scholarly journals Mutational Analysis of Plant Cap-Binding Protein eIF4E Reveals Key Amino Acids Involved in Biochemical Functions and Potyvirus Infection

2008 ◽  
Vol 82 (15) ◽  
pp. 7601-7612 ◽  
Author(s):  
Sylvie German-Retana ◽  
Jocelyne Walter ◽  
Bénédicte Doublet ◽  
Geneviève Roudet-Tavert ◽  
Valérie Nicaise ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Point Mutations ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Natural Resistance ◽  
Initiation Factor ◽  
Amino Acid Residues ◽  
Eukaryotic Translation

ABSTRACT The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo1 1 and mo1 2 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo1 1 or mo1 2 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.

scholarly journals Eukaryotic Translation Initiation Factor 5 Is Critical for Integrity of the Scanning Preinitiation Complex and Accurate Control of GCN4 Translation

2005 ◽  
Vol 25 (13) ◽  
pp. 5480-5491 ◽  
Author(s):  
Chingakham Ranjit Singh ◽  
Cynthia Curtis ◽  
Yasufumi Yamamoto ◽  
Nathan S. Hall ◽  
Dustin S. Kruse ◽  
...  
Keyword(s):  
Amino Acid ◽  
Translation Initiation ◽  
Translational Control ◽  
Point Mutations ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Preinitiation Complex ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal preinitiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for preinitiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts− phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd− phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNAi Met ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn− phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the postassembly process in the 48S complex, likely during the scanning process.

scholarly journals Synthetic mRNAs; Their Analogue Caps and Contribution to Disease

2021 ◽  
Author(s):  
Anthony Kyriakopoulos ◽  
Peter McCullough
Keyword(s):  
Cell Cycle Progression ◽  
Globin Gene ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Transcriptional Level ◽  
Beta Globin ◽  
Eukaryotic Translation ◽  
Successful Design ◽  
Eukaryotic Translation Initiation

The structure of synthetic mRNAs as used in vaccination against cancer and infectious diseases contain specifically designed caps followed by sequences of the 5’ untranslated repeats of β-globin gene. The strategy for successful design of synthetic mRNAs by chemically modifying their caps aims to increase resistance to the enzymatic deccapping complex, offer a higher affinity for binding to the eukaryotic translation initiation factor 4E (elF4E) protein and enforce increased translation of their encoded proteins. However, the cellular homeostasis is finely balanced and obeys to specific laws of thermodynamics conferring balance between complexity and growth rate in evolution. An overwhelming and forced translation even under alarming conditions of the cell during a concurrent viral infection, or when molecular pathways are trying to circumvent precursor events that lead to autoimmunity and cancer, may cause the recipient cells to ignore their differential sensitivities which are essential for keeping normal conditions. The elF4E which is a powerful RNA regulon and a potent oncogene governing cell cycle progression and proliferation at a post-transcriptional level, may then be a great contributor to disease development. The mechanistic target of rapamycin (mTOR) axis manly inhibits the elF4E to proceed with mRNA translation but disturbance in fine balances between mTOR and elF4E action may provide a premature step towards oncogenesis, ignite pre-causal mechanisms of immune deregulation and cause maturation (aging) defects.

scholarly journals Binding of eukaryotic translation initiation factor 4E (eIF4E) to eIF4G represses translation of uncapped mRNA.

1997 ◽  
Vol 17 (12) ◽  
pp. 6876-6886 ◽  
Author(s):  
S Z Tarun ◽  
A B Sachs
Keyword(s):  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Stimulation Of

mRNA translation in crude extracts from the yeast Saccharomyces cerevisiae is stimulated by the cap structure and the poly(A) tail through the binding of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) and the poly(A) tail-binding protein Pab1p. These proteins also bind to the translation initiation factor eIF4G and thereby link the mRNA to the general translational apparatus. In contrast, uncapped, poly(A)-deficient mRNA is translated poorly in yeast extracts, in part because of the absence of eIF4E and Pab1p binding sites on the mRNA. Here, we report that uncapped-mRNA translation is also repressed in yeast extracts due to the binding of eIF4E to eIF4G. Specifically, we find that mutations which weaken the eIF4E binding site on the yeast eIF4G proteins Tif4631p and Tif4632p lead to temperature-sensitive growth in vivo and the stimulation of uncapped-mRNA translation in vitro. A mutation in eIF4E which disturbs its ability to interact with eIF4G also leads to a stimulation of uncapped-mRNA translation in vitro. Finally, overexpression of eIF4E in vivo or the addition of excess eIF4E in vitro reverses these effects of the mutations. These data support the hypothesis that the eIF4G protein can efficiently stimulate translation of exogenous uncapped mRNA in extracts but is prevented from doing so as a result of its association with eIF4E. They also suggest that some mRNAs may be translationally regulated in vivo in response to the amount of free eIF4G in the cell.

scholarly journals PLK1 regulates spindle association of phosphorylated eukaryotic translation initiation factor 4E-binding protein and spindle function in mouse oocytes

2017 ◽  
Vol 313 (5) ◽  
pp. C501-C515 ◽  
Author(s):  
Ashley L. Severance ◽  
Keith E. Latham
Keyword(s):  
Translation Initiation ◽  
Binding Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Spindle Formation ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Chromosome Congression ◽  
Eukaryotic Translation Initiation ◽  
Meiotic Spindles

Oocyte meiotic spindles are associated with spindle-enriched mRNAs, phosphorylated ribosome protein S6, and phosphorylated variants of the key translational regulator, eukaryotic translation initiation factor 4E-binding protein 1 (eIF4E-BP1), consistent with translational control of localized mRNAs by eIF4E-BP1 in facilitating spindle formation and stability. Using specific kinase inhibitors, we determined which kinases regulate phosphorylation status of eIF4E-BP1 associated with meiotic spindles in mouse oocytes and effects of kinase inhibition on chromosome congression and spindle formation. Neither ataxia telangiectasia-mutated kinase nor mechanistic target of rapamycin inhibition significantly affected phosphorylation status of spindle-associated eIF4E-BP1 at the phosphorylation sites examined. Spindle-associated phospho-eIF4E-BP1, spindle formation, and chromosome congression were strongly disrupted by polo-like kinase I (PLK1) inhibition at both metaphase I (MI) and MII. In addition, direct inhibition of eIF4E-BP1 via 4EGI led to spindle defects at MI, indicating a direct role for eIF4E-BP1 phosphorylation in meiotic spindle formation. PLK1 also regulated microtubule dynamics throughout the ooplasm, indicating likely coordination between spindle dynamics and broader ooplasm cytoskeletal dynamics. Because diverse upstream signaling pathways converge on PLK1, these results implicate PLK1 as a major regulatory nexus coupling endogenous and exogenous signals via eIF4E-BP1 to the regulation of spindle formation and stability.

scholarly journals Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein

2012 ◽  
Vol 93 (7) ◽  
pp. 1483-1494 ◽  
Author(s):  
Michelle M. Arnold ◽  
Catie Small Brownback ◽  
Zenobia F. Taraporewala ◽  
John T. Patton
Keyword(s):  
Binding Protein ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Host Protein ◽  
Initiation Factor ◽  
Nuclear Accumulation ◽  
Progeny Virus ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Infected Cells

The rotavirus (RV) non-structural protein NSP3 forms a dimer that has binding domains for the translation initiation factor eIF4G and for a conserved 3′-terminal sequence of viral mRNAs. Through these activities, NSP3 has been proposed to promote viral mRNA translation by directing circularization of viral polysomes. In addition, by disrupting interactions between eIF4G and the poly(A)-binding protein (PABP), NSP3 has been suggested to inhibit translation of host polyadenylated mRNAs and to stimulate relocalization of PABP from the cytoplasm to the nucleus. Herein, we report the isolation and characterization of SA11-4Fg7re, an SA11-4F RV derivative that contains a large sequence duplication initiating within the genome segment (gene 7) encoding NSP3. Our analysis showed that mutant NSP3 (NSP3m) encoded by SA11-4Fg7re is almost twice the size of the wild-type protein and retains the capacity to dimerize. However, in comparison to wild-type NSP3, NSP3m has a decreased capacity to interact with eIF4G and to suppress the translation of polyadenylated mRNAs. In addition, NSP3m fails to induce the nuclear accumulation of PABP in infected cells. Despite the defective activities of NSP3m, the levels of viral protein and progeny virus produced in SA11-4Fg7re- and SA11-4F-infected cells were indistinguishable. Collectively, these data are consistent with a role for NSP3 in suppressing host protein synthesis through antagonism of PABP activity, but also suggest that NSP3 functions may have little or no impact on the efficiency of virus replication in widely used RV-permissive cell lines.

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