaction, we then analyzed the phenotypes resulting from deleting the respective binding domains. Deletion of the Pab1 interaction domain on eRF3 did not affect general mRNA stability or nonsense-mediated mRNA decay (NMD) pathway and induced a decrease in translational readthrough. Furthermore, combined deletions of the respective interacting domains on eRF3 and on Pab1 were viable, did not affect Pab1 function in mRNA stability and harbored an antisuppression phenotype. Our results show that in Saccharomyces cerevisiae the role of the Pab1 C-terminal domain in mRNA stability is independent of eRF3 and the association of these two factors negatively regulates translation termination.

  2. Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae

    PubMed Central

    Roque, Sylvain; Cerciat, Marie; Gaugué, Isabelle; Mora, Liliana; Floch, Aurélie G.; de Zamaroczy, Miklos; Heurgué-Hamard, Valérie

    2015-01-01

    Eukaryotic release factor 3 (eRF3) is implicated in translation termination and also interacts with the poly(A)-binding protein (PABP, Pab1 in yeast), a major player in mRNA metabolism. Despite conservation of this interaction, its precise function remains elusive. First, we showed experimentally that yeast eRF3 does not contain any obvious consensus PAM2 (PABP-interacting motif 2). Thus, in yeast this association is different from the well described interaction between the metazoan factors. To gain insight into the exact function of this interaction, we then analyzed the phenotypes resulting from deleting the respective binding domains. Deletion of the Pab1 interaction domain on eRF3 did not affect general mRNA stability or nonsense-mediated mRNA decay (NMD) pathway and induced a decrease in translational readthrough. Furthermore, combined deletions of the respective interacting domains on eRF3 and on Pab1 were viable, did not affect Pab1 function in mRNA stability and harbored an antisuppression phenotype. Our results show that in Saccharomyces cerevisiae the role of the Pab1 C-terminal domain in mRNA stability is independent of eRF3 and the association of these two factors negatively regulates translation termination. PMID:25411355

  3. DNA immunization with eukaryotic initiation factor-2α of Toxoplasma gondii induces protective immunity against acute and chronic toxoplasmosis in mice.

    PubMed

    Chen, Jia; Huang, Si-Yang; Zhou, Dong-Hui; Li, Zhong-Yuan; Petersen, Eskild; Song, Hui-Qun; Zhu, Xing-Quan

    2013-12-16

    Toxoplasma gondii infection is a serious health problem of humans and animals worldwide. T. gondii eukaryotic initiation factor-2α (TgIF2α) plays a crucial role in parasite viability and is an important virulence factor of T. gondii. To evaluate the vaccine potential of TgIF2α, we constructed a novel eukaryotic plasmid pVAX-IF2α expressing TgIF2α from the RH strain and validated expression and immunogenicity in vitro in the Marc145 cell expression system by indirect immunofluorescence (IFA). Administration of pVAX-IF2α intramuscularly induced specific humoral immune responses including high levels of specific TgIF2α IgG antibody and a mixed IgG1/IgG2a response with a predominance of IgG2a production. The cellular immune response was elicited, showing significant production of IFN-γ and IL-2 associated with Th1 type response, and thus strong cell-mediated cytotoxic activity with increased frequencies of IFN-γ parameters analyzed in both CD4(+) and CD8(+) T cell compartments (CD4(+) IFN-γ(+) T cells and CD8(+) IFN-γ(+) T cells). Immunization resulted in partial protection against acute and chronic toxoplamosis in outbred Kunming mice, demonstrated by a significantly prolonged survival time (15.9±4.6 days) after challenge with the virulent RH strain and significant reduction in brain cysts (44.1%) against chronic infection with PRU cyst in contrast to control mice. Our data suggested that pVAX-IF2α could be used as a DNA vaccine candidate against both acute and chronic T. gondii infection by the activation of effective humoral and cellular immune responses.

  4. Deletion of the eIFiso4G subunit of the Arabidopsis eIFiso4F translation initiation complex impairs health and viability.

    PubMed

    Lellis, Andrew D; Allen, M Leah; Aertker, Alice W; Tran, Jonathan K; Hillis, David M; Harbin, Courtney R; Caldwell, Christian; Gallie, Daniel R; Browning, Karen S

    2010-10-01

    Arabidopsis thaliana knockout lines for the plant-specific eukaryotic translation initiation factors eIFiso4G1 (i4g1) and eIFiso4G2 (i4g2) genes have been obtained. To address the potential for functional redundancy of these genes, homozygous double mutant lines were generated by crossing individual knockout lines. Both single and double mutant plants were analyzed for changes in gross morphology, development, and responses to selected environmental stressors. Single gene knockouts appear to have minimal effect on morphology, germination rate, growth rate, flowering time, or fertility. However, double mutant i4g1/i4g2 knockout plants show reduced germination rates, slow growth rates, moderate chlorosis, impaired fertility and reduced long term seed viability. Double mutant plants also exhibit altered responses to dehydration, salinity, and heat stress. The i4g2 and i4g1/i4g2 double mutant has reduced amounts of chlorophyll a and b suggesting a role in the expression of chloroplast proteins. General protein synthesis did not appear to be affected as the levels of gross protein expression did not appear to change in the mutants. The lack of a phenotype for either of the single mutants suggests there is considerable functional overlap. However, the strong phenotypes observed for the double mutant indicates that the individual gene products may have specialized roles in the expression of proteins involved in plant growth and development.

  5. A Novel Quality Measure and Correction Procedure for the Annotation of Microbial Translation Initiation Sites.

    PubMed

    Overmars, Lex; Siezen, Roland J; Francke, Christof

    2015-01-01

    The identification of translation initiation sites (TISs) constitutes an important aspect of sequence-based genome analysis. An erroneous TIS annotation can impair the identification of regulatory elements and N-terminal signal peptides, and also may flaw the determination of descent, for any particular gene. We have formulated a reference-free method to score the TIS annotation quality. The method is based on a comparison of the observed and expected distribution of all TISs in a particular genome given prior gene-calling. We have assessed the TIS annotations for all available NCBI RefSeq microbial genomes and found that approximately 87% is of appropriate quality, whereas 13% needs substantial improvement. We have analyzed a number of factors that could affect TIS annotation quality such as GC-content, taxonomy, the fraction of genes with a Shine-Dalgarno sequence and the year of publication. The analysis showed that only the first factor has a clear effect. We have then formulated a straightforward Principle Component Analysis-based TIS identification strategy to self-organize and score potential TISs. The strategy is independent of reference data and a priori calculations. A representative set of 277 genomes was subjected to the analysis and we found a clear increase in TIS annotation quality for the genomes with a low quality score. The PCA-based annotation was also compared with annotation with the current tool of reference, Prodigal. The comparison for the model genome of Escherichia coli K12 showed that both methods supplement each other and that prediction agreement can be used as an indicator of a correct TIS annotation. Importantly, the data suggest that the addition of a PCA-based strategy to a Prodigal prediction can be used to 'flag' TIS annotations for re-evaluation and in addition can be used to evaluate a given annotation in case a Prodigal annotation is lacking.

  6. A Novel Quality Measure and Correction Procedure for the Annotation of Microbial Translation Initiation Sites

    PubMed Central

    Overmars, Lex; Siezen, Roland J.; Francke, Christof

    2015-01-01

    The identification of translation initiation sites (TISs) constitutes an important aspect of sequence-based genome analysis. An erroneous TIS annotation can impair the identification of regulatory elements and N-terminal signal peptides, and also may flaw the determination of descent, for any particular gene. We have formulated a reference-free method to score the TIS annotation quality. The method is based on a comparison of the observed and expected distribution of all TISs in a particular genome given prior gene-calling. We have assessed the TIS annotations for all available NCBI RefSeq microbial genomes and found that approximately 87% is of appropriate quality, whereas 13% needs substantial improvement. We have analyzed a number of factors that could affect TIS annotation quality such as GC-content, taxonomy, the fraction of genes with a Shine-Dalgarno sequence and the year of publication. The analysis showed that only the first factor has a clear effect. We have then formulated a straightforward Principle Component Analysis-based TIS identification strategy to self-organize and score potential TISs. The strategy is independent of reference data and a priori calculations. A representative set of 277 genomes was subjected to the analysis and we found a clear increase in TIS annotation quality for the genomes with a low quality score. The PCA-based annotation was also compared with annotation with the current tool of reference, Prodigal. The comparison for the model genome of Escherichia coli K12 showed that both methods supplement each other and that prediction agreement can be used as an indicator of a correct TIS annotation. Importantly, the data suggest that the addition of a PCA-based strategy to a Prodigal prediction can be used to ‘flag’ TIS annotations for re-evaluation and in addition can be used to evaluate a given annotation in case a Prodigal annotation is lacking. PMID:26204119

  7. Structure and dimerization of translation initiation factor aIF5B in solution

    SciTech Connect

    Carø VohlanderRasmussen, Louise; Oliveira, Cristiano Luis Pinto; Byron, Olwyn; Jensen, Janni Mosgaard; Pedersen, Jan Skov; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

    2012-02-07

    Translation initiation factor 5B (IF5B) is required for initiation of protein synthesis. The solution structure of archaeal IF5B (aIF5B) was analysed by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) and was indicated to be in both monomeric and dimeric form. Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) of aIF5B indicated that aIF5B forms irreversible dimers in solution but only to a maximum of 5.0-6.8% dimer. Sedimentation velocity (SV) AUC at higher speed also indicated the presence of two species, and the sedimentation coefficients s{sub 20,w}{sup 0} were determined to be 3.64 and 5.51 {+-} 0.29 S for monomer and dimer, respectively. The atomic resolution (crystallographic) structure of aIF5B (Roll-Mecak et al. [6]) was used to model monomer and dimer, and theoretical sedimentation coefficients for these models were computed (3.89 and 5.63 S, respectively) in good agreement with the sedimentation coefficients obtained from SV analysis. Thus, the structure of aIF5B in solution must be very similar to the atomic resolution structure of aIF5B. SAXS data were acquired in the same buffer with the addition of 2% glycerol to inhibit dimerization, and the resultant monomeric aIF5B in solution did indeed adopt a structure very similar to the one reported earlier for the protein in crystalline form. The p(r) function indicated an elongated conformation supported by a radius of gyration of 37.5 {+-} 0.2 {angstrom} and a maximum dimension of {approx}130 {angstrom}. The effects of glycerol on the formation of dimers are discussed. This new model of aIF5B in solution shows that there are universal structural differences between aIF5B and the homologous protein IF2 from Escherichia coli.

  8. Structure and dimerization of translation initiation factor aIF5B in solution

    SciTech Connect

    Rasmussen, Louise Caroe Vohlander; Oliveira, Cristiano Luis Pinto; Byron, Olwyn; Jensen, Janni Mosgaard; Pedersen, Jan Skov; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer aIF5B forms maximum 5.0-6.8% irreversible dimers in solution. Black-Right-Pointing-Pointer Sedimentation coefficients for monomer and dimer are 3.64 and 5.51 {+-} 0.29 S. Black-Right-Pointing-Pointer Adding only 2% glycerol prevents dimerization. Black-Right-Pointing-Pointer SAXS on aIF5B monomer gave an R{sub g} of 37.5 {+-} 0.2 A and a D{sub max} of {approx}130 A. Black-Right-Pointing-Pointer There are universal structural differences between aIF5B and Escherichia coli IF2. -- Abstract: Translation initiation factor 5B (IF5B) is required for initiation of protein synthesis. The solution structure of archaeal IF5B (aIF5B) was analysed by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) and was indicated to be in both monomeric and dimeric form. Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) of aIF5B indicated that aIF5B forms irreversible dimers in solution but only to a maximum of 5.0-6.8% dimer. Sedimentation velocity (SV) AUC at higher speed also indicated the presence of two species, and the sedimentation coefficients s{sub 20,w}{sup 0} were determined to be 3.64 and 5.51 {+-} 0.29 S for monomer and dimer, respectively. The atomic resolution (crystallographic) structure of aIF5B (Roll-Mecak et al. ) was used to model monomer and dimer, and theoretical sedimentation coefficients for these models were computed (3.89 and 5.63 S, respectively) in good agreement with the sedimentation coefficients obtained from SV analysis. Thus, the structure of aIF5B in solution must be very similar to the atomic resolution structure of aIF5B. SAXS data were acquired in the same buffer with the addition of 2% glycerol to inhibit dimerization, and the resultant monomeric aIF5B in solution did indeed adopt a structure very similar to the one reported earlier for the protein in crystalline form. The p(r) function indicated an elongated conformation supported by a radius of gyration of 37.5 {+-} 0.2 A

  9. The scanning model for translation: an update

    PubMed Central

    1989-01-01

    The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained. PMID:2645293

  10. Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation

    PubMed Central

    Wang, Xin; Xi, Wen; Toomey, Shaun; Chiang, Yueh-Chin; Hasek, Jiri; Laue, Thomas M.; Denis, Clyde L.

    2016-01-01

    Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation. PMID:26953568

  11. Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation.

    PubMed

    Wang, Xin; Xi, Wen; Toomey, Shaun; Chiang, Yueh-Chin; Hasek, Jiri; Laue, Thomas M; Denis, Clyde L

    2016-01-01

    Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation. PMID:26953568

  12. Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation.

    PubMed

    Wang, Xin; Xi, Wen; Toomey, Shaun; Chiang, Yueh-Chin; Hasek, Jiri; Laue, Thomas M; Denis, Clyde L

    2016-01-01

    Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.

  13. Taking aim at translation for tumor therapy

    PubMed Central

    Barnhart, Bryan C.; Simon, M. Celeste

    2007-01-01

    Increased cap-dependent mRNA translation rates are frequently observed in human cancers. Mechanistically, many human tumors often overexpress the cap binding protein eukaryotic translation initiation factor 4E (eIF4E), leading to enhanced translation of numerous tumor-promoting genes. In this issue of the JCI, Graff and colleagues describe potent antitumor effects using second-generation antisense oligonucleotides for eIF4E (see the related article beginning on page 2638). If their results are recapitulated in a clinical setting, this strategy will provide a promising antitumor therapy with broad-reaching applications. PMID:17786234

  14. Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

    PubMed

    Crepin, Thibaut; Shalak, Vyacheslav F; Yaremchuk, Anna D; Vlasenko, Dmytro O; McCarthy, Andrew; Negrutskii, Boris S; Tukalo, Michail A; El'skaya, Anna V

    2014-11-10

    Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.

  15. A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation

    PubMed Central

    Ruehle, Marisa D; Zhang, Haibo; Sheridan, Ryan M; Mitra, Somdeb; Chen, Yuanwei; Gonzalez, Ruben L; Cooperman, Barry S; Kieft, Jeffrey S

    2015-01-01

    Internal ribosome entry sites (IRESs) are powerful model systems to understand how the translation machinery can be manipulated by structured RNAs and for exploring inherent features of ribosome function. The intergenic region (IGR) IRESs from the Dicistroviridae family of viruses are structured RNAs that bind directly to the ribosome and initiate translation by co-opting the translation elongation cycle. These IRESs require an RNA pseudoknot that mimics a codon-anticodon interaction and contains a conformationally dynamic loop. We explored the role of this loop and found that both the length and sequence are essential for translation in different types of IGR IRESs and from diverse viruses. We found that loop 3 affects two discrete elongation factor-dependent steps in the IRES initiation mechanism. Our results show how the IRES directs multiple steps after 80S ribosome placement and highlights the often underappreciated significance of discrete conformationally dynamic elements within the context of structured RNAs. DOI: http://dx.doi.org/10.7554/eLife.08146.001 PMID:26523395

  16. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication.

    PubMed

    Koonin, E V

    1993-06-11

    A new superfamily of (putative) DNA-dependent ATPases is described that includes the ATPase domains of prokaryotic NtrC-related transcription regulators, MCM proteins involved in the initiation of eukaryotic DNA replication, and a group of uncharacterized bacterial and chloroplast proteins. MCM proteins are shown to contain a modified form of the ATP-binding motif and are predicted to mediate ATP-dependent opening of double-stranded DNA in the replication origins. In a second line of investigation, it is demonstrated that the products of unidentified open reading frames from Marchantia mitochondria and from yeast, and a domain of a baculovirus protein involved in viral DNA replication are related to the superfamily III of DNA and RNA helicases that previously has been known to include only proteins of small viruses. Comparison of the multiple alignments showed that the proteins of the NtrC superfamily and the helicases of superfamily III share three related sequence motifs tightly packed in the ATPase domain that consists of 100-150 amino acid residues. A similar array of conserved motifs is found in the family of DnaA-related ATPases. It is hypothesized that the three large groups of nucleic acid-dependent ATPases have similar structure of the core ATPase domain and have evolved from a common ancestor.

  17. The interaction between eukaryotic initiation factor 1A and eIF5 retains eIF1 within scanning preinitiation complexes.

    PubMed

    Luna, Rafael E; Arthanari, Haribabu; Hiraishi, Hiroyuki; Akabayov, Barak; Tang, Leiming; Cox, Christian; Markus, Michelle A; Luna, Lunet E; Ikeda, Yuka; Watanabe, Ryosuke; Bedoya, Edward; Yu, Cathy; Alikhan, Shums; Wagner, Gerhard; Asano, Katsura

    2013-12-31

    Scanning of the mRNA transcript by the preinitiation complex (PIC) requires a panel of eukaryotic initiation factors, which includes eIF1 and eIF1A, the main transducers of stringent AUG selection. eIF1A plays an important role in start codon recognition; however, its molecular contacts with eIF5 are unknown. Using nuclear magnetic resonance, we unveil eIF1A's binding surface on the carboxyl-terminal domain of eIF5 (eIF5-CTD). We validated this interaction by observing that eIF1A does not bind to an eIF5-CTD mutant, altering the revealed eIF1A interaction site. We also found that the interaction between eIF1A and eIF5-CTD is conserved between humans and yeast. Using glutathione S-transferase pull-down assays of purified proteins, we showed that the N-terminal tail (NTT) of eIF1A mediates the interaction with eIF5-CTD and eIF1. Genetic evidence indicates that overexpressing eIF1 or eIF5 suppresses the slow growth phenotype of eIF1A-NTT mutants. These results suggest that the eIF1A-eIF5-CTD interaction during scanning PICs contributes to the maintenance of eIF1 within the open PIC.

  18. Amino acid microsequencing of internal tryptic peptides of heme-regulated eukaryotic initiation factor 2 alpha subunit kinase: homology to protein kinases.

    PubMed Central

    Chen, J J; Pal, J K; Petryshyn, R; Kuo, I; Yang, J M; Throop, M S; Gehrke, L; London, I M

    1991-01-01

    We have purified the heme-regulated eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2 alpha phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)-Tyr-Ser-(Val)-Gly-Val found in catalytic domain IX of protein kinases [Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52]. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. Synthetic HRI peptide P-74 is a very potent inhibitor of eIF-2 alpha phosphorylation by HRI. Since little is known about the function of conserved domain IX, P-74 peptide may be useful in elucidating the role of this domain of protein kinases. Images PMID:1671169

  19. Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2α) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum

    PubMed Central

    Alves, Eduardo; Maluf, Fernando V.; Bueno, Vânia B.; Guido, Rafael V. C.; Oliva, Glaucius; Singh, Maneesh; Scarpelli, Pedro; Costa, Fahyme; Sartorello, Robson; Catalani, Luiz H.; Brady, Declan; Tewari, Rita; Garcia, Celia R. S.

    2016-01-01

    In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2α factor, an eukaryotic initiation factor phosphorylated by eIF2α kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2α phosphorylation pattern as a molecular mechanism for its action. PMID:26915471

  20. Mechanism of Cytoplasmic mRNA Translation

    PubMed Central

    2015-01-01

    Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

  1. Adaptation and Diversification of an RNA Replication System under Initiation- or Termination-Impaired Translational Conditions.

    PubMed

    Mizuuchi, Ryo; Ichihashi, Norikazu; Yomo, Tetsuya

    2016-07-01

    Adaptation to various environments is a remarkable characteristic of life. Is this limited to extant complex living organisms, or is it also possible for a simpler self-replication system to adapt? In this study, we addressed this question by using a translation-coupled RNA replication system that comprised a reconstituted translation system and an RNA "genome" that encoded a replicase gene. We performed RNA replication reactions under four conditions, under which different components of translation were partly inhibited. We found that replication efficiency increased with the number of rounds of replication under all the tested conditions. The types of dominant mutations differed depending on the condition, thus indicating that this simple system adapted to different environments in different ways. This suggests that even a primitive self-replication system composed of a small number of genes on the early earth could have had the ability to adapt to various environments.

  2. RNA polymerase pausing regulates translation initiation by providing additional time for TRAP-RNA interaction.

    PubMed

    Yakhnin, Alexander V; Yakhnin, Helen; Babitzke, Paul

    2006-11-17

    RNA polymerase (RNAP) pause sites have been identified in several prokaryotic genes. Although the presumed biological function of RNAP pausing is to allow synchronization of RNAP position with regulatory factor binding and/or RNA folding, a direct causal link between pausing and changes in gene expression has been difficult to establish. RNAP pauses at two sites in the Bacillus subtilis trpEDCFBA operon leader. Pausing at U107 and U144 participates in transcription attenuation and trpE translation control mechanisms, respectively. Substitution of U144 caused a substantial pausing defect in vitro and in vivo. These mutations led to increased trp operon expression that was suppressed by overproduction of TRAP, indicating that pausing at U144 provides additional time for TRAP to bind to the nascent transcript and promote formation of an RNA structure that blocks translation of trpE. These results establish that pausing is capable of playing a role in regulating translation in bacteria. PMID:17114058

  3. The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas

    SciTech Connect

    Sun, Yu; Wong, Nicholas; Guan, Yinghui; Salamanca, Clara M.; Cheng, Jung Chien; Lee, Jonathan M.; Gray, Joe W.; Auersperg, Nelly

    2008-04-25

    Ovarian epithelial carcinomas (OEC) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In this study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities, and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.

  4. Ribosome traffic on mRNAs maps to gene ontology: genome-wide quantification of translation initiation rates and polysome size regulation.

    PubMed

    Ciandrini, Luca; Stansfield, Ian; Romano, M Carmen

    2013-01-01

    To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function.

  5. Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated.

    PubMed

    Dmitriev, Sergey E; Andreev, Dmitri E; Terenin, Ilya M; Olovnikov, Ivan A; Prassolov, Vladimir S; Merrick, William C; Shatsky, Ivan N

    2007-07-01

    Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.

  6. Efficient Translation Initiation Directed by the 900-Nucleotide-Long and GC-Rich 5′ Untranslated Region of the Human Retrotransposon LINE-1 mRNA Is Strictly Cap Dependent Rather than Internal Ribosome Entry Site Mediated▿

    PubMed Central

    Dmitriev, Sergey E.; Andreev, Dmitri E.; Terenin, Ilya M.; Olovnikov, Ivan A.; Prassolov, Vladimir S.; Merrick, William C.; Shatsky, Ivan N.

    2007-01-01

    Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5′ untranslated region (5′UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5′UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5′UTR-Fluc) or bicistronic (Rluc-L1 5′UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5′UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5′UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5′UTR. Nevertheless, this cap-dependent initiation activity of the L1 5′UTR was unexpectedly high and resembles that of the beta-actin 5′UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5′UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5′UTRs and call into question the conception that every long GC-rich 5′UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event. PMID:17470553

  7. In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins.

    PubMed

    Duszenko, Michael; Redecke, Lars; Mudogo, Celestin Nzanzu; Sommer, Benjamin Philip; Mogk, Stefan; Oberthuer, Dominik; Betzel, Christian

    2015-08-01

    During the last decade, the number of three-dimensional structures solved by X-ray crystallography has increased dramatically. By 2014, it had crossed the landmark of 100 000 biomolecular structures deposited in the Protein Data Bank. This tremendous increase in successfully crystallized proteins is primarily owing to improvements in cloning strategies, the automation of the crystallization process and new innovative approaches to monitor crystallization. However, these improvements are mainly restricted to soluble proteins, while the crystallization and structural analysis of membrane proteins or proteins that undergo major post-translational modifications remains challenging. In addition, the need for relatively large crystals for conventional X-ray crystallography usually prevents the analysis of dynamic processes within cells. Thus, the advent of high-brilliance synchrotron and X-ray free-electron laser (XFEL) sources and the establishment of serial crystallography (SFX) have opened new avenues in structural analysis using crystals that were formerly unusable. The successful structure elucidation of cathepsin B, accomplished by the use of microcrystals obtained by in vivo crystallization in baculovirus-infected Sf9 insect cells, clearly proved that crystals grown intracellularly are very well suited for X-ray analysis. Here, methods by which in vivo crystals can be obtained, isolated and used for structural analysis by novel highly brilliant XFEL and synchrotron-radiation sources are summarized and discussed.

  8. In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins.

    PubMed

    Duszenko, Michael; Redecke, Lars; Mudogo, Celestin Nzanzu; Sommer, Benjamin Philip; Mogk, Stefan; Oberthuer, Dominik; Betzel, Christian

    2015-08-01

    During the last decade, the number of three-dimensional structures solved by X-ray crystallography has increased dramatically. By 2014, it had crossed the landmark of 100 000 biomolecular structures deposited in the Protein Data Bank. This tremendous increase in successfully crystallized proteins is primarily owing to improvements in cloning strategies, the automation of the crystallization process and new innovative approaches to monitor crystallization. However, these improvements are mainly restricted to soluble proteins, while the crystallization and structural analysis of membrane proteins or proteins that undergo major post-translational modifications remains challenging. In addition, the need for relatively large crystals for conventional X-ray crystallography usually prevents the analysis of dynamic processes within cells. Thus, the advent of high-brilliance synchrotron and X-ray free-electron laser (XFEL) sources and the establishment of serial crystallography (SFX) have opened new avenues in structural analysis using crystals that were formerly unusable. The successful structure elucidation of cathepsin B, accomplished by the use of microcrystals obtained by in vivo crystallization in baculovirus-infected Sf9 insect cells, clearly proved that crystals grown intracellularly are very well suited for X-ray analysis. Here, methods by which in vivo crystals can be obtained, isolated and used for structural analysis by novel highly brilliant XFEL and synchrotron-radiation sources are summarized and discussed. PMID:26249677

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

    PubMed

    Chatterjee, Ishita; Gross, Stephane R; Kinzy, Terri Goss; Chen, Kuang Yu

    2006-03-01

    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. PMID:16408210

  10. Posttranscriptional Suppression of Lipopolysaccharide-Stimulated Inflammatory Responses by Macrophages in Middle-Aged Mice: A Possible Role for Eukaryotic Initiation Factor 2 α.

    PubMed

    Shirato, Ken; Imaizumi, Kazuhiko

    2014-01-01

    The intensities of macrophage inflammatory responses to bacterial components gradually decrease with age. Given that a reduced rate of protein synthesis is a common age-related biochemical change, which is partially mediated by increased phosphorylation of eukaryotic initiation factor-2 α (eIF-2 α ), we investigated the mechanism responsible for the deterioration of macrophage inflammatory responses, focusing specifically on the age-related biochemical changes in middle-aged mice. Peritoneal macrophages isolated from 2-month-old (young) and 12-month-old (middle-aged) male BALB/c mice were stimulated with lipopolysaccharide (LPS). Although LPS-stimulated secretion of tumor necrosis factor- α (TNF- α ) by the macrophages from middle-aged mice was significantly lower than that from young mice, LPS caused marked increases in levels of TNF- α mRNA in macrophages from middle-aged as well as young mice. Moreover, LPS evoked similar levels of phosphorylation of c-Jun N-terminal kinase (JNK) and nuclear factor- κ B (NF- κ B) in young and middle-aged mice. In contrast, the basal level of phosphorylated eIF-2 α in macrophages from middle-aged mice was higher than that in macrophages from young mice. Salubrinal, an inhibitor of the phosphatase activity that dephosphorylates eIF-2 α , suppressed the LPS-stimulated inflammatory responses in a murine macrophage cell line RAW264.7. These results suggest that posttranscriptional suppression of macrophage inflammatory responses during middle age requires phosphorylation of eIF-2 α . PMID:24808968

  11. A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E).

    PubMed

    Ruffel, Sandrine; Dussault, Marie-Hélène; Palloix, Alain; Moury, Benoît; Bendahmane, Abdelhafid; Robaglia, Christophe; Caranta, Carole

    2002-12-01

    We show here that the pvr2 locus in pepper, conferring recessive resistance against strains of potato virus Y (PVY), corresponds to a eukaryotic initiation factor 4E (eIF4E) gene. RFLP analysis on the PVY-susceptible and resistant pepper cultivars, using an eIF4E cDNA from tobacco as probe, revealed perfect map co-segregation between a polymorphism in the eIF4E gene and the pvr2 alleles, pvr2(1) (resistant to PVY-0) and pvr2(2) (resistant to PVY-0 and 1). The cloned pepper eIF4E cDNA encoded a 228 amino acid polypeptide with 70-86% nucleotide sequence identity with other plant eIF4Es. The sequences of eIF4E protein from two PVY-susceptible cultivars were identical and differed from the eIF4E sequences of the two PVY-resistant cultivars Yolo Y (YY) (pvr2(1)) and FloridaVR2 (F) (pvr2(2)) at two amino acids, a mutation common to both resistant genotypes and a second mutation specific to each. Complementation experiments were used to show that the eIF4E gene corresponds to pvr2. Thus, potato virus X-mediated transient expression of eIF4E from susceptible cultivar Yolo Wonder (YW) in the resistant genotype YY resulted in loss of resistance to subsequent PVY-0 inoculation and transient expression of eIF4E from YY (resistant to PVY-0; susceptible to PVY-1) rendered genotype F susceptible to PVY-1. Several lines of evidence indicate that interaction between the potyvirus genome-linked protein (VPg) and eIF4E are important for virus infectivity, suggesting that the recessive resistance could be due to incompatibility between the VPg and eIF4E in the resistant genotype. PMID:12492847

  12. Changing perspectives on the origin of eukaryotes.

    PubMed

    Katz, L A

    1998-12-01

    From the initial application of molecular techniques to the study of microbial organisms, three domains of life emerged, with eukaryotes and archaea as sister taxa. However, recent analyses of an expanding molecular data set reveal that the eukaryotic genome is chimeric with respect to archaea and bacteria. Moreover, there is now evidence that the primitive eukaryotic group `Archezoa' once harbored mitochondia. These discoveries have challenged the traditional stepwise model of the evolution of eukaryotes, in which the nucleus and microtubules evolve before the acquisition of mitochondria, and consequently compel a revision of existing models of the origin of eukaryotic cells. PMID:21238406

  13. Residues required for phosphorylation of translation initiation factor eIF2α under diverse stress conditions are divergent between yeast and human

    PubMed Central

    Majumder, Mithu; Mitchell, Daniel; Merkulov, Sergei; Wu, Jing; Guan, Bo-Jhih; Snider, Martin D.; Krokowski, Dawid; Yee, Vivien C.; Hatzoglou, Maria

    2015-01-01

    PERK, PKR, HRI and GCN2 are the four mammalian kinases that phosphorylate the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) on Ser51. This phosphorylation event is conserved among many species and attenuates protein synthesis in response to diverse stress conditions. In contrast, Saccharmyces cerevisiae expresses only the GCN2 kinase. It was demonstrated previously in S. cerevisiae that single point mutations in eIF2α’s N-terminus severely impaired phosphorylation at Ser51. To assess whether similar recognition patterns are present in mammalian eIF2α, we expressed human eIF2α’s with these mutations in mouse embryonic fibroblasts and assessed their phosphorylation under diverse stress conditions. Some of the mutations prevented the stress-induced phosphorylation of eIF2α by all mammalian kinases, thus defining amino acid residues in eIF2α (Gly 30, Leu 50, and Asp 83) that are required for substrate recognition. We also identified residues that were less critical or not required for recognition by the mammalian kinases (Ala 31, Met 44, Lys 79, and Tyr 81), even though they were essential for recognition of the yeast eIF2α by GCN2. We propose that mammalian eIF2α kinases evolved to maximize their interactions with the evolutionarily conserved Ser51 residue of eIF2α in response to diverse stress conditions, thus adding to the complex signaling pathways that mammalian cells have over simpler organisms. PMID:25541374

  14. An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

    PubMed Central

    Jones, R M; Branda, J; Johnston, K A; Polymenis, M; Gadd, M; Rustgi, A; Callanan, L; Schmidt, E V

    1996-01-01

    The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation. PMID:8756633

  15. The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA

    PubMed Central

    Dominissini, Dan; Nachtergaele, Sigrid; Moshitch-Moshkovitz, Sharon; Peer, Eyal; Kol, Nitzan; Ben-Haim, Moshe Shay; Dai, Qing; Di Segni, Ayelet; Salmon-Divon, Mali; Clark, Wesley C.; Zheng, Guanqun; Pan, Tao; Solomon, Oz; Eyal, Eran; Hershkovitz, Vera; Han, Dali; Doré, Louis C.; Amariglio, Ninette; Rechavi, Gideon; He, Chuan

    2016-01-01

    Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N6-methyladenosine (m6A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N1-methyladenosine (m1A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m1A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m1A in promoting translation of methylated mRNA. PMID:26863196

  16. The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA.

    PubMed

    Dominissini, Dan; Nachtergaele, Sigrid; Moshitch-Moshkovitz, Sharon; Peer, Eyal; Kol, Nitzan; Ben-Haim, Moshe Shay; Dai, Qing; Di Segni, Ayelet; Salmon-Divon, Mali; Clark, Wesley C; Zheng, Guanqun; Pan, Tao; Solomon, Oz; Eyal, Eran; Hershkovitz, Vera; Han, Dali; Doré, Louis C; Amariglio, Ninette; Rechavi, Gideon; He, Chuan

    2016-02-25

    Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA. PMID:26863196

  17. Breaking down silos: mapping growth of cross-disciplinary collaboration in a translational science initiative.

    PubMed

    Luke, Douglas A; Carothers, Bobbi J; Dhand, Amar; Bell, Ryan A; Moreland-Russell, Sarah; Sarli, Cathy C; Evanoff, Bradley A

    2015-04-01

    The importance of transdisciplinary collaboration is growing, though not much is known about how to measure collaboration patterns. The purpose of this paper is to present multiple ways of mapping and evaluating the growth of cross-disciplinary partnerships over time. Social network analysis was used to examine the impact of a Clinical and Translational Science Award (CTSA) on collaboration patterns. Grant submissions from 2007 through 2010 and publications from 2007 through 2011 of Institute of Clinical and Translational Sciences (ICTS) members were examined. A Cohort Model examining the first-year ICTS members demonstrated an overall increase in collaborations on grants and publications, as well as an increase in cross-discipline collaboration as compared to within-discipline. A Growth Model that included additional members over time demonstrated the same pattern for grant submissions, but a decrease in cross-discipline collaboration as compared to within-discipline collaboration for publications. ICTS members generally became more cross-disciplinary in their collaborations during the CTSA. The exception of publications for the Growth Model may be due to the time lag between funding and publication, as well as pressure for younger scientists to publish in their own fields. Network analysis serves as a valuable tool for evaluating changes in scientific collaboration.

  18. Initiating tumor banking for translational research: MD Anderson and Liverpool experience.

    PubMed

    Mishra, A; Pandey, A; Shaw, R

    2007-01-01

    The ultimate progress in the cancer diagnosis and therapy has only been possible with the ongoing translational research that is likely to play a very important role in future as well. Hence the importance of such translation from bedside to bench and vis versa cannot be over-emphasized. Accordingly it has become more important to collect tumor samples along with the clinical information in a systematic manner to perform a good basic science research in future. With a population of over a billion and a heavy burden of cancer, India has the 'biggest' potential to establish the 'largest' tumor bank across the globe. Establishing a tumor bank involves money and manpower that may not be feasible across most of the centers in India. Taking into the considering the model of tumor banking of the two leading institutions of the world (MD Anderson Cancer Center, USA and University Hospital Aintree, Liverpool UK), this article presents the salient tips for a center in India to get started with tumor banking with minimal investment. Furthermore a simplified form of ethical consent is presented for the centers to adapt unanimously. PMID:17401220

  19. Sepsis and mechnaical ventilation restrain translation initiation in skeletal muscle by inducing AMPK-associated TSC[2] restriction of mTOR signaling in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In skeletal muscle, AMP-activated protein kinase (AMPK) acts as a cellular energy sensor of AMP: ATP and modulates translation by repressing mammalian target of rapamycin (mTOR) activation. Endotoxin (LPS)-induced sepsis reduces muscle protein synthesis by blunting translation initiation. We hypothe...

  20. The polypyrimidine tract binding protein (PTB) requirement for internal initiation of translation of cardiovirus RNAs is conditional rather than absolute.

    PubMed Central

    Kaminski, A; Jackson, R J

    1998-01-01

    Picornavirus RNAs are translated by an unusual mechanism of internal ribosome entry that requires a substantial segment of the viral 5'-untranslated region, generally known as the internal ribosome entry segment (IRES), and in some circumstances may require cellular trans-acting proteins, particularly polypyrimidine tract binding protein (PTB). It is shown here that for encephalomyocarditis virus (EMCV), the PTB dependence of IRES function in vitro is determined partly by the nature of the reporter cistron, and more especially by the size of an A-rich bulge in the IRES. With a wild-type EMCV IRES (which has a bulge of 6 As), translation is effectively independent of PTB provided the IRES is driving the synthesis of EMCV viral polyprotein. With an enlarged (7A) bulge and heterologous reporters, translation is highly dependent on PTB. Intermediate levels of PTB dependence are seen with a 7A bulge IRES driving viral polyprotein synthesis or a wild-type (6A) bulge IRES linked to a heterologous reporter. None of these parameters influenced the binding of PTB to the high-affinity site in the IRES. These results argue that PTB is not an essential and universal internal initiation factor, but, rather, that when it is required, its binding to the IRES helps to maintain the appropriate higher-order structure and to reverse distortions caused, for example, by an enlarged A-rich bulge. PMID:9622122

  1. Crystal structure of the eukaryotic ribosome.

    PubMed

    Ben-Shem, Adam; Jenner, Lasse; Yusupova, Gulnara; Yusupov, Marat

    2010-11-26

    Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.

  2. Production of eukaryotic cell-free lysate from Leishmania tarentolae.

    PubMed

    Johnston, Wayne A; Alexandrov, Kirill

    2014-01-01

    In this chapter, we describe the production and application of a eukaryotic cell-free expression system based on Leishmania tarentolae. This single-celled flagellate allows straightforward and inexpensive cultivation in flasks or bioreactors. Unlike many other Leishmania species, it is nonpathogenic to humans and does not require special laboratory precautions. An additional reason it is a convenient source organism for cell-free lysate production is that all endogenous protein expression can be suppressed by a single antisense oligonucleotide targeting splice leader sequence on the 5'-end of all protein coding RNAs. We describe simple procedures for cell disruption and lysate processing starting from bioreactor culture. We also describe introduction of genetic information via vectors containing species-independent translation initiation sites (SITS). We consider that such an inexpensive eukaryotic cell-free production system has many advantages when expressing multi-subunit proteins or difficult to express proteins. PMID:24395406

  3. A tertiary structure model of the internal ribosome entry site (IRES) for methionine-independent initiation of translation.

    PubMed Central

    Kanamori, Y; Nakashima, N

    2001-01-01

    Cricket paralysis-like viruses have a dicistronic positive-strand RNA genome. These viruses produce capsid proteins through internal ribosome entry site (IRES)-mediated translation. The IRES element of one of these viruses, Plautia stall intestine virus (PSIV), forms a pseudoknot immediately upstream from the capsid coding sequence, and initiates translation from other than methionine. Previously, we estimated that the IRES element of PSIV consists of seven stem-loops using the program MFOLD; however, experimental evidence of the predicted structures was not shown, except for stem-loop VI, which was responsible for formation of the pseudoknot. To determine the whole structure of the PSIV-IRES element, we introduced compensatory mutations into the upstream MFOLD-predicted helical segments. Mutation analysis showed that stem-loop V exists as predicted, but stem-loop IV is shorter than predicted. The structure of stem-loop III is different from predicted, and stem-loops I and II are not necessary for IRES activity. In addition, we identified two new pseudoknots in the IRES element of PSIV. The complementary sequence segments that are responsible for formation of the two pseudoknots are also observed in cricket paralysis virus (CrPV) and CrPV-like viruses such as Drosophila C virus (DCV), Rhopalosiphum padi virus (RhPV), himetobi P virus (HiPV), Triatoma virus (TrV), and black queen-cell virus (BQCV), although each sequence is distinct in each virus. Considering the three pseudoknots, we constructed a tertiary structure model of the PSIV-IRES element. This structural model is applicable to other CrPV-like viruses, indicating that other CrPV-like viruses can also initiate translation from other than methionine. PMID:11233983

  4. Translational Polarity of a Mutation in the Initiator Aug Codon of the λ cI Gene

    PubMed Central

    Gussin, Gary N.; Brown, Susan; Matz, Karen

    1987-01-01

    A PRM-cI-lacZ fusion inserted into the b2 region of bacteriophage λ was used to isolate mutations affecting expression of both the λ cI gene and the lacZ gene. One such mutation, a change in the cI initiator codon from AUG to AUA, reduces immunity of a λ prophage to superinfection, and causes a 60-70% reduction in β-galactosidase synthesis, even when repressor is supplied in trans. The effect of the mutation on lacZ gene expression is eliminated in a rho- bacterial strain, and the mutation has no effect on transcription initiated at PRM in vitro. Therefore, the effects of the mutation are due to premature ρ-dependent termination of transcription in the absence of translation of the cI gene, as if the mutation were a nonsense polar mutation. PMID:2959588

  5. Two isoforms of TALDO1 generated by alternative translational initiation show differential nucleocytoplasmic distribution to regulate the global metabolic network

    PubMed Central

    Moriyama, Tetsuji; Tanaka, Shu; Nakayama, Yasumune; Fukumoto, Masahiro; Tsujimura, Kenji; Yamada, Kohji; Bamba, Takeshi; Yoneda, Yoshihiro; Fukusaki, Eiichiro; Oka, Masahiro

    2016-01-01

    Transaldolase 1 (TALDO1) is a rate-limiting enzyme involved in the pentose phosphate pathway, which is traditionally thought to occur in the cytoplasm. In this study, we found that the gene TALDO1 has two translational initiation sites, generating two isoforms that differ by the presence of the first 10 N-terminal amino acids. Notably, the long and short isoforms were differentially localised to the cell nucleus and cytoplasm, respectively. Pull-down and in vitro transport assays showed that the long isoform, unlike the short one, binds to importin α and is actively transported into the nucleus in an importin α/β-dependent manner, demonstrating that the 10 N-terminal amino acids are essential for its nuclear localisation. Additionally, we found that these two isoforms can form homo- and/or hetero-dimers with different localisation dynamics. A metabolite analysis revealed that the subcellular localisation of TALDO1 is not crucial for its activity in the pentose phosphate pathway. However, the expression of these two isoforms differentially affected the levels of various metabolites, including components of the tricarboxylic acid cycle, nucleotides, and sugars. These results demonstrate that the nucleocytoplasmic distribution of TALDO1, modulated via alternative translational initiation and dimer formation, plays an important role in a wide range of metabolic networks. PMID:27703206

  6. Genetic analysis of bacteriophage lambda cIII gene: mRNA structural requirements for translation initiation.

    PubMed Central

    Kornitzer, D; Teff, D; Altuvia, S; Oppenheim, A B

    1989-01-01

    The bacteriophage lambda cIII gene product regulates the lysogenic pathway. The cIII gene is located in the leftward operon, which is transcribed from the pL promoter. We have previously shown (S. Altuvia and A. B. Oppenheim, J. Bacteriol. 167:415-419, 1986) that mutations that show elevated expression lie within the cIII coding sequence. We isolated mutants that show decreased CIII activity. All the mutations were found to cause a drastic reduction in the rate of initiation of cIII translation. Several mutations were found to be scattered within the first 40 nucleotides of the cIII coding region. Additional mutations affected the AUG initiation codon, the Shine-Dalgarno sequence, and the upstream RNaseIII processing site. Computer folding of the cIII mRNA suggested the presence of two alternative RNA structures. All the mutations within the coding region that reduce expression reduce the stability of one specific mRNA structure (structure B). Mutations that increase expression lie in the loops of this structure and may in fact stabilize it by interfering with the formation of the alternative structure (structure A). Thus, it appears that a specific mRNA secondary structure at the beginning of the cIII coding region is essential for efficient translation, suggesting that changes in mRNA structure regulate cIII expression. Images PMID:2523380

  7. Effect of silent mutations in Translational Initial Region on the production of recombinant cutinase in Escherichia coli.

    PubMed

    Liu, Zhi-guo; Zhu, Li; Zhu, Kong-liang; Chen, Sheng; Chen, Jian; Wu, Jing

    2011-04-01

    Translational Initial Region (TIR) is the threshold of an intracellular translation process, and tiny alterations in this region are reported to intensely influence the downstream expression. Such property provides a potential utilization in extracellular production of recombinant enzyme. As an esterase, cutinase is an essential catalyst in the process of textile scouring, and has a potential application in food and chemical industry. In the present study, a bacterial cutinase (Tfu_0883) from Thermobifida fusca was expressed in Escherichia coli with pelB as its signal peptide using trc as its promoter. A subsequent TIR degeneracy mutagenesis was then carried out in the initial sequence of pelB. A fast screening method for these mutants was developed and a series of strains with different expression strengths were accordingly obtained. Among these mutants, a high cutinase production level of 38.0 U/ml was achieved, which is three times that of the control group. This study explored the potential utilization of TIR degeneracy mutagenesis in the production of industrial enzymes. PMID:21212953

  8. Translational Research Principles Applied to Education: The Mapping Educational Specialist Knowhow (MESH) Initiative

    ERIC Educational Resources Information Center

    Burden, Kevin; Younie, Sarah; Leask, Marilyn

    2013-01-01

    The Mapping Educational Specialist Knowhow (MESH) Initiative is part of a research project applying knowledge management principles which are well known in other sectors, public and private, to the education sector. The goal is to develop and test out the new ways of working, now possible with digital technologies, which can address long standing…

  9. Translation Sociology and Social Capital in Rural Development Initiatives. A Case Study from the Italian Alps

    ERIC Educational Resources Information Center

    Magnani, Natalia; Struffi, Lauro

    2009-01-01

    This article analyses the results of a European "research and demonstration" project promoting multifunctional and sustainable agriculture in Alpine regions through a participatory approach. It focuses in particular on initiatives undertaken by a local farmers group in the Italian Alpine area of Val di Sole, the purpose being to draw attention to…

  10. Effect of unilateral vestibular deafferentation on the initial human vestibulo-ocular reflex to surge translation

    PubMed Central

    Tian, Jun-Ru; Ishiyama, Akira; Demer, Joseph L.

    2007-01-01

    Transient whole-body surge (fore-aft) translation at 0.5 G peak acceleration was administered to six subjects with unilateral vestibular deafferentation (UVD), and eight age-matched controls. Subjects viewed eccentric targets to determine if linear vestibulo-ocular reflex (LVOR) asymmetry might lateralize otolith deficits. Eye rotation was measured using magnetic search coils. Immediately before surge, subjects viewed a luminous target 50 cm away, centered or displaced 10° horizontally or vertically. The target was extinguished during randomly directed surges. LVOR gain relative to ideal velocity in subjects with UVD for the contralesional horizontally eccentric target (0.59 ± 0.08, mean ± SEM) did not differ significantly from normal (0.50 ± 0.04), but gain for the ipsilesional eccentric target (0.35 ± 0.02) was significantly less than normal (0.48 ± 0.03, P < 0.05). Normal subjects had mean gain asymmetry for horizontally eccentric targets of 0.17 ± 0.03, but asymmetry in UVD was significantly increased to 0.35 ± 0.05 (P < 0.05). Four of six subjects with UVD had maximum gain asymmetry outside normal 95% confidence limits. Asymmetry did not correlate with UVD duration. Gain for 10° vertically eccentric targets averaged 0.38 ± 0.14 for subjects with UVD, insignificantly lower than the normal value of 0.75 ± 0.15 (P > 0.05). Surge LVOR latency was symmetrical in UVD, and did not differ significantly from normal. There was no significant difference in response between dark and visible target conditions until 200 ms after surge onset. Chronic human UVD, on average, significantly impairs the surge LVOR for horizontally eccentric targets placed ipsilesionally, but this asymmetry is small relative to interindividual variation. PMID:16900361

  11. Specific requirement for translation initiation factor 4E or its isoform drives plant host susceptibility to Tobacco etch virus

    PubMed Central

    2014-01-01

    Background In plants, eIF4E translation initiation factors and their eIFiso4E isoforms are essential susceptibility factors for many RNA viruses, including potyviruses. Mutations altering these factors are a major source of resistance to the viruses. The eIF4E allelic series is associated with specific resistance spectra in crops such as Capsicum annum. Genetic evidence shows that potyviruses have a specific requirement for a given 4E isoform that depends on the host plant. For example, Tobacco etch virus (TEV) uses eIF4E1 to infect Capsicum annuum but uses eIFiso4E to infect Arabidopsis thaliana. Here, we investigated how TEV exploits different translation initiation factor isoforms to infect these two plant species. Results A complementation system was set up in Arabidopsis to test the restoration of systemic infection by TEV. Using this system, Arabidopsis susceptibility to TEV was complemented with a susceptible pepper eIF4E1 allele but not with a resistant allele. Therefore, in Arabidopsis, TEV can use the pepper eIF4E1 instead of the endogenous eIFiso4E isoform so is able to switch between translation initiation factor 4E isoform to infect the same host. Moreover, we show that overexpressing the pepper eIF4E1 alleles is sufficient to make Arabidopsis susceptible to an otherwise incompatible TEV strain. Lastly, we show that the resistant eIF4E1 allele is similarly overcome by a resistance-breaking TEV strain as in pepper, confirming that this Arabidopsis TEV-susceptibility complementation system is allele-specific. Conclusion We report here a complementation system in Arabidopsis that makes it possible to assess the role of pepper pvr2-eIF4E alleles in susceptibility to TEV. Heterologous complementation experiments showed that the idiosyncratic properties of the 4E and iso4E proteins create a major checkpoint for viral infection of different hosts. This system could be used to screen natural or induced eIF4E alleles to find and study alleles of interest for

  12. RNA--protein interactions within the internal translation initiation region of encephalomyocarditis virus RNA.

    PubMed Central

    Borovjagin, A V; Ezrokhi, M V; Rostapshov, V M; Ugarova TYu; Bystrova, T F; Shatsky, I N

    1991-01-01

    Various derivatives of the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) RNA have been used to analyze by UV-cross-linking its interaction with mRNA binding proteins from ascites carcinoma Krebs-2 cells. A doublet of proteins with Mr 58 and 60 kD bound to two regions of the IRES. One site is centered at nt 420-421 of EMCV RNA whereas the other is located between nt 315-377. Both sites form hairpin structures, the loops of which contain UCUUU motif, conserved among cardio- and aphthoviruses. The interaction of p58 and p60 with IRES is affected by the integrity of the stem-loop structure proximal to the start AUG codon (nts 680-787), although, under similar conditions, cross-linking of these proteins to this region was not detected. Deletions in the main recognition site of p58 strongly reduce the initiation activity of the IRES in vitro. However, elimination of p58 (p60) binding by these mutations does not completely abolish the ability of the IRES to direct polypeptide synthesis starting from the authentic AUG codon. The IRES can be assembled in vitro from two covalently unlinked transcripts, one containing the target site for p58 and the other encompassing the remaining part of the IRES fused to a reporter gene, resulting in considerable restoration of its activity. Implications of these findings for the mechanism of initiation resulting from internal entry of ribosomes are discussed. Images PMID:1656384

  13. Translation and Initial Validation of the Chinese (Cantonese) Version of Community Integration Measure for Use in Patients with Chronic Stroke

    PubMed Central

    Ng, Shamay S. M.; Ng, Gabriel Y. F.

    2014-01-01

    Objectives. To (1) translate and culturally adapt the English version Community Integration Measure into Chinese (Cantonese), (2) report the results of initial validation of the Chinese (Cantonese) version of CIM (CIM-C) including the content validity, internal consistency, test-retest reliability, and factor structure of CIM-C for use in stroke survivors in a Chinese community setting, and (3) investigate the level of community integration of stroke survivors living in Hong Kong. Design. Cross-sectional study. Setting. University-based rehabilitation centre. Participants. 62 (n = 62) subjects with chronic stroke. Methods. The CIM-C was produced after forward-backward translation, expert panel review, and pretesting. 25 (n = 25) of the same subjects were reassessed after a 1-week interval. Results. The items of the CIM-C demonstrated high internal consistency with a Cronbach's α of 0.84. The CIM-C showed good test-retest reliability with an intraclass correlation coefficient (ICC) of 0.84 (95% confidence interval, 0.64–0.93). A 3-factor structure of the CIM-C including “relationship and engagement,” “sense of knowing,” and “independent living,” was consistent with the original theoretical model. Hong Kong stroke survivors revealed a high level of community integration as measured by the CIM-C (mean (SD): 43.48 (5.79)). Conclusions. The CIM-C is a valid and reliable measure for clinical use. PMID:24995317

  14. Challenges and lessons learned from the translation of evidence-based childhood asthma interventions: a commentary on the MCAN initiative.

    PubMed

    Ohadike, Yvonne U; Malveaux, Floyd J; Lesch, Julie Kennedy

    2011-11-01

    The Merck Childhood Asthma Network (MCAN) used evidence-based interventions (EBIs) for children with asthma to design community-based programs in a wide variety of settings--with varying resource constraints and priorities--that were often determined by the program context. Although challenges were faced, lessons learned strongly suggest that adapting and implementing EBIs is feasible in a variety of settings using a multisite approach. Lessons learned during the MCAN initiative presented unique opportunities to refine best practices that proved to be important to translation of EBIs in community-based settings. The adopted best practices were based on experiential learning during different phases of the project cycle, including monitoring and evaluation, translational research, and implementing policies in local program environments. Throughout this discussion it is important to note the importance of program context in determining the effectiveness of the interventions, opportunities to scale them, their affordability, and the ability to sustain them. Lessons learned from this effort will be important not only to advance science-based approaches to manage childhood asthma but also to assist in closing the gap between intervention development (discovery) and program dissemination and implementation (delivery). PMID:22068365

  15. Poly(A) Binding Protein 1 Enhances Cap-Independent Translation Initiation of Neurovirulence Factor from Avian Herpesvirus

    PubMed Central

    Tahiri-Alaoui, Abdessamad; Zhao, Yuguang; Sadigh, Yashar; Popplestone, James; Kgosana, Lydia; Smith, Lorraine P.; Nair, Venugopal

    2014-01-01

    Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek’s disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a bona fide initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of ‘closed loop’ structure of mRNA. PMID:25503397

  16. 2011 Vascular Research Initiatives Conference: basic foundations of translational research in vascular disease.

    PubMed

    Ziegler, Kenneth R; Dardik, Alan

    2011-07-01

    The Vascular Research Initiatives Conference (VRIC) is an annual conference organized by the Society for Vascular Surgery (SVS). The 2011 VRIC was held in Chicago (IL, USA) to precede and coincide with the first day of the meeting of the Council on Arteriosclerosis, Thrombosis and Vascular Biology (ATVB) of the American Heart Association. The event is designed to present world class vascular research results, encourage collaboration between vascular surgeons and basic scientists in related disciplines, as well as to stimulate interest in research among aspiring academic vascular surgeons. The 2011 VRIC featured plenary sessions addressing peripheral arterial disease, vascular endothelium and thrombosis, aneurysms, and stem cells and tissue engineering. Recipients of the SVS partner grants with the National Institutes of Health K08 awardees presented their progress reports, and keynote addresses were given by Linda Graham and Frank LoGerfo.

  17. 2011 Vascular Research Initiatives Conference: basic foundations of translational research in vascular disease.

    PubMed

    Ziegler, Kenneth R; Dardik, Alan

    2011-07-01

    The Vascular Research Initiatives Conference (VRIC) is an annual conference organized by the Society for Vascular Surgery (SVS). The 2011 VRIC was held in Chicago (IL, USA) to precede and coincide with the first day of the meeting of the Council on Arteriosclerosis, Thrombosis and Vascular Biology (ATVB) of the American Heart Association. The event is designed to present world class vascular research results, encourage collaboration between vascular surgeons and basic scientists in related disciplines, as well as to stimulate interest in research among aspiring academic vascular surgeons. The 2011 VRIC featured plenary sessions addressing peripheral arterial disease, vascular endothelium and thrombosis, aneurysms, and stem cells and tissue engineering. Recipients of the SVS partner grants with the National Institutes of Health K08 awardees presented their progress reports, and keynote addresses were given by Linda Graham and Frank LoGerfo. PMID:21809965

  18. Haloferax volcanii, a Prokaryotic Species that Does Not Use the Shine Dalgarno Mechanism for Translation Initiation at 5′-UTRs

    PubMed Central

    Kramer, Piet; Gäbel, Katrin; Pfeiffer, Friedhelm; Soppa, Jörg

    2014-01-01

    It was long assumed that translation initiation in prokaryotes generally occurs via the so-called Shine Dalgarno (SD) mechanism. Recently, it became clear that translation initiation in prokaryotes is more heterogeneous. In the haloarchaeon Haloferax volcanii, the majority of transcripts is leaderless and most transcripts with a 5′-UTR lack a SD motif. Nevertheless, a bioinformatic analysis predicted that 20–30% of all genes are preceded by a SD motif in haloarchaea. To analyze the importance of the SD mechanism for translation initiation in haloarchaea experimentally the monocistronic sod gene was chosen, which contains a 5′-UTR with an extensive SD motif of seven nucleotides and a length of 19 nt, the average length of 5′UTRs in this organism. A translational fusion of part of the sod gene with the dhfr reporter gene was constructed. A mutant series was generated that matched the SD motif from zero to eight positions, respectively. Surprisingly, there was no correlation between the base pairing ability between transcripts and 16S rRNA and translational efficiency in vivo under several different growth conditions. Furthermore, complete replacement of the SD motif by three unrelated sequences did not reduce translational efficiency. The results indicate that H. volcanii does not make use of the SD mechanism for translation initiation in 5′-UTRs. A genome analysis revealed that while the number of SD motifs in 5′-UTRs is rare, their fraction within open reading frames is high. Possible biological functions for intragenic SD motifs are discussed, including re-initiation of translation at distal genes in operons. PMID:24733188

  19. Predictive combinatorial design of mRNA translation initiation regions for systematic optimization of gene expression levels

    PubMed Central

    Seo, Sang Woo; Yang, Jae-Seong; Cho, Han-Saem; Yang, Jina; Kim, Seong Cheol; Park, Jong Moon; Kim, Sanguk; Jung, Gyoo Yeol

    2014-01-01

    Balancing the amounts of enzymes is one of the important factors to achieve optimum performance of a designed metabolic pathway. However, the random mutagenesis approach is impractical since it requires searching an unnecessarily large number of variants and often results in searching a narrow range of expression levels which are out of optimal level. Here, we developed a predictive combinatorial design method, called UTR Library Designer, which systematically searches a large combinatorial space of expression levels. It accomplishes this by designing synthetic translation initiation region of mRNAs in a predictive way based on a thermodynamic model and genetic algorithm. Using this approach, we successfully enhanced lysine and hydrogen production in Escherichia coli. Our method significantly reduced the number of variants to be explored for covering large combinatorial space and efficiently enhanced pathway efficiency, thereby facilitating future efforts in metabolic engineering and synthetic biology. PMID:24682040

  20. Poliovirus Switches to an eIF2-Independent Mode of Translation during Infection▿

    PubMed Central

    White, James P.; Reineke, Lucas C.; Lloyd, Richard E.

    2011-01-01

    Inhibition of translation is an integral component of the innate antiviral response and is largely accomplished via interferon-activated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). To successfully infect a host, a virus must overcome this blockage by either controlling eIF2α phosphorylation or by utilizing a noncanonical mode of translation initiation. Here we show that enterovirus RNA is sensitive to translation inhibition resulting from eIF2α phosphorylation, but it becomes resistant as infection progresses. Further, we show that the cleavage of initiation factor eIF5B during enteroviral infection, along with the viral internal ribosome entry site, plays a role in mediating viral translation under conditions that are nonpermissive for host cell translation. Together, these results provide a mechanism by which enteroviruses evade the antiviral response and provide insight into a noncanonical mechanism of translation initiation. PMID:21697471

  1. Salubrinal protects cardiomyocytes against apoptosis in a rat myocardial infarction model via suppressing the dephosphorylation of eukaryotic translation initiation factor 2α.

    PubMed

    Li, Rui-Jun; He, Kun-Lun; Li, Xin; Wang, Li-Li; Liu, Chun-Lei; He, Yun-Yun

    2015-07-01

    The aim of the present study was to examine the role of eIF2α in cardiomyocyte apoptosis and evaluate the cardioprotective role of salubrinal in a rat myocardial infarction (MI) model. Rat left anterior descending coronary arteries were ligated and the classical proteins involved in the endoplasmic reticulum stress (ERS)-induced apoptotic pathway were analyzed using quantitative polymerase chain reaction and western blot analysis. Salubrinal was administered to the rats and cardiomyocyte apoptosis and infarct size were evaluated by a specific staining method. Compared with the sham surgery group, the rate of cardiomyocyte apoptosis in the MI group was increased with the development of the disease. It was also demonstrated that the mRNA and protein levels of GRP78, caspase-12, CHOP and the protein expression of p-eIF2α were increased in the MI group. Furthermore, the results showed that treatment with salubrinal can decrease cardiomyocyte apoptosis and infarct size by increasing eIF2α phosphorylation and decreasing the expression of caspase-12 and CHOP. The present study suggests that salubrinal protects against ER stress-induced rat cadiomyocyte apoptosis via suppressing the dephosphorylation of eIF2α in the ERS-associated pathway. PMID:25816071

  2. Tandem translation of E. coli initiation factor IF2 beta: purification and characterization in vitro of two active forms.

    PubMed

    Nyengaard, N R; Mortensen, K K; Lassen, S F; Hershey, J W; Sperling-Petersen, H U

    1991-12-31

    Two forms of E. coli initiation factor IF2, IF2 alpha and IF2 beta, have been known for several years. Both forms are products of the gene infB with translational initiation at codon 1 (AUG) and codon 158 (GUG) in the same reading frame. In this work we demonstrate that IF2 beta exists in two forms, IF2 beta and IF2 beta' with initiation codons 158 (GUG) and 165 (AUG) and molecular masses of 79.7 kDa and 78.8 kDa respectively. We have recently described a fast purification method for IF2 alpha, using an FPLC procedure consisting of ion-exchange liquid chromatography on Q Sepharose HP, Mono Q and Mono S. After the Mono Q step, an apparently homogeneous IF2 beta was observed when analyzed by SDS-PAGE. However the chromatography on Mono S results in the elution of two peaks containing IF2 beta. The N-terminal amino acid sequence of the two proteins identified the first peak to be IF2 beta and the second as a protein which we term IF2 beta' starting seven residues downstream at the AUG codon 165. The activity in vitro of the two purified forms of IF2 beta was tested by measuring the stimulation of binding of the initiator fMet-tRNA(fMet) to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger-RNA. In this assay no difference in activity is detected.

  3. Translation of the shallot virus X TGB3 gene depends on non-AUG initiation and leaky scanning.

    PubMed

    Lezzhov, Alexander A; Gushchin, Vladimir A; Lazareva, Ekaterina A; Vishnichenko, Valery K; Morozov, Sergey Y; Solovyev, Andrey G

    2015-10-01

    Triple gene block (TGB), a conserved gene module found in the genomes of many filamentous and rod-shaped plant viruses, encodes three proteins, TGB1, TGB2 and TGB3, required for viral cell-to-cell movement through plasmodesmata and systemic transport via the phloem. The genome of Shallot virus X, the type species of the genus Allexivirus, includes TGB1 and TGB2 genes, but contains no canonical ORF for TGB3 protein. However, a TGB3-like protein-encoding sequence lacking an AUG initiator codon has been found in the shallot virus X (ShVX) genome in a position typical for TGB3 genes. This putative TGB3 gene is conserved in all allexiviruses. Here, we carried out sequence analysis to predict possible non-AUG initiator codons in the ShVX TGB3-encoding sequence. We further used an agroinfiltration assay in Nicotiana benthamiana to confirm this prediction. Site-directed mutagenesis was used to demonstrate that the ShVX TGB3 could be translated on a bicistronic mRNA template via a leaky scanning mechanism.

  4. RatA (YfjG), An Escherichia Coli Toxin, Inhibits 70S Ribosome Association to Block Translation Initiation

    PubMed Central

    Zhang, Yonglong; Inouye, Masayori

    2011-01-01

    Summary RatA (YfjG) is a toxin encoded by the ratA-ratB (yfjG-yfjF) operon on the Escherichia coli genome. Induction of RatA led to the inhibition of protein synthesis, while DNA and RNA synthesis was not affected. The stability of mRNAs was also unchanged as judged by in vivo primer extension experiments and by Northern blotting analysis. The ribosome profile of the cells overexpressing RatA showed that 70S ribosomes as well as polysomes significantly decreased with concomitant increase of 50S and 30S subunits. The addition of purified RatA to a cell-free system inhibited the formation of 70S ribosomes even in the presence of 6 mM Mg2+. RatA was specifically associated with 50S subunits, indicating that it binds to 50S subunits to block its association with 30S subunits leading to the inhibition of formation of 70S ribosomes. However, RatA did not cause dissociation of 70S ribosomes and its anti-association activity was blocked by paromomycin, an inhibitor for IF3, an essential initiation factor, having 21% sequence homology with RatA. Here we demonstrate that RatA is a new E. coli toxin, which effectively blocks the translation initiation step. We propose that this toxin of previously unknown function be renamed as RatA (Ribosome association toxin A). PMID:21323758

  5. Translation of the shallot virus X TGB3 gene depends on non-AUG initiation and leaky scanning.

    PubMed

    Lezzhov, Alexander A; Gushchin, Vladimir A; Lazareva, Ekaterina A; Vishnichenko, Valery K; Morozov, Sergey Y; Solovyev, Andrey G

    2015-10-01

    Triple gene block (TGB), a conserved gene module found in the genomes of many filamentous and rod-shaped plant viruses, encodes three proteins, TGB1, TGB2 and TGB3, required for viral cell-to-cell movement through plasmodesmata and systemic transport via the phloem. The genome of Shallot virus X, the type species of the genus Allexivirus, includes TGB1 and TGB2 genes, but contains no canonical ORF for TGB3 protein. However, a TGB3-like protein-encoding sequence lacking an AUG initiator codon has been found in the shallot virus X (ShVX) genome in a position typical for TGB3 genes. This putative TGB3 gene is conserved in all allexiviruses. Here, we carried out sequence analysis to predict possible non-AUG initiator codons in the ShVX TGB3-encoding sequence. We further used an agroinfiltration assay in Nicotiana benthamiana to confirm this prediction. Site-directed mutagenesis was used to demonstrate that the ShVX TGB3 could be translated on a bicistronic mRNA template via a leaky scanning mechanism. PMID:26296665

  6. Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

    PubMed

    Carvajal, Felipe; Vallejos, Maricarmen; Walters, Beth; Contreras, Nataly; Hertz, Marla I; Olivares, Eduardo; Cáceres, Carlos J; Pino, Karla; Letelier, Alejandro; Thompson, Sunnie R; López-Lastra, Marcelo

    2016-07-01

    The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5' leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis. PMID:27191820

  7. Translation initiation in Saccharomyces cerevisiae mitochondria: functional interactions among mitochondrial ribosomal protein Rsm28p, initiation factor 2, methionyl-tRNA-formyltransferase and novel protein Rmd9p.

    PubMed

    Williams, Elizabeth H; Butler, Christine A; Bonnefoy, Nathalie; Fox, Thomas D

    2007-03-01

    Rsm28p is a dispensable component of the mitochondrial ribosomal small subunit in Saccharomyces cerevisiae that is not related to known proteins found in bacteria. It was identified as a dominant suppressor of certain mitochondrial mutations that reduced translation of the COX2 mRNA. To explore further the function of Rsm28p, we isolated mutations in other genes that caused a synthetic respiratory defective phenotype together with rsm28Delta. These mutations identified three nuclear genes: IFM1, which encodes the mitochondrial translation initiation factor 2 (IF2); FMT1, which encodes the methionyl-tRNA-formyltransferase; and RMD9, a gene of unknown function. The observed genetic interactions strongly suggest that the ribosomal protein Rsm28p and Ifm1p (IF2) have similar and partially overlapping functions in yeast mitochondrial translation initiation. Rmd9p, bearing a TAP-tag, was localized to mitochondria and exhibited roughly equal distribution in soluble and membrane-bound fractions. A small fraction of the Rmd9-TAP sedimented together with presumed monosomes, but not with either individual ribosomal subunit. Thus, Rmd9 is not a ribosomal protein, but may be a novel factor associated with initiating monosomes. The poorly respiring rsm28Delta, rmd9-V363I double mutant did not have a strong translation-defective phenotype, suggesting that Rmd9p may function upstream of translation initiation, perhaps at the level of localization of mitochondrially coded mRNAs.

  8. The isoforms generated by alternative translation initiation adopt similar conformation in the selectivity filter in TREK-2.

    PubMed

    Zhuo, Ren-Gong; Peng, Peng; Liu, Xiao-Yan; Zhang, Shu-Zhuo; Xu, Jiang-Ping; Zheng, Jian-Quan; Wei, Xiao-Li; Ma, Xiao-Yun

    2015-12-01

    TREK-2 (TWIK-related K(+) channel-2), a member of two-pore domain potassium (K2P) channel family, tunes cellular excitability via conducting leak or background currents. In TREK-2, the isoforms generated by alternative translation initiation (ATI) mechanism exhibit large divergence in unitary conductance, but similar in selectivity to K(+). Up to now, the structural basis for this similarity in ion selectivity is unknown. Here, we report that externally applied Ba(2+) inhibits the currents of TREK-2 in a concentration- and time-dependent manner. The blocking effect is blunted by elevated extracellular K(+) or mutation of S4 K(+) binding site, which suggests that the inhibitory mechanism of Ba(2+) is due to its competitive docking properties within the selectivity filter (SF). Next, we demonstrate that all the ATI isoforms exhibit analogous behaviors upon the application of Ba(2+) and alteration of extracellular pH (pHo), which acts on the outer position of the SF. These results strongly support the notion that all the ATI isoforms of TREK-2 possess resembled SF conformation in S4 site and the position defined by pHo, which implicates that neither the role of N-terminus (Nt) nor the unitary conductance is associated with SF conformation. Our findings might help to understand the detail gating mechanism of TREK-2 and K2P channels. PMID:26271386

  9. uORFdb—a comprehensive literature database on eukaryotic uORF biology

    PubMed Central

    Wethmar, Klaus; Barbosa-Silva, Adriano; Andrade-Navarro, Miguel A.; Leutz, Achim

    2014-01-01

    Approximately half of all human transcripts contain at least one upstream translational initiation site that precedes the main coding sequence (CDS) and gives rise to an upstream open reading frame (uORF). We generated uORFdb, publicly available at http://cbdm.mdc-berlin.de/tools/uorfdb, to serve as a comprehensive literature database on eukaryotic uORF biology. Upstream ORFs affect downstream translation by interfering with the unrestrained progression of ribosomes across the transcript leader sequence. Although the first uORF-related translational activity was observed >30 years ago, and an increasing number of studies link defective uORF-mediated translational control to the development of human diseases, the features that determine uORF-mediated regulation of downstream translation are not well understood. The uORFdb was manually curated from all uORF-related literature listed at the PubMed database. It categorizes individual publications by a variety of denominators including taxon, gene and type of study. Furthermore, the database can be filtered for multiple structural and functional uORF-related properties to allow convenient and targeted access to the complex field of eukaryotic uORF biology. PMID:24163100

  10. Post-transcriptional Boolean computation by combining aptazymes controlling mRNA translation initiation and tRNA activation.

    PubMed

    Klauser, Benedikt; Saragliadis, Athanasios; Ausländer, Simon; Wieland, Markus; Berthold, Michael R; Hartig, Jörg S

    2012-09-01

    In cellular systems environmental and metabolic signals are integrated for the conditional control of gene expression. On the other hand, artificial manipulation of gene expression is of high interest for metabolic and genetic engineering. Especially the reprogramming of gene expression patterns to orchestrate cellular responses in a predictable fashion is considered to be of great importance. Here we introduce a highly modular RNA-based system for performing Boolean logic computation at a post-transcriptional level in Escherichia coli. We have previously shown that artificial riboswitches can be constructed by utilizing ligand-dependent Hammerhead ribozymes (aptazymes). Employing RNA self-cleavage as the expression platform-mechanism of an artificial riboswitch has the advantage that it can be applied to control several classes of RNAs such as mRNAs, tRNAs, and rRNAs. Due to the highly modular and orthogonal nature of these switches it is possible to combine aptazyme regulation of activating a suppressor tRNA with the regulation of mRNA translation initiation. The different RNA classes can be controlled individually by using distinct aptamers for individual RNA switches. Boolean logic devices are assembled by combining such switches in order to act on the expression of a single mRNA. In order to demonstrate the high modularity, a series of two-input Boolean logic operators were constructed. For this purpose, we expanded our aptazyme toolbox with switches comprising novel behaviours with respect to the small molecule triggers thiamine pyrophosphate (TPP) and theophylline. Then, individual switches were combined to yield AND, NOR, and ANDNOT gates. This study demonstrates that post-transcriptional aptazyme-based switches represent versatile tools for engineering advanced genetic devices and circuits without the need for regulatory protein cofactors. PMID:22777205

  11. Structural and functional similarities between the central eukaryotic initiation factor (eIF)4A-binding domain of mammalian eIF4G and the eIF4A-binding domain of yeast eIF4G.

    PubMed Central

    Dominguez, D; Kislig, E; Altmann, M; Trachsel, H

    2001-01-01

    The translation eukaryotic initiation factor (eIF)4G of the yeast Saccharomyces cerevisiae interacts with the RNA helicase eIF4A (a member of the DEAD-box protein family; where DEAD corresponds to Asp-Glu-Ala-Asp) through a C-terminal domain in eIF4G (amino acids 542-883). Mammalian eIF4G has two interaction domains for eIF4A, a central domain and a domain close to the C-terminus. This raises the question of whether eIF4A binding to eIF4G is conserved between yeast and mammalian cells or whether it is different. We isolated eIF4G1 mutants defective in eIF4A binding and showed that these mutants are strongly impaired in translation and growth. Extracts from mutants displaying a temperature-sensitive phenotype for growth have low in vitro translation activity, which can be restored by addition of the purified eIF4G1-eIF4E complex, but not by eIF4E alone. Analysis of mutant eIF4G(542-883) proteins defective in eIF4A binding shows that the interaction of yeast eIF4A with eIF4G1 depends on amino acid motifs that are conserved between the yeast eIF4A-binding site and the central eIF4A-binding domain of mammalian eIF4G. We show that mammalian eIF4A binds tightly to yeast eIF4G1 and, furthermore, that mutant yeast eIF4G(542-883) proteins, which do not bind yeast eIF4A, do not interact with mammalian eIF4A. Despite the conservation of the eIF4A-binding site in eIF4G and the strong sequence conservation between yeast and mammalian eIF4A (66% identity; 82% similarity at the amino acid level) mammalian eIF4A does not substitute for the yeast factor in vivo and is not functional in a yeast in vitro translation system. PMID:11256967

  12. Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA–like structural element in the ribosomal decoding center

    PubMed Central

    Koh, Cha San; Brilot, Axel F.; Grigorieff, Nikolaus; Korostelev, Andrei A.

    2014-01-01

    In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a cap-independent manner. We report two structures of the ribosome-bound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA–like structure in the A site, whereas the 40S P site remains unoccupied during this initial step. PMID:24927574

  13. The structure and function of the eukaryotic ribosome.

    PubMed

    Wilson, Daniel N; Doudna Cate, Jamie H

    2012-05-01

    Structures of the bacterial ribosome have provided a framework for understanding universal mechanisms of protein synthesis. However, the eukaryotic ribosome is much larger than it is in bacteria, and its activity is fundamentally different in many key ways. Recent cryo-electron microscopy reconstructions and X-ray crystal structures of eukaryotic ribosomes and ribosomal subunits now provide an unprecedented opportunity to explore mechanisms of eukaryotic translation and its regulation in atomic detail. This review describes the X-ray crystal structures of the Tetrahymena thermophila 40S and 60S subunits and the Saccharomyces cerevisiae 80S ribosome, as well as cryo-electron microscopy reconstructions of translating yeast and plant 80S ribosomes. Mechanistic questions about translation in eukaryotes that will require additional structural insights to be resolved are also presented.

  14. AtLa1 protein initiates IRES-dependent translation of WUSCHEL mRNA and regulates the stem cell homeostasis of Arabidopsis in response to environmental hazards.

    PubMed

    Cui, Yuchao; Rao, Shaofei; Chang, Beibei; Wang, Xiaoshuang; Zhang, Kaidian; Hou, Xueliang; Zhu, Xueyi; Wu, Haijun; Tian, Zhaoxia; Zhao, Zhong; Yang, Chengwei; Huang, Tao

    2015-10-01

    Plant stem cells are hypersensitive to environmental hazards throughout their life cycle, but the mechanism by which plants safeguard stem cell homeostasis in response to environmental hazards is largely unknown. The homeodomain transcription factor WUSCHEL (WUS) protein maintains the stem cell pool in the shoot apical meristem of Arabidopsis. Here, we demonstrate that the translation of WUS mRNA is directed by an internal ribosomal entry site (IRES) located in the 5'-untranslated region. The AtLa1 protein, an RNA-binding factor, binds to the 5'-untranslated region and initiates the IRES-dependent translation of WUS mRNA. Knockdown of AtLa1 expression represses the WUS IRES-dependent translation and leads to the arrest of growth and development. The AtLa1 protein is mainly located in the nucleoplasm. However, environmental hazards promote the nuclear-to-cytoplasmic translocation of the AtLa1 protein, which further enhances the IRES-dependent translation of WUS mRNA. Genetic evidence indicates that the WUS protein increases the tolerance of the shoot apical meristem to environmental hazards. Based on these results, we conclude that the stem cell niche in Arabidopsis copes with environmental hazards by enhancing the IRES-dependent translation of WUS mRNA under the control of the AtLa1 protein.

  15. Differential contribution of the m7G-cap to the 5' end-dependent translation initiation of mammalian mRNAs.

    PubMed

    Andreev, Dmitri E; Dmitriev, Sergey E; Terenin, Ilya M; Prassolov, Vladimir S; Merrick, William C; Shatsky, Ivan N

    2009-10-01

    Many mammalian mRNAs possess long 5' UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5' UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5' UTRs with so-called 'cellular IRESes' demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5' UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5' UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.

  16. Differential contribution of the m7G-cap to the 5′ end-dependent translation initiation of mammalian mRNAs

    PubMed Central

    Andreev, Dmitri E.; Dmitriev, Sergey E.; Terenin, Ilya M.; Prassolov, Vladimir S.; Merrick, William C.; Shatsky, Ivan N.

    2009-01-01

    Many mammalian mRNAs possess long 5′ UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5′ UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5′ UTRs with so-called ‘cellular IRESes’ demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5′ UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5′ UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated. PMID:19696074

  17. Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding

    PubMed Central

    Fabbretti, Attilio; Schedlbauer, Andreas; Brandi, Letizia; Kaminishi, Tatsuya; Giuliodori, Anna Maria; Garofalo, Raffaella; Ochoa-Lizarralde, Borja; Takemoto, Chie; Yokoyama, Shigeyuki; Connell, Sean R.; Gualerzi, Claudio O.

    2016-01-01

    In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon–anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state. PMID:27071098

  18. Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding.

    PubMed

    Fabbretti, Attilio; Schedlbauer, Andreas; Brandi, Letizia; Kaminishi, Tatsuya; Giuliodori, Anna Maria; Garofalo, Raffaella; Ochoa-Lizarralde, Borja; Takemoto, Chie; Yokoyama, Shigeyuki; Connell, Sean R; Gualerzi, Claudio O; Fucini, Paola

    2016-04-19

    In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state. PMID:27071098

  19. Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations.

    PubMed

    Peixeiro, Isabel; Inácio, Ângela; Barbosa, Cristina; Silva, Ana Luísa; Liebhaber, Stephen A; Romão, Luísa

    2012-02-01

    Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.

  20. Emerging Therapeutics Targeting mRNA Translation

    PubMed Central

    Malina, Abba; Mills, John R.; Pelletier, Jerry

    2012-01-01

    A defining feature of many cancers is deregulated translational control. Typically, this occurs at the level of recruitment of the 40S ribosomes to the 5′-cap of cellular messenger RNAs (mRNAs), the rate-limiting step of protein synthesis, which is controlled by the heterotrimeric eukaryotic initiation complex eIF4F. Thus, eIF4F in particular, and translation initiation in general, represent an exploitable vulnerability and unique opportunity for therapeutic intervention in many transformed cells. In this article, we discuss the development, mode of action and biological activity of a number of small-molecule inhibitors that interrupt PI3K/mTOR signaling control of eIF4F assembly, as well as compounds that more directly block eIF4F activity. PMID:22474009

  1. Translational control by eIF2α kinases in long-lasting synaptic plasticity and long-term memory.

    PubMed

    Trinh, Mimi A; Klann, Eric

    2013-10-01

    Although the requirement for new protein synthesis in synaptic plasticity and memory has been well established, recent genetic, molecular, electrophysiological, and pharmacological studies have broadened our understanding of the translational control mechanisms that are involved in these processes. One of the critical translational control points mediating general and gene-specific translation depends on the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) by four regulatory kinases. Here, we review the literature highlighting the important role for proper translational control via regulation of eIF2α phosphorylation by its kinases in long-lasting synaptic plasticity and long-term memory.

  2. Acidocalcisomes of eukaryotes.

    PubMed

    Docampo, Roberto; Huang, Guozhong

    2016-08-01

    Acidocalcisomes are organelles rich in polyphosphate and cations and acidified by proton pumps. Although they have also been described in prokaryotes they have been better characterized in unicellular and multicellular eukaryotes. Eukaryotic acidocalcisomes belong to the group of lysosome-related organelles. They have a variety of functions, from the storage of cations and phosphorus to calcium signaling, autophagy, osmoregulation, blood coagulation, and inflammation. Acidocalcisomes of several unicellular eukaryotes possess a variety of transporters, channels and pumps implying a large energetic requirement for their maintenance and suggesting other important functions waiting to be discovered. PMID:27125677

  3. Pharmacogenetic diversification by alternative translation initiation: background channels to the fore: Commentary on Kisselbach et al., Br J Pharmacol 171: 5182–5194

    PubMed Central

    Abbott, G W

    2015-01-01

    Linked Article This article is a Commentary on Kisselbach J, Seyler C, Schweizer PA, Gerstberger R, Becker R, Katus HA and Thomas D (2014). Modulation of K2P2.1 and K2P10.1 K+ channel sensitivity to carvedilol by alternative mRNA translation initiation. Br J Pharmacol 171: 5182–5194. doi: 10.1111/bph.12596 PMID:24528336

  4. The tree of eukaryotes.

    PubMed

    Keeling, Patrick J; Burger, Gertraud; Durnford, Dion G; Lang, B Franz; Lee, Robert W; Pearlman, Ronald E; Roger, Andrew J; Gray, Michael W

    2005-12-01

    Recent advances in resolving the tree of eukaryotes are converging on a model composed of a few large hypothetical 'supergroups', each comprising a diversity of primarily microbial eukaryotes (protists, or protozoa and algae). The process of resolving the tree involves the synthesis of many kinds of data, including single-gene trees, multigene analyses, and other kinds of molecular and structural characters. Here, we review the recent progress in assembling the tree of eukaryotes, describing the major evidence for each supergroup, and where gaps in our knowledge remain. We also consider other factors emerging from phylogenetic analyses and comparative genomics, in particular lateral gene transfer, and whether such factors confound our understanding of the eukaryotic tree.

  5. The ribosomal protein Asc1/RACK1 is required for efficient translation of short mRNAs

    PubMed Central

    Thompson, Mary K; Rojas-Duran, Maria F; Gangaramani, Paritosh; Gilbert, Wendy V

    2016-01-01

    Translation is a core cellular process carried out by a highly conserved macromolecular machine, the ribosome. There has been remarkable evolutionary adaptation of this machine through the addition of eukaryote-specific ribosomal proteins whose individual effects on ribosome function are largely unknown. Here we show that eukaryote-specific Asc1/RACK1 is required for efficient translation of mRNAs with short open reading frames that show greater than average translational efficiency in diverse eukaryotes. ASC1 mutants in S. cerevisiae display compromised translation of specific functional groups, including cytoplasmic and mitochondrial ribosomal proteins, and display cellular phenotypes consistent with their gene-specific translation defects. Asc1-sensitive mRNAs are preferentially associated with the translational ‘closed loop’ complex comprised of eIF4E, eIF4G, and Pab1, and depletion of eIF4G mimics the translational defects of ASC1 mutants. Together our results reveal a role for Asc1/RACK1 in a length-dependent initiation mechanism optimized for efficient translation of genes with important housekeeping functions. DOI: http://dx.doi.org/10.7554/eLife.11154.001 PMID:27117520

  6. Features of a leader peptide coding region that regulate translation initiation for the anti-TRAP protein of B. subtilis.

    PubMed

    Chen, Guangnan; Yanofsky, Charles

    2004-03-12

    The rtpA gene of Bacillus subtilis encodes the Anti-TRAP protein, AT. AT can bind and inhibit the TRAP regulatory protein, preventing TRAP from promoting transcription termination in the trpEDCFBA operon leader region. AT synthesis is upregulated transcriptionally and translationally in response to the accumulation of uncharged tRNA(Trp). Here we analyze AT's translational regulation by rtpLP, a 10 residue leader peptide coding region located immediately preceding the rtpA Shine-Dalgarno sequence. Our findings suggest that, whenever the charged tRNA(Trp) level is sufficient to allow the ribosome translating rtpLP to reach its stop codon, it blocks the adjacent rtpA Shine-Dalgarno sequence, inhibiting AT synthesis. However, when there is a charged tRNA(Trp) deficiency, the translating ribosome presumably stalls at one of three adjacent rtpLP Trp codons. This stalling exposes the rtpA Shine-Dalgarno sequence, permitting AT synthesis. RNA-RNA pairing may also influence AT synthesis. Production of AT would inactivate TRAP, thereby increasing trp operon expression. PMID:15023340

  7. Overexpression of eIF5 or its protein mimic 5MP perturbs eIF2 function and induces ATF4 translation through delayed re-initiation

    PubMed Central

    Kozel, Caitlin; Thompson, Brytteny; Hustak, Samantha; Moore, Chelsea; Nakashima, Akio; Singh, Chingakham Ranjit; Reid, Megan; Cox, Christian; Papadopoulos, Evangelos; Luna, Rafael E.; Anderson, Abbey; Tagami, Hideaki; Hiraishi, Hiroyuki; Slone, Emily Archer; Yoshino, Ken-ichi; Asano, Masayo; Gillaspie, Sarah; Nietfeld, Jerome; Perchellet, Jean-Pierre; Rothenburg, Stefan; Masai, Hisao; Wagner, Gerhard; Beeser, Alexander; Kikkawa, Ushio; Fleming, Sherry D.; Asano, Katsura

    2016-01-01

    ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila. The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions. PMID:27325740

  8. Structural disorder in eukaryotes.

    PubMed

    Pancsa, Rita; Tompa, Peter

    2012-01-01

    Based on early bioinformatic studies on a handful of species, the frequency of structural disorder of proteins is generally thought to be much higher in eukaryotes than in prokaryotes. To refine this view, we present here a comparative prediction study and analysis of 194 fully described eukaryotic proteomes and 87 reference prokaryotes for structural disorder. We found that structural disorder does distinguish eukaryotes from prokaryotes, but its frequency spans a very wide range in the two superkingdoms that largely overlap. The number of disordered binding regions and different Pfam domain types also contribute to distinguish eukaryotes from prokaryotes. Unexpectedly, the highest levels--and highest variability--of predicted disorder is found in protists, i.e. single-celled eukaryotes, often surpassing more complex eukaryote organisms, plants and animals. This trend contrasts with that of the number of domain types, which increases rather monotonously toward more complex organisms. The level of structural disorder appears to be strongly correlated with lifestyle, because some obligate intracellular parasites and endosymbionts have the lowest levels, whereas host-changing parasites have the highest level of predicted disorder. We conclude that protists have been the evolutionary hot-bed of experimentation with structural disorder, in a period when structural disorder was actively invented and the major functional classes of disordered proteins established.

  9. Casein kinase 1ε promotes cell proliferation by regulating mRNA translation.

    PubMed

    Shin, Sejeong; Wolgamott, Laura; Roux, Philippe P; Yoon, Sang-Oh

    2014-01-01

    Deregulation of translation initiation factors contributes to many pathogenic conditions, including cancer. Here, we report the definition of a novel regulatory pathway for translational initiation with possible therapeutic import in cancer. Specifically, we found that casein kinase 1ε (CK1ε) is highly expressed in breast tumors and plays a critical role in cancer cell proliferation by controlling mRNA translation. Eukaryotic translation initiation factor eIF4E, an essential component of the translation initiation complex eIF4F, is downregulated by binding the negative-acting factor 4E-BP1. We found that genetic or pharmacologic inhibition of CK1ε attenuated 4E-BP1 phosphorylation, thereby increasing 4E-BP1 binding to eIF4E and inhibiting mRNA translation. Mechanistic investigations showed that CK1ε interacted with and phosphorylated 4E-BP1 at two novel sites T41 and T50, which were essential for 4E-BP1 inactivation along with increased mRNA translation and cell proliferation. In summary, our work identified CK1ε as a pivotal regulator of mRNA translation and cell proliferation that acts by inhibiting 4E-BP1 function. As CK1ε is highly expressed in breast tumors, these findings offer an initial rationale to explore CK1ε blockade as a therapeutic strategy to treat cancers driven by deregulated mRNA translation.

  10. The RNA Helicase eIF4A Is Required for Sapovirus Translation

    PubMed Central

    Hosmillo, Myra; Sweeney, Trevor R.; Chaudhry, Yasmin; Leen, Eoin; Curry, Stephen

    2016-01-01

    The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5′ untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5′ UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation. PMID:26937032

  11. An analysis of vertebrate mRNA sequences: intimations of translational control

    PubMed Central

    1991-01-01

    Five structural features in mRNAs have been found to contribute to the fidelity and efficiency of initiation by eukaryotic ribosomes. Scrutiny of vertebrate cDNA sequences in light of these criteria reveals a set of transcripts--encoding oncoproteins, growth factors, transcription factors, and other regulatory proteins--that seem designed to be translated poorly. Thus, throttling at the level of translation may be a critical component of gene regulation in vertebrates. An alternative interpretation is that some (perhaps many) cDNAs with encumbered 5' noncoding sequences represent mRNA precursors, which would imply extensive regulation at a posttranscriptional step that precedes translation. PMID:1955461

  12. Translational control at the synapse: role of RNA regulators

    PubMed Central

    Iacoangeli, Anna; Tiedge, Henri

    2012-01-01

    Translational control of gene expression is instrumental in the regulation of eukaryotic cellular form and function. Neurons in particular rely on this form of control as their numerous synaptic connections need to be independently modulated in an input-specific manner. Brain cytoplasmic (BC) RNAs implement translational control at neuronal synapses. BC RNAs regulate protein synthesis by interacting with eIF4 translation initiation factors. Recent evidence suggests that such regulation is required to control synaptic strength, and that dysregulation of local protein synthesis precipitates neuronal hyperexcitability and a propensity for epileptogenic responses. A similar phenotype results from lack of fragile X mental retardation protein (FMRP), indicating that BC RNAs and FMRP use overlapping and convergent modes of action in neuronal translational regulation. PMID:23218750

  13. Shwachman–Bodian–Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs

    PubMed Central

    In, Kyungmin; Zaini, Mohamad A.; Müller, Christine; Warren, Alan J.; von Lindern, Marieke; Calkhoven, Cornelis F.

    2016-01-01

    Mutations in the Shwachman–Bodian–Diamond Syndrome (SBDS) gene cause Shwachman–Diamond Syndrome (SDS), a rare congenital disease characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of the C/EBPα and -β mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPβ-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5′ untranslated regions (5′ UTRs) of both mRNAs. Furthermore, we show that as a consequence of the C/EBPα and -β deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation by loss of SBDS function may contribute to the development of the SDS phenotype. PMID:26762974

  14. Shwachman-Bodian-Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs.

    PubMed

    In, Kyungmin; Zaini, Mohamad A; Müller, Christine; Warren, Alan J; von Lindern, Marieke; Calkhoven, Cornelis F

    2016-05-19

    Mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene cause Shwachman-Diamond Syndrome (SDS), a rare congenital disease characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of the C/EBPα and -β mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPβ-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5' untranslated regions (5' UTRs) of both mRNAs. Furthermore, we show that as a consequence of the C/EBPα and -β deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation by loss of SBDS function may contribute to the development of the SDS phenotype. PMID:26762974

  15. Topology of mRNA chain in isolated eukaryotic double-row polyribosomes.

    PubMed

    Afonina, Zh A; Myasnikov, A G; Khabibullina, N F; Belorusova, A Yu; Menetret, J-F; Vasiliev, V D; Klaholz, B P; Shirokov, V A; Spirin, A S

    2013-05-01

    In the process of protein synthesis, the translating ribosomes of eukaryotic cells form polyribosomes that are found to be multiplex functional complexes possessing elements of ordered spatial organization. As revealed by a number of electron microscopy studies, the predominant visible configurations of the eukaryotic polyribosomes are circles (circular polyribosomes) and two-stranded formations (so-called double-row polyribosomes). The "long" (i.e. heavy loaded) polyribosomes are usually represented by double-row structures, which can be interpreted as either topologically circular ("collapsed rings"), or topologically linear (zigzags or helices). In the present work we have analyzed the mRNA path within the eukaryotic polyribosomes, isolated from a wheat germ cell-free translation system, by integrating two approaches: the visualization of mRNA ends in polyribosomes by marking them with gold nanoparticles (3'-end) and initiating 40S subunits (5'-end), as well as by the cryoelectron tomography. Examination of the location of the mRNA markers in polyribosomes and mutual orientation of ribosomes in them has shown that the double-row polyribosomes of the same sample can have both circular and linear arrangements of their mRNA.

  16. Translational Control during Calicivirus Infection

    PubMed Central

    Royall, Elizabeth; Locker, Nicolas

    2016-01-01

    In this review, we provide an overview of the strategies developed by caliciviruses to subvert or regulate the host protein synthesis machinery to their advantage. As intracellular obligate parasites, viruses strictly depend on the host cell resources to produce viral proteins. Thus, many viruses have developed strategies that regulate the function of the host protein synthesis machinery, often leading to preferential translation of viral mRNAs. Caliciviruses lack a 5′ cap structure but instead have a virus-encoded VPg protein covalently linked to the 5′ end of their mRNAs. Furthermore, they encode 2–4 open reading frames within their genomic and subgenomic RNAs. Therefore, they use alternative mechanisms for translation whereby VPg interacts with eukaryotic initiation factors (eIFs) to act as a proteinaceous cap-substitute, and some structural proteins are produced by reinitiation of translation events. This review discusses our understanding of these key mechanisms during caliciviruses infection as well as recent insights into the global regulation of eIF4E activity. PMID:27104553

  17. Translational Control during Calicivirus Infection.

    PubMed

    Royall, Elizabeth; Locker, Nicolas

    2016-04-01

    In this review, we provide an overview of the strategies developed by caliciviruses to subvert or regulate the host protein synthesis machinery to their advantage. As intracellular obligate parasites, viruses strictly depend on the host cell resources to produce viral proteins. Thus, many viruses have developed strategies that regulate the function of the host protein synthesis machinery, often leading to preferential translation of viral mRNAs. Caliciviruses lack a 5' cap structure but instead have a virus-encoded VPg protein covalently linked to the 5' end of their mRNAs. Furthermore, they encode 2-4 open reading frames within their genomic and subgenomic RNAs. Therefore, they use alternative mechanisms for translation whereby VPg interacts with eukaryotic initiation factors (eIFs) to act as a proteinaceous cap-substitute, and some structural proteins are produced by reinitiation of translation events. This review discusses our understanding of these key mechanisms during caliciviruses infection as well as recent insights into the global regulation of eIF4E activity. PMID:27104553

  18. Regulation of transcription attenuation and translation initiation by allosteric control of an RNA-binding protein: the Bacillus subtilis TRAP protein.

    PubMed

    Babitzke, Paul

    2004-04-01

    Tryptophan allosterically controls the 11-subunit trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis. When activated by tryptophan, TRAP binds to multiple trinucleotide repeats in target transcripts. TRAP is responsible for the decision to terminate transcription in the leader region of the trpEDCFBA operon or to allow transcription to proceed into the structural genes. TRAP also regulates translation of trpE by promoting formation of an RNA structure that prevents ribosome binding. In addition, bound TRAP regulates translation initiation of pabA, trpP and ycbK by directly blocking ribosome binding. The anti-TRAP protein inhibits TRAP activity by competing with RNA for the RNA binding surface of TRAP. PMID:15063849

  19. Promotion of viral internal ribosomal entry site-mediated translation under amino acid starvation.

    PubMed

    Licursi, Maria; Komatsu, Yumiko; Pongnopparat, Theerawat; Hirasawa, Kensuke

    2012-05-01

    Cap-dependent and internal ribosomal entry site (IRES)-mediated translation are regulated differently within cells. Viral IRES-mediated translation often remains active when cellular cap-dependent translation is severely impaired under cellular stresses induced by virus infection. To investigate how cellular stresses influence the efficiency of viral IRES-mediated translation, we used a bicistronic luciferase reporter construct harbouring IRES elements from the following viruses: encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV) or human rhinovirus (HRV). NIH3T3 cells transfected with these bicistronic reporter constructs were subjected to different cellular stresses. Increased translation initiation was only observed under amino acid starvation when EMCV or FMDV IRES elements were present. To identify cellular mechanisms that promoted viral IRES-mediated translation, we tested the involvement of eukaryotic initiation factor 4E-binding protein (4E-BP), general control non-depressed 2 (GCN2) and eukaryotic initiation factor 2B (eIF2B), as these are known to be modulated under amino acid starvation. Knockdown of 4E-BP1 impaired the promotion of EMCV and FMDV IRES-mediated translation under amino acid starvation, whereas GCN2 and eIF2B were not involved. To further investigate how 4E-BP1 regulates translation initiated by EMCV and FMDV IRES elements, we used a phosphoinositide kinase-3 inhibitor (LY294002), an mTOR inhibitor (Torin1) or leucine starvation to mimic 4E-BP1 dephosphorylation induced by amino acid starvation. 4E-BP1 dephosphorylation induced by the treatments was not sufficient to promote viral IRES-mediated translation. These results suggest that 4E-BP1 regulates EMCV and FMDV IRES-mediated translation under amino acid starvation, but not via its dephosphorylation. PMID:22302880

  20. Bean common mosaic virus Isolate Exhibits a Novel Pathogenicity Profile in Common Bean, Overcoming the bc-3 Resistance Allele Coding for the Mutated eIF4E Translation Initiation Factor.

    PubMed

    Feng, Xue; Myers, James R; Karasev, Alexander V

    2015-11-01

    Resistance against Bean common mosaic virus (BCMV) in Phaseolus vulgaris is governed by six recessive resistance alleles at four loci. One of these alleles, bc-3, is able to protect P. vulgaris against all BCMV strains and against other potyviruses; bc-3 was identified as the eIF4E allele carrying mutated eukaryotic translation initiation factor gene. Here, we characterized a novel BCMV isolate 1755a that was able to overcome bc-2 and bc-3 alleles in common bean. Thus, it displayed a novel pattern of interactions with resistance genes in P. vulgaris, and was assigned to a new pathogroup, PG-VIII. The IVT7214 cultivar supporting the replication of BCMV-1755a was found to have the intact homozygous bc-3 cleaved amplified polymorphic sequences marker and corresponding mutations in the eIF4E allele that confer resistance to BCMV isolates from all other pathogroups as well as to other potyviruses. The VPg protein of 1755a had seven amino acid substitutions relative to VPgs of other BCMV isolates unable to overcome bc-3. The 1755a genome was found to be a recombinant between NL1, US1 (both PG-I), and a yet unknown BCMV strain. Analysis of the recombination patterns in the genomes of NL1 and US1 (PG-I), NY15P (PG-V), US10 and RU1-OR (PG-VII), and 1755a (PG-VIII), indicated that P1/HC-Pro cistrons of BCMV strains may interact with most resistance genes. This is the first report of a BCMV isolate able to overcome the bc-3 resistance allele, suggesting that the virus has evolved mechanisms to overcome multiple resistance genes available in common bean. PMID:26196181

  1. Mimivirus gene promoters exhibit an unprecedented conservation among all eukaryotes.

    PubMed

    Suhre, Karsten; Audic, Stéphane; Claverie, Jean-Michel

    2005-10-11

    The initial analysis of the recently sequenced genome of Acanthamoeba polyphaga Mimivirus, the largest known double-stranded DNA virus, predicted a proteome of size and complexity more akin to small parasitic bacteria than to other nucleocytoplasmic large DNA viruses and identified numerous functions never before described in a virus. It has been proposed that the Mimivirus lineage could have emerged before the individualization of cellular organisms from the three domains of life. An exhaustive in silico analysis of the noncoding moiety of all known viral genomes now uncovers the unprecedented perfect conservation of an AAAATTGA motif in close to 50% of the Mimivirus genes. This motif preferentially occurs in genes transcribed from the predicted leading strand and is associated with functions required early in the viral infectious cycle, such as transcription and protein translation. A comparison with the known promoter of unicellular eukaryotes, amoebal protists in particular, strongly suggests that the AAAATTGA motif is the structural equivalent of the TATA box core promoter element. This element is specific to the Mimivirus lineage and may correspond to an ancestral promoter structure predating the radiation of the eukaryotic kingdoms. This unprecedented conservation of core promoter regions is another exceptional feature of Mimivirus that again raises the question of its evolutionary origin.

  2. Organelle fission in eukaryotes.

    PubMed

    Osteryoung, K W

    2001-12-01

    The cellular machineries that power chloroplast and mitochondrial division in eukaryotes carry out the topologically challenging job of constricting and severing these double-membraned organelles. Consistent with their endosymbiotic origins, mitochondria in protists and chloroplasts in photosynthetic eukaryotes have evolved organelle-targeted forms of FtsZ, the prokaryotic ancestor of tubulin, as key components of their fission complexes. In fungi, animals and plants, mitochondria no longer utilize FtsZ for division, but several mitochondrial division proteins that localize to the outer membrane and intermembrane space, including two related to the filament-forming dynamins, have been identified in yeast and animals. Although the reactions that mediate organelle division are not yet understood, recent progress in uncovering the constituents of the organelle division machineries promises rapid advancement in our understanding of the biochemical mechanisms underlying the distinct but related processes of chloroplast and mitochondrial division in eukaryotes.

  3. The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with eIF1 and eIF2β.

    PubMed

    Luna, Rafael E; Arthanari, Haribabu; Hiraishi, Hiroyuki; Nanda, Jagpreet; Martin-Marcos, Pilar; Markus, Michelle A; Akabayov, Barak; Milbradt, Alexander G; Luna, Lunet E; Seo, Hee-Chan; Hyberts, Sven G; Fahmy, Amr; Reibarkh, Mikhail; Miles, David; Hagner, Patrick R; O'Day, Elizabeth M; Yi, Tingfang; Marintchev, Assen; Hinnebusch, Alan G; Lorsch, Jon R; Asano, Katsura; Wagner, Gerhard

    2012-06-28

    Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2β. This study provides mechanistic insight into the role of eIF5-CTD's dynamic interplay with eIF1 and eIF2β in switching PICs from an open to a closed state at start codons.

  4. Molecular phylogeny of eukaryotes.

    PubMed

    Schlegel, M

    1994-09-01

    Comparisons of ribosomal RNAs and various protein coding genes have contributed to a new view of eukaryote phylogeny. Analyses of paralogous protein coding genes suggest that archaebacteria and eukaryotes are sistergroups. Sequence diversity of small subunit rRNAs in protists by far exceeds that of any multicellular or prokaryote taxon. Remarkably, a group of taxa that lack mitochondria first branches off in the small subunit rRNA tree. The later radiations are formed by a series of clades that were once thought to be more ancestral. Furthermore, tracing of the evolutionary origin of secondary endobiontic events is now possible with sequence comparisons.

  5. Eukaryotic Cell Panorama

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2011-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. This report describes the scientific results that support an illustration of a eukaryotic cell, enlarged by one million times to show the distribution and arrangement of macromolecules. The panoramic cross section includes eight panels that extend…

  6. Prokaryote and eukaryote evolvability.

    PubMed

    Poole, Anthony M; Phillips, Matthew J; Penny, David

    2003-05-01

    The concept of evolvability covers a broad spectrum of, often contradictory, ideas. At one end of the spectrum it is equivalent to the statement that evolution is possible, at the other end are untestable post hoc explanations, such as the suggestion that current evolutionary theory cannot explain the evolution of evolvability. We examine similarities and differences in eukaryote and prokaryote evolvability, and look for explanations that are compatible with a wide range of observations. Differences in genome organisation between eukaryotes and prokaryotes meets this criterion. The single origin of replication in prokaryote chromosomes (versus multiple origins in eukaryotes) accounts for many differences because the time to replicate a prokaryote genome limits its size (and the accumulation of junk DNA). Both prokaryotes and eukaryotes appear to switch from genetic stability to genetic change in response to stress. We examine a range of stress responses, and discuss how these impact on evolvability, particularly in unicellular organisms versus complex multicellular ones. Evolvability is also limited by environmental interactions (including competition) and we describe a model that places limits on potential evolvability. Examples are given of its application to predator competition and limits to lateral gene transfer. We suggest that unicellular organisms evolve largely through a process of metabolic change, resulting in biochemical diversity. Multicellular organisms evolve largely through morphological changes, not through extensive changes to cellular biochemistry. PMID:12689728

  7. Quantitative studies of mRNA recruitment to the eukaryotic ribosome.

    PubMed

    Fraser, Christopher S

    2015-07-01

    The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5' UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5' UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome.

  8. Quantitative studies of mRNA recruitment to the eukaryotic ribosome

    PubMed Central

    Fraser, Christopher S.

    2015-01-01

    The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5′ UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5′ UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome. PMID:25742741

  9. Non-canonical translation in RNA viruses

    PubMed Central

    Brierley, Ian

    2012-01-01

    Viral protein synthesis is completely dependent upon the translational machinery of the host cell. However, many RNA virus transcripts have marked structural differences from cellular mRNAs that preclude canonical translation initiation, such as the absence of a 5′ cap structure or the presence of highly structured 5′UTRs containing replication and/or packaging signals. Furthermore, whilst the great majority of cellular mRNAs are apparently monocistronic, RNA viruses must often express multiple proteins from their mRNAs. In addition, RNA viruses have very compact genomes and are under intense selective pressure to optimize usage of the available sequence space. Together, these features have driven the evolution of a plethora of non-canonical translational mechanisms in RNA viruses that help them to meet these challenges. Here, we review the mechanisms utilized by RNA viruses of eukaryotes, focusing on internal ribosome entry, leaky scanning, non-AUG initiation, ribosome shunting, reinitiation, ribosomal frameshifting and stop-codon readthrough. The review will highlight recently discovered examples of unusual translational strategies, besides revisiting some classical cases. PMID:22535777

  10. Machine Translation Project

    NASA Technical Reports Server (NTRS)

    Bajis, Katie

    1993-01-01

    The characteristics and capabilities of existing machine translation systems were examined and procurement recommendations were developed. Four systems, SYSTRAN, GLOBALINK, PC TRANSLATOR, and STYLUS, were determined to meet the NASA requirements for a machine translation system. Initially, four language pairs were selected for implementation. These are Russian-English, French-English, German-English, and Japanese-English.

  11. The others: our biased perspective of eukaryotic genomes.

    PubMed

    del Campo, Javier; Sieracki, Michael E; Molestina, Robert; Keeling, Patrick; Massana, Ramon; Ruiz-Trillo, Iñaki

    2014-05-01

    Understanding the origin and evolution of the eukaryotic cell and the full diversity of eukaryotes is relevant to many biological disciplines. However, our current understanding of eukaryotic genomes is extremely biased, leading to a skewed view of eukaryotic biology. We argue that a phylogeny-driven initiative to cover the full eukaryotic diversity is needed to overcome this bias. We encourage the community: (i) to sequence a representative of the neglected groups available at public culture collections, (ii) to increase our culturing efforts, and (iii) to embrace single cell genomics to access organisms refractory to propagation in culture. We hope that the community will welcome this proposal, explore the approaches suggested, and join efforts to sequence the full diversity of eukaryotes.

  12. PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes.

    PubMed

    Burgui, Idoia; Aragón, Tomás; Ortín, Juan; Nieto, Amelia

    2003-12-01

    It has previously been shown that influenza virus NS1 protein enhances the translation of viral but not cellular mRNAs. This enhancement occurs by increasing the rate of translation initiation and requires the 5'UTR sequence, common to all viral mRNAs. In agreement with these findings, we show here that viral mRNAs, but not cellular mRNAs, are associated with NS1 during virus infection. We have previously reported that NS1 interacts with the translation initiation factor eIF4GI, next to its poly(A)-binding protein 1 (PABP1)-interacting domain and that NS1 and eIF4GI are associated in influenza virus-infected cells. Here we show that NS1, although capable of binding poly(A), does not compete with PABP1 for association with eIF4GI and, furthermore, that NS1 and PABP1 interact both in vivo and in vitro in an RNA-independent manner. The interaction maps between residues 365 and 535 in PABP1 and between residues 1 and 81 in NS1. These mapping studies, together with those previously reported for NS1-eIF4GI and PABP1-eIF4GI interactions, imply that the binding of all three proteins would be compatible. Collectively, these and previously published data suggest that NS1 interactions with eIF4GI and PABP1, as well as with viral mRNAs, could promote the specific recruitment of 43S complexes to the viral mRNAs.

  13. Lateral gene transfer in eukaryotes.

    PubMed

    Andersson, J O

    2005-06-01

    Lateral gene transfer -- the transfer of genetic material between species -- has been acknowledged as a major mechanism in prokaryotic genome evolution for some time. Recently accumulating data indicate that the process also occurs in the evolution of eukaryotic genomes. However, there are large rate variations between groups of eukaryotes; animals and fungi seem to be largely unaffected, with a few exceptions, while lateral gene transfer frequently occurs in protists with phagotrophic lifestyles, possibly with rates comparable to prokaryotic organisms. Gene transfers often facilitate the acquisition of functions encoded in prokaryotic genomes by eukaryotic organisms, which may enable them to colonize new environments. Transfers between eukaryotes also occur, mainly into larger phagotrophic eukaryotes that ingest eukaryotic cells, but also between plant lineages. These findings have implications for eukaryotic genomic research in general, and studies of the origin and phylogeny of eukaryotes in particular.

  14. 15-Deoxyspergualin inhibits eukaryotic protein synthesis through eIF2α phosphorylation

    PubMed Central

    Ramya, T. N. C.; Surolia, Namita; Surolia, Avadhesha

    2006-01-01

    DSG (15-deoxyspergualin), an immunosuppressant with tumoricidal properties, binds potently to the regulatory C-terminal ‘EEVD’ motif of Hsps (heat-shock proteins). In the present study we demonstrate that DSG inhibits eukaryotic protein synthesis by sequestering Hsp70 which is required for maintaining HRI (haem-regulated inhibitor), a kinase of the eIF2α (eukaryotic initiation factor 2α), inactive. DSG stalled initiation of protein synthesis through phosphorylation of HRI and eIF2α. Addition of a recombinant eIF2α (S51A) protein, which lacks the phosphorylation site, lowered the inhibitory potential of DSG in reticulocyte lysate. The inhibitory effect of DSG was also attenuated in HRI knockdown cells. Moreover, exogenous addition of Hsp70 or the peptide ‘EEVD’ reversed the inhibitory effect of DSG. Interestingly, the inhibitory effect of DSG in different mammalian cancer cells was found to negatively correlate with the amount of Hsp70 expressed in the cells, emphasizing the link with Hsp70 in DSG inhibition of eukaryotic translation. PMID:16952278

  15. 1H, 13C and 15N resonance assignments and secondary structure analysis of translation initiation factor 1 from Pseudomonas aeruginosa

    PubMed Central

    Bernal, Alejandra; Hu, Yanmei; Palmer, Stephanie O.; Silva, Aaron; Bullard, James; Zhang, Yonghong

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of infection in humans. P. aeruginosa can acquire resistance against multiple groups of antimicrobial agents, including β-lactams, aminoglycosides and fluoroquinolones, and multidrug resistance is increasing in this organism which makes treatment of the infections difficult and expensive. This has led to the unmet need for discovery of new compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Translation initiation factor 1 from P. aeruginosa (Pa-IF1) is the smallest of the three initiation factors that acts to establish the 30S initiation complex to initiate translation during protein biosynthesis, and its structure is unknown. Here we report the 1H, 13C and 15N chemical shift assignments of Pa-IF1 as the basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified five β-strands with an unusually extended β-strand at the C-terminal end of the protein and one short α-helix arranged in the sequential order β1–β2–β3–α1–β4–β5. This is further supported by 15N–{1H} hetero NOEs. These secondary structure elements suggest the Pa-IF1 adopts the typical β-barrel structure and is composed of an oligomer-binding motif. PMID:26983940

  16. The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes.

    PubMed Central

    Fletcher, Simon P; Ali, Iraj K; Kaminski, Ann; Digard, Paul; Jackson, Richard J

    2002-01-01

    Classical swine fever virus (CSFV) is a member of the pestivirus family, which shares many features in common with hepatitis C virus (HCV). It is shown here that CSFV has an exceptionally efficient cis-acting internal ribosome entry segment (IRES), which, like that of HCV, is strongly influenced by the sequences immediately downstream of the initiation codon, and is optimal with viral coding sequences in this position. Constructs that retained 17 or more codons of viral coding sequence exhibited full IRES activity, but with only 12 codons, activity was approximately 66% of maximum in vitro (though close to maximum in transfected BHK cells), whereas with just 3 codons or fewer, the activity was only approximately 15% of maximum. The minimal coding region elements required for high activity were exchanged between HCV and CSFV. Although maximum activity was observed in each case with the homologous combination of coding region and 5' UTR, the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the 5' UTR and coding sequences. On the other hand, inversion of the coding sequences resulted in low IRES activity, particularly with the HCV coding sequences. RNA structure probing showed that the efficiency of internal initiation of these chimeric constructs correlated most closely with the degree of single-strandedness of the region around and immediately downstream of the initiation codon. The low activity IRESs could not be rescued by addition of supplementary eIF4A (the initiation factor with ATP-dependent RNA helicase activity). The extreme sensitivity to secondary structure around the initiation codon is likely to be due to the fact that the eIF4F complex (which has eIF4A as one of its subunits) is not required for and does not participate in initiation on these IRESs. PMID:12515388

  17. Structural insights into eukaryotic aquaporin regulation.

    PubMed

    Törnroth-Horsefield, Susanna; Hedfalk, Kristina; Fischer, Gerhard; Lindkvist-Petersson, Karin; Neutze, Richard

    2010-06-18

    Aquaporin-mediated water transport across cellular membranes is an ancient, ubiquitous mechanism within cell biology. This family of integral membrane proteins includes both water selective pores (aquaporins) and transport facilitators of other small molecules such as glycerol and urea (aquaglyceroporins). Eukaryotic aquaporins are frequently regulated post-translationally by gating, whereby the rate of flux through the channel is controlled, or by trafficking, whereby aquaporins are shuttled from intracellular storage sites to the plasma membrane. A number of high-resolution X-ray structures of eukaryotic aquaporins have recently been reported and the new structural insights into gating and trafficking that emerged from these studies are described. Basic structural themes reoccur, illustrating how the problem of regulation in diverse biological contexts builds upon a limited set of possible solutions. PMID:20416297

  18. The Iron Metallome in Eukaryotic Organisms

    PubMed Central

    Dlouhy, Adrienne C.; Outten, Caryn E.

    2013-01-01

    This chapter is focused on the iron metallome in eukaryotes at the cellular and subcellular level, including properties, utilization in metalloproteins, trafficking, storage, and regulation of these processes. Studies in the model eukaryote Saccharomyces cerevisiae and mammalian cells will be highlighted. The discussion of iron properties will center on the speciation and localization of intracellular iron as well as the cellular and molecular mechanisms for coping with both low iron bioavailability and iron toxicity. The section on iron metalloproteins will emphasize heme, iron-sulfur cluster, and non-heme iron centers, particularly their cellular roles and mechanisms of assembly. The section on iron uptake, trafficking, and storage will compare methods used by yeast and mammalian cells to import iron, how this iron is brought into various organelles, and types of iron storage proteins. Regulation of these processes will be compared between yeast and mammalian cells at the transcriptional, post-transcriptional, and post-translational levels. PMID:23595675

  19. Reinitiation enhances reliable transcriptional responses in eukaryotes.

    PubMed

    Liu, Bo; Yuan, Zhanjiang; Aihara, Kazuyuki; Chen, Luonan

    2014-08-01

    Gene transcription is a noisy process carried out by the transcription machinery recruited to the promoter. Noise reduction is a fundamental requirement for reliable transcriptional responses which in turn are crucial for signal transduction. Compared with the relatively simple transcription initiation in prokaryotes, eukaryotic transcription is more complex partially owing to its additional reinitiation mechanism. By theoretical analysis, we showed that reinitiation reduces noise in eukaryotic transcription independent of the transcription level. Besides, a higher reinitiation rate enables a stable scaffold complex an advantage in noise reduction. Finally, we showed that the coupling between scaffold formation and transcription can further reduce transcription noise independent of the transcription level. Furthermore, compared with the reinitiation mechanism, the noise reduction effect of the coupling can be of more significance in the case that the transcription level is low and the intrinsic noise dominates. Our results uncover a mechanistic route which eukaryotes may use to facilitate a more reliable response in the noisy transcription process. PMID:24850905

  20. Evolution of proteasome regulators in eukaryotes.

    PubMed

    Fort, Philippe; Kajava, Andrey V; Delsuc, Fredéric; Coux, Olivier

    2015-05-01

    All living organisms require protein degradation to terminate biological processes and remove damaged proteins. One such machine is the 20S proteasome, a specialized barrel-shaped and compartmentalized multicatalytic protease. The activity of the 20S proteasome generally requires the binding of regulators/proteasome activators (PAs), which control the entrance of substrates. These include the PA700 (19S complex), which assembles with the 20S and forms the 26S proteasome and allows the efficient degradation of proteins usually labeled by ubiquitin tags, PA200 and PA28, which are involved in proteolysis through ubiquitin-independent mechanisms and PI31, which was initially identified as a 20S inhibitor in vitro. Unlike 20S proteasome, shown to be present in all Eukaryotes and Archaea, the evolutionary history of PAs remained fragmentary. Here, we made a comprehensive survey and phylogenetic analyses of the four types of regulators in 17 clades covering most of the eukaryotic supergroups. We found remarkable conservation of each PA700 subunit in all eukaryotes, indicating that the current complex PA700 structure was already set up in the last eukaryotic common ancestor (LECA). Also present in LECA, PA200, PA28, and PI31 showed a more contrasted evolutionary picture, because many lineages have subsequently lost one or two of them. The paramount conservation of PA700 composition in all eukaryotes and the dynamic evolution of PA200, PA28, and PI31 are discussed in the light of current knowledge on their physiological roles.

  1. Evolution of Proteasome Regulators in Eukaryotes

    PubMed Central

    Fort, Philippe; Kajava, Andrey V.; Delsuc, Fredéric; Coux, Olivier

    2015-01-01

    All living organisms require protein degradation to terminate biological processes and remove damaged proteins. One such machine is the 20S proteasome, a specialized barrel-shaped and compartmentalized multicatalytic protease. The activity of the 20S proteasome generally requires the binding of regulators/proteasome activators (PAs), which control the entrance of substrates. These include the PA700 (19S complex), which assembles with the 20S and forms the 26S proteasome and allows the efficient degradation of proteins usually labeled by ubiquitin tags, PA200 and PA28, which are involved in proteolysis through ubiquitin-independent mechanisms and PI31, which was initially identified as a 20S inhibitor in vitro. Unlike 20S proteasome, shown to be present in all Eukaryotes and Archaea, the evolutionary history of PAs remained fragmentary. Here, we made a comprehensive survey and phylogenetic analyses of the four types of regulators in 17 clades covering most of the eukaryotic supergroups. We found remarkable conservation of each PA700 subunit in all eukaryotes, indicating that the current complex PA700 structure was already set up in the last eukaryotic common ancestor (LECA). Also present in LECA, PA200, PA28, and PI31 showed a more contrasted evolutionary picture, because many lineages have subsequently lost one or two of them. The paramount conservation of PA700 composition in all eukaryotes and the dynamic evolution of PA200, PA28, and PI31 are discussed in the light of current knowledge on their physiological roles. PMID:25943340

  2. IRES interaction with translation initiation factors: functional characterization of novel RNA contacts with eIF3, eIF4B, and eIF4GII.

    PubMed

    López de Quinto, S; Lafuente, E; Martínez-Salas, E

    2001-09-01

    Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.

  3. Stress-induced Start Codon Fidelity Regulates Arsenite-inducible Regulatory Particle-associated Protein (AIRAP) Translation*

    PubMed Central

    Zach, Lolita; Braunstein, Ilana; Stanhill, Ariel

    2014-01-01

    Initial steps in protein synthesis are highly regulated processes as they define the reading frame of the translation machinery. Eukaryotic translation initiation is a process facilitated by numerous factors (eIFs), aimed to form a “scanning” mechanism toward the initiation codon. Translation initiation of the main open reading frame (ORF) in an mRNA transcript has been reported to be regulated by upstream open reading frames (uORFs) in a manner of re-initiation. This mode of regulation is governed by the phosphorylation status of eIF2α and controlled by cellular stresses. Another mode of translational initiation regulation is leaky scanning, and this regulatory process has not been extensively studied. We have identified arsenite-inducible regulatory particle-associated protein (AIRAP) transcript to be translationally induced during arsenite stress conditions. AIRAP transcript contains a single uORF in a poor-kozak context. AIRAP translation induction is governed by means of leaky scanning and not re-initiation. This induction of AIRAP is solely dependent on eIF1 and the uORF kozak context. We show that eIF1 is phosphorylated under specific conditions that induce protein misfolding and have biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning like re-initiation is responsive to stress conditions and that leaky scanning can induce ORF translation by bypassing poor kozak context of a single uORF transcript. PMID:24898249

  4. Nucleotide and RNA metabolism prime translational initiation in the earliest events of mitochondrial biogenesis during Arabidopsis germination.

    PubMed

    Law, Simon R; Narsai, Reena; Taylor, Nicolas L; Delannoy, Etienne; Carrie, Chris; Giraud, Estelle; Millar, A Harvey; Small, Ian; Whelan, James

    2012-04-01

    Mitochondria play a crucial role in germination and early seedling growth in Arabidopsis (Arabidopsis thaliana). Morphological observations of mitochondria revealed that mitochondrial numbers, typical size, and oval morphology were evident after 12 h of imbibition in continuous light (following 48 h of stratification). The transition from a dormant to an active metabolic state was punctuated by an early molecular switch, characterized by a transient burst in the expression of genes encoding mitochondrial proteins. Factors involved in mitochondrial transcription and RNA processing were overrepresented among these early-expressed genes. This was closely followed by an increase in the transcript abundance of genes encoding proteins involved in mitochondrial DNA replication and translation. This burst in the expression of factors implicated in mitochondrial RNA and DNA metabolism was accompanied by an increase in transcripts encoding components required for nucleotide biosynthesis in the cytosol and increases in transcript abundance of specific members of the mitochondrial carrier protein family that have previously been associated with nucleotide transport into mitochondria. Only after these genes peaked in expression and largely declined were typical mitochondrial numbers and morphology observed. Subsequently, there was an increase in transcript abundance for various bioenergetic and metabolic functions of mitochondria. The coordination of nucleus- and organelle-encoded gene expression was also examined by quantitative reverse transcription-polymerase chain reaction, specifically for components of the mitochondrial electron transport chain and the chloroplastic photosynthetic machinery. Analysis of protein abundance using western-blot analysis and mass spectrometry revealed that for many proteins, patterns of protein and transcript abundance changes displayed significant positive correlations. A model for mitochondrial biogenesis during germination is proposed, in

  5. Circular code motifs in genomes of eukaryotes.

    PubMed

    El Soufi, Karim; Michel, Christian J

    2016-11-01

    A set X of 20 trinucleotides was identified in genes of bacteria, eukaryotes, plasmids and viruses, which has in average the highest occurrence in reading frame compared to its two shifted frames (Michel, 2015; Arquès and Michel, 1996). This set X has an interesting mathematical property as X is a circular code (Arquès and Michel, 1996). Thus, the motifs from this circular code X, called X motifs, have the property to always retrieve, synchronize and maintain the reading frame in genes. In this paper, we develop several statistical analyzes of X motifs in 138 available complete genomes of eukaryotes in which genes as well as non-gene regions are examined. Large X motifs (with lengths of at least 15 consecutive trinucleotides of X and compositions of at least 10 different trinucleotides of X among 20) have the highest occurrence in genomes of eukaryotes compared to its 23 large bijective motifs, its two large permuted motifs and large random motifs. The largest X motifs identified in eukaryotic genomes are presented, e.g. an X motif in a non-gene region of the genome Solanum pennellii with a length of 155 trinucleotides (465 nucleotides) and an expectation E=10(-71). In the human genome, the largest X motif occurs in a non-gene region of the chromosome 13 with a length of 36 trinucleotides and an expectation E=10(-11). X motifs in non-gene regions of genomes could be evolutionary relics of primitive genes using the circular code for translation. However, the proportion of X motifs (with lengths of at least 10 consecutive trinucleotides of X and compositions of at least 5 different trinucleotides of X among 20) in genes/non-genes of the 138 complete eukaryotic genomes is about 8. Thus, the X motifs occur preferentially in genes, as expected from the previous works of 20 years.

  6. Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus.

    PubMed Central

    Ross, T K; Achberger, E C; Braymer, H D

    1989-01-01

    The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine. Images PMID:2649480

  7. 5'-Terminal AUGs in Escherichia coli mRNAs with Shine-Dalgarno Sequences: Identification and Analysis of Their Roles in Non-Canonical Translation Initiation.

    PubMed

    Beck, Heather J; Fleming, Ian M C; Janssen, Gary R

    2016-01-01

    Analysis of the Escherichia coli transcriptome identified a unique subset of messenger RNAs (mRNAs) that contain a conventional untranslated leader and Shine-Dalgarno (SD) sequence upstream of the gene's start codon while also containing an AUG triplet at the mRNA's 5'- terminus (5'-uAUG). Fusion of the coding sequence specified by the 5'-terminal putative AUG start codon to a lacZ reporter gene, as well as primer extension inhibition assays, reveal that the majority of the 5'-terminal upstream open reading frames (5'-uORFs) tested support some level of lacZ translation, indicating that these mRNAs can function both as leaderless and canonical SD-leadered mRNAs. Although some of the uORFs were expressed at low levels, others were expressed at levels close to that of the respective downstream genes and as high as the naturally leaderless cI mRNA of bacteriophage λ. These 5'-terminal uORFs potentially encode peptides of varying lengths, but their functions, if any, are unknown. In an effort to determine whether expression from the 5'-terminal uORFs impact expression of the immediately downstream cistron, we examined expression from the downstream coding sequence after mutations were introduced that inhibit efficient 5'-uORF translation. These mutations were found to affect expression from the downstream cistrons to varying degrees, suggesting that some 5'-uORFs may play roles in downstream regulation. Since the 5'-uAUGs found on these conventionally leadered mRNAs can function to bind ribosomes and initiate translation, this indicates that canonical mRNAs containing 5'-uAUGs should be examined for their potential to function also as leaderless mRNAs. PMID:27467758

  8. 5’-Terminal AUGs in Escherichia coli mRNAs with Shine-Dalgarno Sequences: Identification and Analysis of Their Roles in Non-Canonical Translation Initiation

    PubMed Central

    Beck, Heather J.; Fleming, Ian M. C.

    2016-01-01

    Analysis of the Escherichia coli transcriptome identified a unique subset of messenger RNAs (mRNAs) that contain a conventional untranslated leader and Shine-Dalgarno (SD) sequence upstream of the gene’s start codon while also containing an AUG triplet at the mRNA’s 5’- terminus (5’-uAUG). Fusion of the coding sequence specified by the 5’-terminal putative AUG start codon to a lacZ reporter gene, as well as primer extension inhibition assays, reveal that the majority of the 5’-terminal upstream open reading frames (5’-uORFs) tested support some level of lacZ translation, indicating that these mRNAs can function both as leaderless and canonical SD-leadered mRNAs. Although some of the uORFs were expressed at low levels, others were expressed at levels close to that of the respective downstream genes and as high as the naturally leaderless cI mRNA of bacteriophage λ. These 5’-terminal uORFs potentially encode peptides of varying lengths, but their functions, if any, are unknown. In an effort to determine whether expression from the 5’-terminal uORFs impact expression of the immediately downstream cistron, we examined expression from the downstream coding sequence after mutations were introduced that inhibit efficient 5’-uORF translation. These mutations were found to affect expression from the downstream cistrons to varying degrees, suggesting that some 5’-uORFs may play roles in downstream regulation. Since the 5’-uAUGs found on these conventionally leadered mRNAs can function to bind ribosomes and initiate translation, this indicates that canonical mRNAs containing 5’-uAUGs should be examined for their potential to function also as leaderless mRNAs. PMID:27467758

  9. The Translation Initiation Factor eIF4E Regulates the Sex-Specific Expression of the Master Switch Gene Sxl in Drosophila melanogaster

    PubMed Central

    Graham, Patricia L.; Yanowitz, Judith L.; Penn, Jill K. M.; Deshpande, Girish; Schedl, Paul

    2011-01-01

    In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors—the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d—that have been directly implicated in Sxl splicing regulation. PMID:21829374

  10. EJC core component MLN51 interacts with eIF3 and activates translation

    PubMed Central

    Chazal, Pierre-Etienne; Daguenet, Elisabeth; Wendling, Corinne; Ulryck, Nathalie; Tomasetto, Catherine; Sargueil, Bruno; Le Hir, Hervé

    2013-01-01

    The multiprotein exon junction complex (EJC), deposited by the splicing machinery, is an important constituent of messenger ribonucleoprotein particles because it participates to numerous steps of the mRNA lifecycle from splicing to surveillance via nonsense-mediated mRNA decay pathway. By an unknown mechanism, the EJC also stimulates translation efficiency of newly synthesized mRNAs. Here, we show that among the four EJC core components, the RNA-binding protein metastatic lymph node 51 (MLN51) is a translation enhancer. Overexpression of MLN51 preferentially increased the translation of intron-containing reporters via the EJC, whereas silencing MLN51 decreased translation. In addition, modulation of the MLN51 level in cell-free translational extracts confirmed its direct role in protein synthesis. Immunoprecipitations indicated that MLN51 associates with translation-initiating factors and ribosomal subunits, and in vitro binding assays revealed that MLN51, alone or as part of the EJC, interacts directly with the pivotal eukaryotic translation initiation factor eIF3. Taken together, our data define MLN51 as a translation activator linking the EJC and the translation machinery. PMID:23530232

  11. Polyamines in Eukaryotes, Bacteria, and Archaea.

    PubMed

    Michael, Anthony J

    2016-07-15

    Polyamines are primordial polycations found in most cells and perform different functions in different organisms. Although polyamines are mainly known for their essential roles in cell growth and proliferation, their functions range from a critical role in cellular translation in eukaryotes and archaea, to bacterial biofilm formation and specialized roles in natural product biosynthesis. At first glance, the diversity of polyamine structures in different organisms appears chaotic; however, biosynthetic flexibility and evolutionary and ecological processes largely explain this heterogeneity. In this review, I discuss the biosynthetic, evolutionary, and physiological processes that constrain or expand polyamine structural and functional diversity.

  12. An inhibitor of eIF2 activity in the sRNA pool of eukaryotic cells.

    PubMed

    Centrella, Michael; Porter, David L; McCarthy, Thomas L

    2011-08-15

    Eukaryotic protein synthesis is a multi-step and highly controlled process that includes an early initiation complex containing eukaryotic initiation factor 2 (eIF2), GTP, and methionine-charged initiator methionyl-tRNA (met-tRNAi). During studies to reconstruct formation of the ternary complex containing these molecules, we detected a potent inhibitor in low molecular mass RNA (sRNA) preparations of eukaryotic tRNA. The ternary complex inhibitor (TCI) was retained in the total sRNA pool after met-tRNAi was charged by aminoacyl tRNA synthetase, co-eluted with sRNA by size exclusion chromatography, but resolved from met-tRNAi by ion exchange chromatography. The adverse effect of TCI was not overcome by high GTP or magnesium omission and was independent of GTP regeneration. Rather, TCI suppressed the rate of ternary complex formation, and disrupted protein synthesis and the accumulation of heavy polymeric ribosomes in reticulocyte lysates in vitro. Lastly, a component or components in ribosome depleted cell lysate significantly reversed TCI activity. Since assembly of the met-tRNAi/eIF2/GTP ternary complex is integral to protein synthesis, awareness of TCI is important to avoid confusion in studies of translation initiation. A clear definition of TCI may also allow a better appreciation of physiologic or pathologic situations, factors, and events that control protein synthesis in vivo.

  13. Vaccinia Virus Protein Synthesis Has a Low Requirement for the Intact Translation Initiation Factor eIF4F, the Cap-Binding Complex, within Infected Cells

    PubMed Central

    Mulder, Jacqueline; Robertson, Morwenna E. M.; Seamons, Rachael A.; Belsham, Graham J.

    1998-01-01

    The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both β-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (β-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5′ noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5′ and 3′ termini. PMID:9765426

  14. Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome

    PubMed Central

    Armache, Jean-Paul; Jarasch, Alexander; Anger, Andreas M.; Villa, Elizabeth; Becker, Thomas; Bhushan, Shashi; Jossinet, Fabrice; Habeck, Michael; Dindar, Gülcin; Franckenberg, Sibylle; Marquez, Viter; Mielke, Thorsten; Thomm, Michael; Berninghausen, Otto; Beatrix, Birgitta; Söding, Johannes; Westhof, Eric; Wilson, Daniel N.; Beckmann, Roland

    2010-01-01

    Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74/80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus. PMID:20974910

  15. Translational initiation factor expression and ribosomal protein gene expression are repressed coordinately but by different mechanisms in murine lymphosarcoma cells treated with glucocorticoids.

    PubMed Central

    Huang, S; Hershey, J W

    1989-01-01

    P1798 murine lymphosarcoma cells cease to proliferate upon exposure to 10(-7) M dexamethasone and exhibit a dramatic inhibition of rRNA and ribosomal protein synthesis (O. Meyuhas, E. Thompson, Jr., and R. P. Perry, Mol. Cell Biol. 7:2691-2699, 1987). These workers demonstrated that ribosomal protein synthesis is regulated primarily at the level of translation, since dexamethasone did not alter mRNA levels but shifted the mRNAs from active polysomes into inactive messenger ribonucleoproteins. We have examined the effects of dexamethasone on the biosynthesis of initiation factor proteins in the same cell line. The relative protein synthesis rates of eIF-4A and eIF-2 alpha were inhibited by about 70% by the hormone, a reduction comparable to that for ribosomal proteins. The mRNA levels of eIF-4A, eIF-4D, and eIF-2 alpha also were reduced by 60 to 70%, indicating that synthesis rates are proportional to mRNA concentrations. Analysis of polysome profiles showed that the average number of ribosomes per initiation factor polysome was only slightly reduced by dexamethasone, and little or no mRNA was present in messenger ribonucleoproteins. The results indicate that initiation factor gene expression is coordinately regulated with ribosomal protein synthesis but is controlled primarily by modulating mRNA levels rather than mRNA efficiency. Images PMID:2779563

  16. A Network of Hydrophobic Residues Impeding Helix αC Rotation Maintains Latency of Kinase Gcn2, Which Phosphorylates the α Subunit of Translation Initiation Factor 2▿

    PubMed Central

    Gárriz, Andrés; Qiu, Hongfang; Dey, Madhusudan; Seo, Eun-Joo; Dever, Thomas E.; Hinnebusch, Alan G.

    2009-01-01

    Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the α subunit of translation initiation factor 2 (eIF2α). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix αC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2α phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of αC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of αC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation. PMID:19114556

  17. Endosymbiotic theories for eukaryote origin.

    PubMed

    Martin, William F; Garg, Sriram; Zimorski, Verena

    2015-09-26

    For over 100 years, endosymbiotic theories have figured in thoughts about the differences between prokaryotic and eukaryotic cells. More than 20 different versions of endosymbiotic theory have been presented in the literature to explain the origin of eukaryotes and their mitochondria. Very few of those models account for eukaryotic anaerobes. The role of energy and the energetic constraints that prokaryotic cell organization placed on evolutionary innovation in cell history has recently come to bear on endosymbiotic theory. Only cells that possessed mitochondria had the bioenergetic means to attain eukaryotic cell complexity, which is why there are no true intermediates in the prokaryote-to-eukaryote transition. Current versions of endosymbiotic theory have it that the host was an archaeon (an archaebacterium), not a eukaryote. Hence the evolutionary history and biology of archaea increasingly comes to bear on eukaryotic origins, more than ever before. Here, we have compiled a survey of endosymbiotic theories for the origin of eukaryotes and mitochondria, and for the origin of the eukaryotic nucleus, summarizing the essentials of each and contrasting some of their predictions to the observations. A new aspect of endosymbiosis in eukaryote evolution comes into focus from these considerations: the host for the origin of plastids was a facultative anaerobe.

  18. Endosymbiotic theories for eukaryote origin

    PubMed Central

    Martin, William F.; Garg, Sriram; Zimorski, Verena

    2015-01-01

    For over 100 years, endosymbiotic theories have figured in thoughts about the differences between prokaryotic and eukaryotic cells. More than 20 different versions of endosymbiotic theory have been presented in the literature to explain the origin of eukaryotes and their mitochondria. Very few of those models account for eukaryotic anaerobes. The role of energy and the energetic constraints that prokaryotic cell organization placed on evolutionary innovation in cell history has recently come to bear on endosymbiotic theory. Only cells that possessed mitochondria had the bioenergetic means to attain eukaryotic cell complexity, which is why there are no true intermediates in the prokaryote-to-eukaryote transition. Current versions of endosymbiotic theory have it that the host was an archaeon (an archaebacterium), not a eukaryote. Hence the evolutionary history and biology of archaea increasingly comes to bear on eukaryotic origins, more than ever before. Here, we have compiled a survey of endosymbiotic theories for the origin of eukaryotes and mitochondria, and for the origin of the eukaryotic nucleus, summarizing the essentials of each and contrasting some of their predictions to the observations. A new aspect of endosymbiosis in eukaryote evolution comes into focus from these considerations: the host for the origin of plastids was a facultative anaerobe. PMID:26323761

  19. FrameD: a flexible program for quality check and gene prediction in prokaryotic genomes and noisy matured eukaryotic sequences

    PubMed Central

    Schiex, Thomas; Gouzy, Jérôme; Moisan, Annick; de Oliveira, Yannick

    2003-01-01

    We describe FrameD, a program that predicts coding regions in prokaryotic and matured eukaryotic sequences. Initially targeted at gene prediction in bacterial GC rich genomes, the gene model used in FrameD also allows to predict genes in the presence of frameshifts and partially undetermined sequences which makes it also very suitable for gene prediction and frameshift correction in unfinished sequences such as EST and EST cluster sequences. Like recent eukaryotic gene prediction programs, FrameD also includes the ability to take into account protein similarity information both in its prediction and its graphical output. Its performances are evaluated on different bacterial genomes. The web site (http://genopole.toulouse.inra.fr/bioinfo/FrameD/FD) allows direct prediction, sequence correction and translation and the ability to learn new models for new organisms. PMID:12824407

  20. FrameD: A flexible program for quality check and gene prediction in prokaryotic genomes and noisy matured eukaryotic sequences.

    PubMed

    Schiex, Thomas; Gouzy, Jérôme; Moisan, Annick; de Oliveira, Yannick

    2003-07-01

    We describe FrameD, a program that predicts coding regions in prokaryotic and matured eukaryotic sequences. Initially targeted at gene prediction in bacterial GC rich genomes, the gene model used in FrameD also allows to predict genes in the presence of frameshifts and partially undetermined sequences which makes it also very suitable for gene prediction and frameshift correction in unfinished sequences such as EST and EST cluster sequences. Like recent eukaryotic gene prediction programs, FrameD also includes the ability to take into account protein similarity information both in its prediction and its graphical output. Its performances are evaluated on different bacterial genomes. The web site (http://genopole.toulouse.inra.fr/bioinfo/FrameD/FD) allows direct prediction, sequence correction and translation and the ability to learn new models for new organisms. PMID:12824407

  1. The Diversity of Eukaryotes.

    PubMed

    Patterson

    1999-10-01

    The discipline of evolutionary protistology has emerged in the past 30 yr. There is as yet no agreed view of how protists are interrelated or how they should be classified. The foundations of a stable taxonomic superstructure for the protists and other eukaryotes lie in cataloging the diversity of the major monophyletic lineages of these organisms. The use of common patterns of cell organization (ultrastructural identity) seems to provide us with the most robust hypotheses of such lineages. These lineages are placed in 71 groups without identifiable sister taxa. These groups are here referred to as "major building blocks." For the first time, the compositions, ultrastructural identities, synapomorphies (where available), and subgroups of the major building blocks are summarized. More than 200 further lineages without clear identities are listed. This catalog includes all known major elements of the comprehensive evolutionary tree of protists and eukaryotes. Different approaches among protistologists to issues of nomenclature, ranking, and definitions of these groups are discussed, with particular reference to two groups-the stramenopiles and the Archezoa. The concept of "extended in-group" is introduced to refer to in-groups and the most proximate sister group and to assist in identifying the hierarchical location of taxa.

  2. virG, an Agrobacterium tumefaciens transcriptional activator, initiates translation at a UUG codon and is a sequence-specific DNA-binding protein.

    PubMed Central

    Pazour, G J; Das, A

    1990-01-01

    The Agrobacterium tumefaciens Ti plasmid virG locus, in conjunction with virA and acetosyringone, activates transcription of the virulence (vir) genes. Insertional and deoxyoligonucleotide-directed mutagenesis studies showed that both octopine and nopaline Ti plasmid virG genes initiate translation at a UUG codon. VirG protein initiated at this UUG codon was found to be 241 amino acid residues in length and had an apparent molecular mass of 27.1 kilodaltons. A Salmonella typhimurium trp-virG transcriptional fusion was constructed to overproduce VirG. Agrobacterium cells containing this gene fusion showed a large increase in virG activity in the presence of virA and acetosyringone. Since the trp promoter is not under virA-virG control, this result indicates that modification of VirG is necessary for its full activity. VirG overproduced in Escherichia coli was purified from inclusion bodies. It was found to be a DNA-binding protein that preferentially bound DNA fragments containing the 5' nontranscribed regions of the virA, -B, -C, -D, and -G operons. Significant specific binding to the 5' nontranscribed region sequences of virE was not detected. DNase I footprinting of the upstream regions of virC-virD and virG showed that VirG binds to sequences around the vir box region. Images FIG. 1 FIG. 2 FIG. 3 PMID:2307647

  3. The enactment of knowledge translation: a study of the Collaborations for Leadership in Applied Health Research and Care initiative within the English National Health Service

    PubMed Central

    D’Andreta, Daniela; Scarbrough, Harry; Evans, Sarah

    2014-01-01

    Objectives We contribute to existing knowledge translation (KT) literature by developing the notion of ‘enactment’ and illustrate this through an interpretative, comparative case-study analysis of three Collaborations for Leadership in Applied Health Research and Care (CLAHRC) initiatives. We argue for a focus on the way in which the CLAHRC model has been ‘enacted’ as central to the different KT challenges and capabilities encountered. Methods A comparative, mixed method study created a typology of enactments (Classical, Home-grown and Imported) using qualitative analysis and social network analysis. Results We identify systematic differences in the enactment of the CLAHRC model. The sources of these different enactments are subsequently related to variation in formative interpretations and leadership styles, the implementation of different governance structures, and the relative epistemic differences between the professional groups involved. Conclusions Enactment concerns the creative agency of individuals and groups in constituting a particular context for their work through their local interpretation of a particular KT model. Our theory of enactment goes beyond highlighting variation between CLAHRCs, to explore the mechanisms that influence the way a particular model is interpreted and acted upon. We thus encourage less focus on conceptual models and more on the formative role played by leaders of KT initiatives. PMID:24048695

  4. The Sec translocon mediated protein transport in prokaryotes and eukaryotes.

    PubMed

    Denks, Kärt; Vogt, Andreas; Sachelaru, Ilie; Petriman, Narcis-Adrian; Kudva, Renuka; Koch, Hans-Georg

    2014-01-01

    Protein transport via the Sec translocon represents an evolutionary conserved mechanism for delivering cytosolically-synthesized proteins to extra-cytosolic compartments. The Sec translocon has a three-subunit core, termed Sec61 in Eukaryotes and SecYEG in Bacteria. It is located in the endoplasmic reticulum of Eukaryotes and in the cytoplasmic membrane of Bacteria where it constitutes a channel that can be activated by multiple partner proteins. These partner proteins determine the mechanism of polypeptide movement across the channel. During SRP-dependent co-translational targeting, the ribosome threads the nascent protein directly into the Sec channel. This pathway is in Bacteria mainly dedicated for membrane proteins but in Eukaryotes also employed by secretory proteins. The alternative pathway, leading to post-translational translocation across the Sec translocon engages an ATP-dependent pushing mechanism by the motor protein SecA in Bacteria and a ratcheting mechanism by the lumenal chaperone BiP in Eukaryotes. Protein transport and biogenesis is also assisted by additional proteins at the lateral gate of SecY/Sec61α and in the lumen of the endoplasmic reticulum or in the periplasm of bacterial cells. The modular assembly enables the Sec complex to transport a vast array of substrates. In this review we summarize recent biochemical and structural information on the prokaryotic and eukaryotic Sec translocons and we describe the remarkably complex interaction network of the Sec complexes.

  5. Thyroid hormone increases bulk histones expression by enhancing translational efficiency.

    PubMed

    Zambrano, Alberto; García-Carpizo, Verónica; Villamuera, Raquel; Aranda, Ana

    2015-01-01

    The expression of canonical histones is normally coupled to DNA synthesis during the S phase of the cell cycle. Replication-dependent histone mRNAs do not contain a poly(A) tail at their 3' terminus, but instead possess a stem-loop motif, the binding site for the stem-loop binding protein (SLBP), which regulates mRNA processing, stability, and relocation to polysomes. Here we show that the thyroid hormone can increase the levels of canonical histones independent of DNA replication. Incubation of mouse embryonic fibroblasts with T3 increases the total levels of histones, and expression of the thyroid hormone receptor β induces a further increase. This is not restricted to mouse embryonic fibroblasts, because T3 also raises histone expression in other cell lines. T3 does not increase histone mRNA or SLBP levels, suggesting that T3 regulates histone expression by a posttranscriptional mechanism. Indeed, T3 enhanced translational efficiency, inducing relocation of histone mRNA to heavy polysomes. Increased translation was associated with augmented transcription of the eukaryotic translation initiation factor 4 γ2 (EIF4G2). T3 induced EIF4G2 protein and mRNA levels and the thyroid hormone receptor bound to the promoter region of the Eif4g2 gene. Induction of EIF4G2 was essential for T3-dependent histone induction, because depletion of this factor abolished histone increase. These results point out the importance of the thyroid hormones on the posttranscriptional regulation of histone biosynthesis in a cell cycle-independent manner and also suggest the potential regulation of eukaryotic translation by the modulation of the initiation factor EIF4G2, which also operates in the translation of canonical mRNAs.

  6. Molecular paleontology and complexity in the last eukaryotic common ancestor.

    PubMed

    Koumandou, V Lila; Wickstead, Bill; Ginger, Michael L; van der Giezen, Mark; Dacks, Joel B; Field, Mark C

    2013-01-01

    Eukaryogenesis, the origin of the eukaryotic cell, represents one of the fundamental evolutionary transitions in the history of life on earth. This event, which is estimated to have occurred over one billion years ago, remains rather poorly understood. While some well-validated examples of fossil microbial eukaryotes for this time frame have been described, these can provide only basic morphology and the molecular machinery present in these organisms has remained unknown. Complete and partial genomic information has begun to fill this gap, and is being used to trace proteins and cellular traits to their roots and to provide unprecedented levels of resolution of structures, metabolic pathways and capabilities of organisms at these earliest points within the eukaryotic lineage. This is essentially allowing a molecular paleontology. What has emerged from these studies is spectacular cellular complexity prior to expansion of the eukaryotic lineages. Multiple reconstructed cellular systems indicate a very sophisticated biology, which by implication arose following the initial eukaryogenesis event but prior to eukaryotic radiation and provides a challenge in terms of explaining how these early eukaryotes arose and in understanding how they lived. Here, we provide brief overviews of several cellular systems and the major emerging conclusions, together with predictions for subsequent directions in evolution leading to extant taxa. We also consider what these reconstructions suggest about the life styles and capabilities of these earliest eukaryotes and the period of evolution between the radiation of eukaryotes and the eukaryogenesis event itself.

  7. Molecular paleontology and complexity in the last eukaryotic common ancestor

    PubMed Central

    Koumandou, V. Lila; Wickstead, Bill; Ginger, Michael L.; van der Giezen, Mark; Dacks, Joel B.

    2013-01-01

    Eukaryogenesis, the origin of the eukaryotic cell, represents one of the fundamental evolutionary transitions in the history of life on earth. This event, which is estimated to have occurred over one billion years ago, remains rather poorly understood. While some well-validated examples of fossil microbial eukaryotes for this time frame have been described, these can provide only basic morphology and the molecular machinery present in these organisms has remained unknown. Complete and partial genomic information has begun to fill this gap, and is being used to trace proteins and cellular traits to their roots and to provide unprecedented levels of resolution of structures, metabolic pathways and capabilities of organisms at these earliest points within the eukaryotic lineage. This is essentially allowing a molecular paleontology. What has emerged from these studies is spectacular cellular complexity prior to expansion of the eukaryotic lineages. Multiple reconstructed cellular systems indicate a very sophisticated biology, which by implication arose following the initial eukaryogenesis event but prior to eukaryotic radiation and provides a challenge in terms of explaining how these early eukaryotes arose and in understanding how they lived. Here, we provide brief overviews of several cellular systems and the major emerging conclusions, together with predictions for subsequent directions in evolution leading to extant taxa. We also consider what these reconstructions suggest about the life styles and capabilities of these earliest eukaryotes and the period of evolution between the radiation of eukaryotes and the eukaryogenesis event itself. PMID:23895660

  8. An archaeal origin of eukaryotes supports only two primary domains of life.

    PubMed

    Williams, Tom A; Foster, Peter G; Cox, Cymon J; Embley, T Martin

    2013-12-12

    The discovery of the Archaea and the proposal of the three-domains 'universal' tree, based on ribosomal RNA and core genes mainly involved in protein translation, catalysed new