scholarly journals Eukaryotic Translation Initiation Factor 3 (eIF3) and eIF2 Can Promote mRNA Binding to 40S Subunits Independently of eIF4G in Yeast

2006 ◽  
Vol 26 (4) ◽  
pp. 1355-1372 ◽  
Author(s):  
Antonina V. Jivotovskaya ◽  
Leoš Valášek ◽  
Alan G. Hinnebusch ◽  
Klaus H. Nielsen
Keyword(s):  
Ternary Complex ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Initiation Factors ◽  
Eukaryotic Translation Initiation ◽  
Mrna Binding ◽  
Rate Limiting

ABSTRACT Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAi Met ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.

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 Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

2007 ◽  
Vol 27 (6) ◽  
pp. 2384-2397 ◽  
Author(s):  
Jeanne M. Fringer ◽  
Michael G. Acker ◽  
Christie A. Fekete ◽  
Jon R. Lorsch ◽  
Thomas E. Dever
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Cell Extracts ◽  
Eukaryotic Translation Initiation ◽  
Subunit Joining

ABSTRACT The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

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

2002 ◽  
Vol 22 (13) ◽  
pp. 4499-4511 ◽  
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.

scholarly journals Cytoplasmic eIF6 Promotes OSCC Malignant Behavior Through AKT Pathway

2021 ◽  
Author(s):  
Zechen Zhao ◽  
Weming Chu ◽  
Yang Zheng ◽  
Chao Wang ◽  
Yuemei Yang ◽  
...  
Keyword(s):  
Western Blot ◽  
Direct Interaction ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Migration And Invasion ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Mesenchymal Transformation

Abstract Background: Eukaryotic translation initiation factor 6 (eIF6), also known as integrin β4 binding protein, is involved in the formation and translation of ribosomes and acts as an anti-association factor. It is also essential for the growth and reproduction of cells, including tumor cells. Yet, its role in oral squamous cell carcinoma (OSCC) remains unclear. Methods: The expression characteristics of eIF6 in 233 samples were comprehensively analyzed by immumohistochemical staining (IHC). Effects of eIF6 over-expression and knockdown on cell proliferation, migration and invasion were determined by CCK-8, wound healing and Transwell assays. Western blot, immunofluorescence (IF) and co-immunoprecipitation (co-IP) were performed for mechanism verification.Results: We found that cytoplasmic eIF6 was abnormally highly expressed in OSCC tissues, and its expression was associated with tumor size and the clinical grade. Amplification of eIF6 promoted the growth, migration, and invasion capabilities of OSCC cell lines in vitro and tumor growth in vivo. Through Western blot analysis, we further discovered that eIF6 significantly promotes epithelial-mesenchymal transformation (EMT) in OSCC cells, while depletion of eIF6 can reverse this process. Mechanistically, eIF6 promotes tumor progression by activating the AKT signaling pathway. By performing co-immunoprecipitation, we discovered a direct interaction between endogenous eIF6 and AKT protein in the cytoplasm. Conclusions: This was the first report on the role and mechanism of eIF6 in OSCC. These results demonstrated that eIF6 could be a new therapeutic target in OSCC, thus providing a new basis for the prognosis of OSCC patients in the future.

scholarly journals The β Subunit of Eukaryotic Translation Initiation Factor 2 Binds mRNA through the Lysine Repeats and a Region Comprising the C2-C2 Motif

1999 ◽  
Vol 19 (1) ◽  
pp. 173-181 ◽  
Author(s):  
Jomar P. Laurino ◽  
Glória M. Thompson ◽  
Eliza Pacheco ◽  
Beatriz A. Castilho
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Initiation Factor 2 ◽  
Lysine Residues ◽  
Eukaryotic Translation Initiation ◽  
Mrna Binding ◽  
Translation Initiation Factor 2

ABSTRACT Eukaryotic translation initiation factor 2 (eIF2) has been implicated in the selection of the AUG codon as the start site for eukaryotic translation initiation, since mutations in its three subunits in yeast that allow the recognition of a UUG codon by the anticodon of the initiator Met-tRNAMet have been identified. All such mutations in the beta subunit of eIF2 (eIF2β) mapped to a region containing a putative zinc finger structure of the C2-C2 type, indicating that these sequences could be involved in RNA recognition. Another feature of eIF2β that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three repeats of seven lysine residues which are highly conserved in other species. We show here the ability of eIF2β, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro. Through a deletion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C2-C2motif. Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run. We further show that only one run of lysine residues is sufficient for the in vivo function of eIF2β, probably through charge interaction, since its replacement by arginines did not impair cell viability, whereas substitution for alanines resulted in inviable cells. mRNA binding, but not GTP-dependent initiator Met-tRNAMet binding, by the eIF2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit.

scholarly journals Eukaryotic Translation Initiation Factor 3 Subunit B Is a Promoter in the Development and Progression of Pancreatic Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Haoyuan Ren ◽  
Gang Mai ◽  
Yong Liu ◽  
Rongchao Xiang ◽  
Chong Yang ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 3 ◽  
Subunit B

Background: Pancreatic cancer (PC) is a malignant tumor with hidden incidence, high degree of malignancy, rapid disease progression, and poor prognosis. Eukaryotic translation initiation factor 3 subunit B (EIF3B) is necessary for tumor growth, which is an alternative therapeutic target for many cancers. However, little is known about the relationship between EIF3B and PC.Methods: The expression of EIF3B in PC was detected by immunohistochemistry. EIF3B knockdown cell models were constructed by lentivirus infection. The MTT assay, the wound-healing assay, the transwell assay, the flow cytometry, and the Human Apoptosis Antibody Array was used to detect the effects of EIF3B knockdown on cell proliferation, cell migration, cell apoptosis, and cell cycle in vitro. Also, the effects of EIF3B knockdown on the tumor growth of PC were determined in vivo.Results: This study showed that the expression level of EIF3B was significantly up-regulated in PC tumor tissues and associated with pathological grade. In vitro, EIF3B knockdown inhibited the PC cell proliferation and migration, and the apoptosis levels were obviously promoted by regulating apoptosis-related proteins including Bcl-2, HSP27, HSP60, Survivin, sTNF-R2, TNF-α, TNF-β, TRAILR-3, TRAILR-4, and XIAP. Furthermore, the tumor growth of PC was inhibited after the knockdown of EIF3B in vivo.Conclusion: EIF3B was up-regulated in PC and was a promoter in the development and progression of PC, which could be considered as a therapeutic target for the treatment of PC.

scholarly journals DENR/MCTS1 knockdown modulates repeat-associated non-AUG translation

2021 ◽  
Author(s):  
Katelyn M Green ◽  
Shannon L Miller ◽  
Indranil Malik ◽  
Peter K Todd
Keyword(s):  
Inhibitory Effect ◽  
Translation Initiation Factor ◽  
Model Systems ◽  
Initiation Factor ◽  
Cgg Repeat ◽  
Eukaryotic Translation ◽  
Initiation Factors ◽  
Eukaryotic Translation Initiation ◽  
Ran Translation

Repeat associated non-AUG (RAN) translation of mRNAs containing repeat-expansion mutations produces toxic peptides in neurons of patients suffering from neurodegenerative diseases. Recent findings indicate that RAN translation in diverse model systems is not inhibited by cellular stressors that impair global translation through phosphorylation of the alpha subunit of eIF2, the essential eukaryotic translation initiation factor that brings the initiator tRNA to the 40S ribosome. Using in vitro, cell-based, and Drosophila models, we examined the role of alternative ternary complex factors that may function in place of eIF2, including eIF2A, eIF2D, and DENR/MCTS1. Among these factors, DENR knockdown had the greatest inhibitory effect on RAN translation of expanded GGGGCC and CGG repeat reporters, and its reduction improved survival of Drosophila expressing expanded GGGGCC repeats. Taken together, these data support a role for alternative initiation factors in RAN translation and suggest they may serve as novel therapeutic targets in neurodegenerative disease.

scholarly journals Cytoplasmic eIF6 Promotes OSCC Malignant Behavior Through AKT Pathway

2021 ◽  
Author(s):  
Zechen Zhao ◽  
Weiming Chu ◽  
Yang Zheng ◽  
Chao Wang ◽  
Yuemei Yang ◽  
...  
Keyword(s):  
Western Blot ◽  
Direct Interaction ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Migration And Invasion ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Mesenchymal Transformation

Abstract Background: Eukaryotic translation initiation factor 6 (eIF6), also known as integrin β4 binding protein, is involved in the formation and translation of ribosomes and acts as an anti-association factor. It is also essential for the growth and reproduction of cells, including tumor cells. Yet, its role in oral squamous cell carcinoma (OSCC) remains unclear. Methods: The expression characteristics of eIF6 in 233 samples were comprehensively analyzed by immumohistochemical staining (IHC). Effects of eIF6 over-expression and knockdown on cell proliferation, migration and invasion were determined by CCK-8, wound healing and Transwell assays. Western blot, immunofluorescence (IF) and co-immunoprecipitation (co-IP) were performed for mechanism verification.Results: We found that cytoplasmic eIF6 was abnormally highly expressed in OSCC tissues, and its expression was associated with tumor size and the clinical grade. Amplification of eIF6 promoted the growth, migration, and invasion capabilities of OSCC cell lines in vitro and tumor growth in vivo. Through Western blot analysis, we further discovered that eIF6 significantly promotes epithelial-mesenchymal transformation (EMT) in OSCC cells, while depletion of eIF6 can reverse this process. Mechanistically, eIF6 promotes tumor progression by activating the AKT signaling pathway. By performing co-immunoprecipitation, we discovered a direct interaction between endogenous eIF6 and AKT protein in the cytoplasm. Conclusion: This was the first report on the role and mechanism of eIF6 in OSCC. These results demonstrated that eIF6 could be a new therapeutic target in OSCC, thus providing a new basis for the prognosis of OSCC patients in the future.

Preclinical Studies Using Polyethylenimine (PEI) Nanoparticles Complexed with Eukaryotic Translation Initiation Factor 5A (eIF5A) siRNA and eIF5AK50R Plasmid DNA Demonstrates Significant Anti-Myeloma Activity in Vitro and in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 249-249
Author(s):  
John A. Lust ◽  
John E. Thompson ◽  
Catherine A. Taylor ◽  
Bin Ye ◽  
Zhongda Liu ◽  
...  
Keyword(s):  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Group 3 ◽  
Group 2 ◽  
Pei Nanoparticles ◽  
Sirna Group

Abstract Background: Hypusine is a unique amino acid present in all eukaryotes but found in only one highly conserved protein, eukaryotic translation initiation factor 5A. It is formed during a two-step post-translational modification of eIF5A. eIF5A has been identified as a critical factor for cell proliferation in yeast, as a modulator of p53, and recent studies indicate that the unhypusinated form of eIF5A is a pro-apoptotic protein. Because myeloma remains incurable, we hypothesized that induction of apoptosis by modulation of eIF5A may be a useful therapeutic approach. In this study we utilized PEI nanoparticles to deliver plasmid DNA and siRNA to myeloma tumor cells in vitro and in vivo. Methods: An siRNA was used to suppress levels of endogenous hypusinated eIF5A in tumors while an RNAi-resistant plasmid expressing a mutant of eIF5A (eIF5AK50R), that cannot be hypusinated, was used to increase levels of unhypusinated eIF5A which is proapoptotic. In vitro, KAS-6/1 myeloma cells were incubated with control siRNA/control plasmid, eIF5A siRNA alone, or eIF5A siRNA/eIF5AK50R for 72 hours in the presence or absence of IL-6. Apoptosis was quantitated using flow cytometry to detect annexin V/propidium iodide stained cells; results were corrected for percent apoptosis of the mock control to enable comparison between experiments. In vivo, SCID mice (3/group) were injected with 10 million KAS-6/1 cells SQ in the right flank and approximately 6 weeks later injected intratumorally twice weekly as follows: Group 1 control mice received 20 μg empty vector and 10 μg control siRNA. Group 2 received 20 μg eIF5AK50R plasmid and 10 μg control siRNA. Group 3 received 20 μg eIF5AK50R plasmid and 10 μg eIF5A siRNA. Tumor volumes were quantitated in mm3 using a2b/2 where a is the smaller dimension. Results: The in vitro results showed the following % apoptosis: Absence of IL-6 - Control: 6.5%, eIF5A siRNA/eIF5AK50R 30.3%. Presence of IL-6–Control:8.2%; eIF5A siRNA alone 36.5%; eIF5A siRNA/eIF5AK50R 45.2%. Western analysis demonstrated that the siRNA successfully inhibited the production of endogenous eIF5A. In vivo, after 24 days of monitoring tumor volume: group 1 mice showed that the KAS-6/1 cells grew from a mean tumor volume of 7±1 up to 102±16 mm3; tumors in group 2 mice remained relatively stable in size from 21±9 to 26±10 mm3. Group 3 mice showed a mean 92% tumor regression from 99±21 down to 7±1 mm3. Conclusions: Delivery of eIF5A siRNA and eIF5AK50R plasmid by PEI nanoparticles results in significant anti-tumoral reponses in vitro and in vivo. These studies may provide a novel therapy for myeloma patients with end stage disease that have failed standard treatments.

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