scholarly journals Specific Isoforms of Translation Initiation Factor 4GI Show Differences in Translational Activity

2006 ◽  
Vol 26 (22) ◽  
pp. 8448-8460 ◽  
Author(s):  
Mark J. Coldwell ◽  
Simon J. Morley
Keyword(s):  
Translation Initiation ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Critical Role ◽  
Translation Initiation Factor ◽  
Protein Isoforms ◽  
Initiation Factor ◽  
Functional Studies ◽  
Eif2α Phosphorylation ◽  
Translational Activity

ABSTRACT The eukaryotic initiation factor (eIF) 4GI gene locus (eIF4GI) contains three identified promoters, generating alternately spliced mRNAs, yielding a total of five eIF4GI protein isoforms. Although eIF4GI plays a critical role in mRNA recruitment to the ribosomes, little is known about the functions of the different isoforms, their partner binding capacities, or the role of the homolog, eIF4GII, in translation initiation. To directly address this, we have used short interfering RNAs (siRNAs) expressed from DNA vectors to silence the expression of eIF4GI in HeLa cells. Here we show that reduced levels of specific mRNA and eIF4GI isoforms in HeLa cells promoted aberrant morphology and a partial inhibition of translation. The latter reflected dephosphorylation of 4E-BP1 and decreased eIF4F complex levels, with no change in eIF2α phosphorylation. Expression of siRNA-resistant Myc-tagged eIF4GI isoforms has allowed us to show that the different isoforms exhibit significant differences in their ability to restore translation rates. Here we quantify the efficiency of eIF4GI promoter usage in mammalian cells and demonstrate that even though the longest isoform of eIF4GI (eIF4GIf) was relatively poorly expressed when reintroduced, it was more efficient at promoting the translation of cellular mRNAs than the more highly expressed shorter isoforms used in previous functional studies.

scholarly journals Inhibition of a constitutive translation initiation factor 2α phosphatase, CReP, promotes survival of stressed cells

2003 ◽  
Vol 163 (4) ◽  
pp. 767-775 ◽  
Author(s):  
Céline Jousse ◽  
Seiichi Oyadomari ◽  
Isabel Novoa ◽  
Phoebe Lu ◽  
Yuhong Zhang ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Translation Initiation ◽  
Mammalian Cells ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Regulatory Subunit ◽  
Gene Encoding ◽  
Eukaryotic Translation ◽  
Eif2α Phosphorylation ◽  
Stressed Cells

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) on serine 51 is effected by specific stress-activated protein kinases. eIF2α phosphorylation inhibits translation initiation promoting a cytoprotective gene expression program known as the integrated stress response (ISR). Stress-induced activation of GADD34 feeds back negatively on this pathway by promoting eIF2α dephosphorylation, however, GADD34 mutant cells retain significant eIF2α-directed phosphatase activity. We used a somatic cell genetic approach to identify a gene encoding a novel regulatory subunit of a constitutively active holophosphatase complex that dephosphorylates eIF2α. RNAi of this gene, which we named constitutive repressor of eIF2α phosphorylation (CReP, or PPP1R15B), repressed the constitutive eIF2α-directed phosphatase activity and activated the ISR. CReP RNAi strongly protected mammalian cells against oxidative stress, peroxynitrite stress, and more modestly against accumulation of malfolded proteins in the endoplasmic reticulum. These findings suggest that therapeutic inhibition of eIF2α dephosphorylation by targeting the CReP-protein–phosphatase-1 complex may be used to access the salubrious qualities of the ISR.

scholarly journals Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation

2020 ◽  
Vol 117 (20) ◽  
pp. 10935-10945 ◽  
Author(s):  
Shanta Karki ◽  
Kathrina Castillo ◽  
Zhaolan Ding ◽  
Olivia Kerr ◽  
Teresa M. Lamb ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nutrient Metabolism ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase ◽  
The Impact

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.

scholarly journals mRNA Decay during Herpes Simplex Virus (HSV) Infections: Protein-Protein Interactions Involving the HSV Virion Host Shutoff Protein and Translation Factors eIF4H and eIF4A

2005 ◽  
Vol 79 (15) ◽  
pp. 9651-9664 ◽  
Author(s):  
Pinghui Feng ◽  
David N. Everly ◽  
G. Sullivan Read
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Translation Initiation ◽  
Mammalian Cells ◽  
Translation Initiation Factor ◽  
Site Directed Mutagenesis ◽  
Initiation Factor ◽  
Virion Host Shutoff ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein of herpes simplex virus accelerates the degradation of both host and viral mRNAs. In so doing, it helps redirect the cell from host to viral protein synthesis and facilitates the sequential expression of different viral genes. Vhs interacts with the cellular translation initiation factor eIF4H, and several point mutations that abolish its mRNA degradative activity also abrogate its ability to bind eIF4H. In addition, a complex containing bacterially expressed Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein has RNase activity. eIF4H shares a region of sequence homology with eIF4B, and it appears to be functionally similar in that both stimulate the RNA helicase activity of eIF4A, a component of the mRNA cap-binding complex eIF4F. We show that eIF4H interacts physically with eIF4A in the yeast two-hybrid system and in GST pull-down assays and that the two proteins can be coimmunoprecipitated from mammalian cells. Vhs also interacts with eIF4A in GST pull-down and coimmunoprecipitation assays. Site-directed mutagenesis of Vhs and eIF4H revealed residues of each that are important for their mutual interaction, but not for their interaction with eIF4A. Thus, Vhs, eIF4H, and eIF4A comprise a group of proteins, each of which is able to interact directly with the other two. Whether they interact simultaneously as a tripartite complex or sequentially is unclear. The data suggest a mechanism for linking the degradation of an mRNA to its translation and for targeting Vhs to mRNAs and to regions of translation initiation.

scholarly journals Substrate recognition determinants of human eIF2α phosphatases

2021 ◽  
Author(s):  
George Hodgson ◽  
Antonina Andreeva ◽  
Anne Bertolotti
Keyword(s):  
Mammalian Cells ◽  
Biological Significance ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type ◽  
Binding Region ◽  
Antagonistic Action ◽  
Catalytic Subunits ◽  
Eif2α Phosphorylation ◽  
Cellular Defence

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Attempts at reconstituting recombinant holophosphatases have generated two models, one proposing that substrate recruitment requires the addition of actin, whilst the second proposes that this function is encoded by R15s. The biological relevance of actin in substrate recruitment has not been evaluated. Here we generated a series of truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A325-554 and R15B340-639. Actin does not bind these regions establishing that it is not required for substrate recruitment. Activity assays in cells showed that R15A325-674 and R15B340-713, encompassing the substrate-binding region and the PP1 binding-region, exhibit wild-type activity. This study identifies essential regions of R15s and demonstrates they function as substrate receptors. This work will guide the design of future structural studies with biological significance.

scholarly journals Efficient Translation of Rotavirus mRNA Requires Simultaneous Interaction of NSP3 with the Eukaryotic Translation Initiation Factor eIF4G and the mRNA 3′ End

2000 ◽  
Vol 74 (15) ◽  
pp. 7064-7071 ◽  
Author(s):  
Patrice Vende ◽  
Maria Piron ◽  
Nathalie Castagné ◽  
Didier Poncet
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Consensus Sequence ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Mrna ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT In contrast to the vast majority of cellular proteins, rotavirus proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically binds the 3′-end consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G. Here we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between the cap structure and the 3′ end of rotavirus mRNA was observed in NSP3-expressing cells. The enhancement of viral mRNA translation by NSP3 was also observed in a rabbit reticulocyte lysate translation system supplemented with recombinant NSP3. The use of NSP3 mutants indicates that its RNA- and eIF4G-binding domains are both required to enhance the translation of viral mRNA. The results reported here show that NSP3 forms a link between viral mRNA and the cellular translation machinery and hence is a functional analogue of cellular poly(A)-binding protein.

scholarly journals Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

2016 ◽  
Author(s):  
Colin Echeverría Aitken ◽  
Petra Beznosková ◽  
Vladislava Vlčkova ◽  
Wen-Ling Chiu ◽  
Fujun Zhou ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Critical Role ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Functional Variants ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 3

AbstractEukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2·GTP·Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncover a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

scholarly journals Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Colin Echeverría Aitken ◽  
Petra Beznosková ◽  
Vladislava Vlčkova ◽  
Wen-Ling Chiu ◽  
Fujun Zhou ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Critical Role ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Functional Variants ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 3

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

scholarly journals Snf1 Promotes Phosphorylation of the α Subunit of Eukaryotic Translation Initiation Factor 2 by Activating Gcn2 and Inhibiting Phosphatases Glc7 and Sit4

2010 ◽  
Vol 30 (12) ◽  
pp. 2862-2873 ◽  
Author(s):  
Vera Cherkasova ◽  
Hongfang Qiu ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Activation Loop ◽  
Α Subunit ◽  
Eukaryotic Translation ◽  
Eif2α Phosphorylation ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 2

ABSTRACT Snf1 is the ortholog of mammalian AMP-activated kinase and is responsible for activation of glucose-repressed genes at low glucose levels in budding yeast. We show that Snf1 promotes the formation of phosphorylated α subunit of eukaryotic translation initiation factor 2 (eIF2α-P), a regulator of general and gene-specific translation, by stimulating the function of eIF2α kinase Gcn2 during histidine starvation of glucose-grown cells. Thus, eliminating Snf1 or mutating its activation loop lowers Gcn2 kinase activity, reducing the autophosphorylation of Thr-882 in the Gcn2 activation loop, and decreases eIF2α-P levels in starved cells. Consistently, eliminating Reg1, a negative regulator of Snf1, provokes Snf1-dependent hyperphosphorylation of both Thr-882 and eIF2α. Interestingly, Snf1 also promotes eIF2α phosphorylation in the nonpreferred carbon source galactose, but this occurs by inhibition of protein phosphatase 1α (PP1α; Glc7) and the PP2A-like enzyme Sit4, rather than activation of Gcn2. Both Glc7 and Sit4 physically interact with eIF2α in cell extracts, supporting their direct roles as eIF2α phosphatases. Our results show that Snf1 modulates the level of eIF2α phosphorylation by different mechanisms, depending on the kind of nutrient deprivation existing in cells.

scholarly journals mRNA Decay during Herpesvirus Infections: Interaction between a Putative Viral Nuclease and a Cellular Translation Factor

2001 ◽  
Vol 75 (21) ◽  
pp. 10272-10280 ◽  
Author(s):  
Pinghui Feng ◽  
David N. Everly ◽  
G. Sullivan Read
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Mrnas ◽  
Cellular Translation ◽  
Simplex Virus

ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5′ end of mRNAs prior to the 3′ end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using theSaccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathioneS-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.

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