Effect of cleavage of the p220 subunit of eukaryotic translation initiation factor eIF-4F on protein synthesis in vitro

Eukaryotic translation initiation factor 2α (eIF-2α), a target molecule of the interferon-inducible double-stranded-RNA-dependent protein kinase (PKR), was cleaved in apoptotic Saos-2 cells on treatment with poly(I)˙poly(C) or tumour necrosis factor α. This cleavage occurred with a time course similar to that of poly(ADP-ribose) polymerase, a well-known caspase substrate. In addition, eIF-2α was cleaved by recombinant active caspase-3 in vitro. By site-directed mutagenesis, the cleavage site was mapped to an Ala-Glu-Val-Asp300 ↓ Gly301 sequence located in the C-terminal portion of eIF-2α. PKR phosphorylates eIF-2α on Ser51, resulting in the suppression of protein synthesis. PKR-mediated translational suppression was repressed when the C-terminally cleaved product of eIF-2α was overexpressed in Saos-2 cells, even though PKR can phosphorylate this cleaved product. These results suggest that caspase-3 or related protease(s) can modulate the efficiency of protein synthesis by cleaving the α subunit of eIF-2, a key component in the initiation of translation.

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

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.6876 ◽

1997 ◽

Vol 17 (12) ◽

pp. 6876-6886 ◽

Cited By ~ 52

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.

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Implications of the Use of Eukaryotic Translation Initiation Factor 5A (eIF5A) for Prognosis and Treatment of Hepatocellular Carcinoma

International Journal of Hepatology ◽

10.1155/2012/760928 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6 ◽

Cited By ~ 14

Author(s):

Felix H. Shek ◽

Sarwat Fatima ◽

Nikki P. Lee

Keyword(s):

Hepatocellular Carcinoma ◽

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Initiation Of Translation ◽

Eukaryotic Translation Initiation ◽

Hcc Cells

Hepatocellular carcinoma (HCC) is a primary liver malignancy and accounts for most of the total liver cancer cases. Lack of treatment options and late diagnosis contribute to high mortality rate of HCC. In eukaryotes, translation of messenger RNA (mRNA) to protein is a key process in protein biosynthesis in which initiation of translation involves interaction of different eukaryotic translation initiation factors (eIFs), ribosome subunits and mRNAs. Eukaryotic translation initiation factor 5A (eIF5A) is one of the eIFs involved in translation initiation and eIF5A2, one of its isoforms, is upregulated in various cancers including HCC as a result of chromosomal instability, where it resides. In HCC, eIF5A2 expression is associated with adverse prognosis such as presence of tumor metastasis and venous infiltration. Based on eIF5A2 functional studies, suppressing eIF5A2 expression by short interfering RNA alleviates the tumorigenic properties of HCC cellsin vitrowhile ectopic expression of eIF5A2 enhances the aggressiveness of HCC cellsin vivoandin vitroby inducing epithelial-mesenchymal transition. In conclusion, eIF5A2 is a potential prognostic marker as well as a therapeutic target for HCC.

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The 39-kilodalton subunit of eukaryotic translation initiation factor 3 is essential for the complex's integrity and for cell viability in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.17.1.145 ◽

1997 ◽

Vol 17 (1) ◽

pp. 145-153 ◽

Cited By ~ 33

Author(s):

T Naranda ◽

M Kainuma ◽

S E MacMillan ◽

J W Hershey

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Synthesis ◽

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Initiation Phase ◽

Eukaryotic Translation Initiation ◽

Translation Initiation Factor 3

Eukaryotic translation initiation factor 3 (eIF3) in the yeast Saccharomyces cerevisiae comprises about eight polypeptides and plays a central role in the binding of methionyl-tRNAi and mRNA to the 40S ribosomal subunit. The fourth largest subunit, eIF3-p39, was gel purified, and a 12-amino-acid tryptic peptide was sequenced, enabling the cloning of the TIF34 gene. TIF34 encodes a 38,753-Da protein that corresponds to eIF3-p39 in size and antigenicity. Disruption of TIF34 is lethal, and depletion of eIF3-p39 by glucose repression of TIF34 expressed from a GAL promoter results in cessation of cell growth. As eIF3-p39 levels fall, polysomes become smaller, indicating a role for eIF3-p39 in the initiation phase of protein synthesis. Unexpectedly, depletion results in degradation of all of the subunit proteins of eIF3 at a rate much faster than the normal turnover rates of these proteins. eIF3-p39 has 46% sequence identity with the p36 subunit of human eIF3. Both proteins are members of the WD-repeat family of proteins, possessing five to seven repeat elements. Taken together, the results indicate that eIF3-p39 plays an important, although not necessarily direct, role in the initiation phase of protein synthesis and suggest that it may be required for the assembly and maintenance of the eIF3 complex in eukaryotic cells.

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Generation of Multiple Isoforms of Eukaryotic Translation Initiation Factor 4GI by Use of Alternate Translation Initiation Codons

Molecular and Cellular Biology ◽

10.1128/mcb.22.13.4499-4511.2002 ◽

2002 ◽

Vol 22 (13) ◽

pp. 4499-4511 ◽

Cited By ~ 58

Author(s):

Marshall P. Byrd ◽

Miguel Zamora ◽

Richard E. Lloyd

Keyword(s):

Translation Initiation ◽

Cdna Clone ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation ◽

Ires Element

ABSTRACT Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5′ end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5′ sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5′ untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.

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Correction for Vosler et al., Ischemia-induced calpain activation causes eukaryotic (translation) initiation factor 4G1 (eIF4GI) degradation, protein synthesis inhibition, and neuronal death

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1202401109 ◽

2012 ◽

Vol 109 (10) ◽

pp. 4020-4020 ◽

Cited By ~ 1

Keyword(s):

Protein Synthesis ◽

Translation Initiation ◽

Neuronal Death ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Protein Synthesis Inhibition ◽

Eukaryotic Translation Initiation Factor ◽

Synthesis Inhibition ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation

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

10.1101/072702 ◽

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.

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

eLife ◽

10.7554/elife.20934 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 30

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.

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