scholarly journals Activation of a GPCR leads to eIF4G phosphorylation at the 5′ cap and to IRES-dependent translation

2014 ◽  
Vol 52 (3) ◽  
pp. 373-382 ◽  
Author(s):  
Kelly León ◽  
Thomas Boulo ◽  
Astrid Musnier ◽  
Julia Morales ◽  
Christophe Gauthier ◽  
...  
Keyword(s):  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Hek293 Cells ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Translational Machinery ◽  
Phosphorylation Event ◽  
Eukaryotic Initiation Factor 4G ◽  
Molecular Bases

The control of mRNA translation has been mainly explored in response to activated tyrosine kinase receptors. In contrast, mechanistic details on the translational machinery are far less available in the case of ligand-bound G protein-coupled receptors (GPCRs). In this study, using the FSH receptor (FSH-R) as a model receptor, we demonstrate that part of the translational regulations occurs by phosphorylation of the translation pre-initiation complex scaffold protein, eukaryotic initiation factor 4G (eIF4G), in HEK293 cells stably expressing the FSH-R. This phosphorylation event occurred when eIF4G was bound to the mRNA 5′ cap, and probably involves mammalian target of rapamycin. This regulation might contribute to cap-dependent translation in response to FSH. The cap-binding protein eIF4E also had its phosphorylation level enhanced upon FSH stimulation. We also show that FSH-induced signaling not only led to cap-dependent translation but also to internal ribosome entry site (IRES)-dependent translation of some mRNA. These data add detailed information on the molecular bases underlying the regulation of selective mRNA translation by a GPCR, and a topological model recapitulating these mechanisms is proposed.

scholarly journals Cap-Independent Translation Conferred by the 5′ Leader of Tobacco Etch Virus Is Eukaryotic Initiation Factor 4G Dependent

2001 ◽  
Vol 75 (24) ◽  
pp. 12141-12152 ◽  
Author(s):  
Daniel R. Gallie
Keyword(s):  
Large Subunit ◽  
Tobacco Etch Virus ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Initiation Factor 4G ◽  
Independent Translation

ABSTRACT The 5′ leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.

Manipulation of the host translation initiation complex eIF4F by DNA viruses

2010 ◽  
Vol 38 (6) ◽  
pp. 1511-1516 ◽  
Author(s):  
Derek Walsh
Keyword(s):  
Translation Initiation ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Viral Proteins ◽  
Mitogen Activated Protein Kinase ◽  
Scaffolding Protein ◽  
Initiation Factor ◽  
Viral Protein Synthesis ◽  
Dna Viruses ◽  
Translational Machinery

In the absence of their own translational machinery, all viruses must gain access to host cell ribosomes to synthesize viral proteins and replicate. Ribosome recruitment and scanning of capped host mRNAs is facilitated by the multisubunit eIF (eukaryotic initiation factor) 4F, which consists of a cap-binding protein, eIF4E and an RNA helicase, eIF4A, assembled on a large scaffolding protein, eIF4G. Although inactivated by many viruses to inhibit host translation, a growing number of DNA viruses are being found to employ diverse strategies to stimulate eIF4F activity in infected cells and maximize viral protein synthesis. These strategies include stimulation of cellular mTOR (mammalian target of rapamycin) signalling to inactivate 4E-BPs (eIF4E-binding proteins), a family of translational repressors that limit eIF4E availability and eIF4F complex formation, together with modulating the activity of the eIF4E kinase Mnk (mitogen-activated protein kinase signal-integrating kinase) in a variety of manners to regulate both host and viral mRNA translation. In some cases, specific viral proteins that mediate these signalling events have been identified, whereas others have been shown to interact with host translation initiation factors or complexes and modify their activity and/or subcellular localization. The present review outlines current understanding of the role of eIF4F in the life cycle of various DNA viruses and discusses its potential as a therapeutic target to suppress viral infection.

scholarly journals A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2Apro: Effects on Cellular and Viral Gene Expression

2000 ◽  
Vol 74 (5) ◽  
pp. 2383-2392 ◽  
Author(s):  
Angel Barco ◽  
Elena Feduchi ◽  
Luis Carrasco
Keyword(s):  
Hela Cell ◽  
Cell Line ◽  
Cell Clone ◽  
Morphological Changes ◽  
Viral Gene Expression ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Viral Gene ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2Apro) under the control of tetracycline has been obtained. Synthesis of 2Apro induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2Apro cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2Apro, prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2Apro still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2Apro. Moreover, synthesis of 2Apro in 2A7d cells complements the translational defect of a poliovirus 2Apro-defective variant. These results show that poliovirus 2Apro expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2Aprofunctions, to complement poliovirus 2Apro mutants, and to test antiviral compounds.

scholarly journals Multiple Mechanisms Control Phosphorylation of PHAS-I in Five (S/T)P Sites That Govern Translational Repression

2000 ◽  
Vol 20 (10) ◽  
pp. 3558-3567 ◽  
Author(s):  
Isabelle Mothe-Satney ◽  
Daqing Yang ◽  
Patrick Fadden ◽  
Timothy A. J. Haystead ◽  
John C. Lawrence
Keyword(s):  
Mrna Translation ◽  
Hek293 Cells ◽  
Initiation Factor ◽  
Translational Repression ◽  
Phosphorylation Sites ◽  
Translational Repressor ◽  
Eukaryotic Initiation Factor 4E ◽  
Dependent Processes ◽  
Constitutive Phosphorylation

ABSTRACT Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr → Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

scholarly journals 4E-BP2–dependent translation in parvalbumin neurons controls epileptic seizure threshold

2021 ◽  
Vol 118 (15) ◽  
pp. e2025522118
Author(s):  
Vijendra Sharma ◽  
Rapita Sood ◽  
Danning Lou ◽  
Tzu-Yu Hung ◽  
Maxime Lévesque ◽  
...  
Keyword(s):  
Animal Models ◽  
Mammalian Target Of Rapamycin ◽  
Neuronal Cell ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Inhibitory Neurons ◽  
Initiation Factor ◽  
Seizure Threshold ◽  
Parvalbumin Neurons

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals to regulate critical cellular processes such as mRNA translation, lipid biogenesis, and autophagy. Germline and somatic mutations in mTOR and genes upstream of mTORC1, such as PTEN, TSC1/2, AKT3, PIK3CA, and components of GATOR1 and KICSTOR complexes, are associated with various epileptic disorders. Increased mTORC1 activity is linked to the pathophysiology of epilepsy in both humans and animal models, and mTORC1 inhibition suppresses epileptogenesis in humans with tuberous sclerosis and animal models with elevated mTORC1 activity. However, the role of mTORC1-dependent translation and the neuronal cell types mediating the effect of enhanced mTORC1 activity in seizures remain unknown. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 2 (4E-BP2) are translational repressors downstream of mTORC1. Here we show that the ablation of 4E-BP2, but not 4E-BP1, in mice increases the sensitivity to pentylenetetrazole (PTZ)- and kainic acid (KA)–induced seizures. We demonstrate that the deletion of 4E-BP2 in inhibitory, but not excitatory neurons, causes an increase in the susceptibility to PTZ-induced seizures. Moreover, mice lacking 4E-BP2 in parvalbumin, but not somatostatin or VIP inhibitory neurons exhibit a lowered threshold for seizure induction and reduced number of parvalbumin neurons. A mouse model harboring a human PIK3CA mutation that enhances the activity of the PI3K-AKT pathway (Pik3caH1047R-Pvalb) selectively in parvalbumin neurons shows susceptibility to PTZ-induced seizures. Our data identify 4E-BP2 as a regulator of epileptogenesis and highlight the central role of increased mTORC1-dependent translation in parvalbumin neurons in the pathophysiology of epilepsy.

scholarly journals Factor-Independent Assembly of Elongation-Competent Ribosomes by an Internal Ribosome Entry Site Located in an RNA Virus That Infects Penaeid Shrimp

2005 ◽  
Vol 79 (2) ◽  
pp. 677-683 ◽  
Author(s):  
Randal C. Cevallos ◽  
Peter Sarnow
Keyword(s):  
Internal Ribosome Entry Site ◽  
Intergenic Region ◽  
Rna Virus ◽  
Penaeid Shrimp ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Functional Conservation

ABSTRACT The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.

scholarly journals Dengue Virus Utilizes a Novel Strategy for Translation Initiation When Cap-Dependent Translation Is Inhibited

2006 ◽  
Vol 80 (6) ◽  
pp. 2976-2986 ◽  
Author(s):  
Dianna Edgil ◽  
Charlotta Polacek ◽  
Eva Harris
Keyword(s):  
Dengue Virus ◽  
Translation Initiation ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Initiation Factor 4E ◽  
Cellular Translation ◽  
Cellular Mrnas ◽  
Novel Strategy

ABSTRACT Viruses have developed numerous mechanisms to usurp the host cell translation apparatus. Dengue virus (DEN) and other flaviviruses, such as West Nile and yellow fever viruses, contain a 5′ m7GpppN-capped positive-sense RNA genome with a nonpolyadenylated 3′ untranslated region (UTR) that has been presumed to undergo translation in a cap-dependent manner. However, the means by which the DEN genome is translated effectively in the presence of capped, polyadenylated cellular mRNAs is unknown. This report demonstrates that DEN replication and translation are not affected under conditions that inhibit cap-dependent translation by targeting the cap-binding protein eukaryotic initiation factor 4E, a key regulator of cellular translation. We further show that under cellular conditions in which translation factors are limiting, DEN can alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that appears not to require a functional m7G cap. This DEN noncanonical translation is not mediated by an internal ribosome entry site but requires the interaction of the DEN 5′ and 3′ UTRs for activity, suggesting a novel strategy for translation of animal viruses.

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