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 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 A C. elegans model of C9orf72-associated ALS/FTD uncovers a conserved role for eIF2D in RAN translation

2020 ◽  
Author(s):  
Yoshifumi Sonobe ◽  
Jihad Aburas ◽  
Priota Islam ◽  
Tania F. Gendron ◽  
André E.X. Brown ◽  
...  
Keyword(s):  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
C Elegans ◽  
Hexanucleotide Repeat ◽  
Eukaryotic Translation Initiation ◽  
Hexanucleotide Repeat Expansion ◽  
Lateral Sclerosis ◽  
Ran Translation

ABSTRACTA hexanucleotide repeat expansion GGGGCC in the noncoding region of C9orf72 is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Potentially toxic dipeptide repeats (DPRs) are synthesized from the GGGGCC sequence via repeat associated non-AUG (RAN) translation. We developed C. elegans models that express, either ubiquitously or exclusively in neurons, a transgene with 75 GGGGCC repeats flanked by intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly-glycine-proline [poly-GP]) and display neurodegeneration, locomotor and lifespan defects. Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats blocked poly-GA production and ameliorated disease, suggesting poly-GA is pathogenic. Importantly, eukaryotic translation initiation factor 2D (eif-2D/eIF2D) was necessary for RAN translation. Genetic removal of eif-2D increased lifespan in both C. elegans models. In vitro findings in human cells demonstrated a conserved role for eif-2D/eIF2D in RAN translation that could be exploited for ALS and FTD therapy.

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

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
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.

scholarly journals The role of hypusine depletion in cytostasis induced by S-adenosyl-l-methionine decarboxylase inhibition: new evidence provided by 1-methylspermidine and 1,12-dimethylspermine

1994 ◽  
Vol 303 (2) ◽  
pp. 363-368 ◽  
Author(s):  
T L Byers ◽  
J R Lakanen ◽  
J K Coward ◽  
A E Pegg
Keyword(s):  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Decarboxylase Inhibitor ◽  
Deoxyhypusine Synthase ◽  
Eukaryotic Translation ◽  
Polyamine Analogues ◽  
L1210 Cells ◽  
Eukaryotic Translation Initiation ◽  
New Evidence

The abilities of the natural polyamines, spermidine and spermine, and of the synthetic analogues, 1-methylspermidine and 1,12-dimethylspermine, to reverse the effects of the S-adenosyl-L-methionine decarboxylase inhibitor 5′-([(Z)-4-aminobut-2-enyl]methylamino)-5′-deoxyadenosine (AbeAdo) on L1210-cell growth were studied. L1210 cells were exposed to AbeAdo for 12 days to induce cytostasis and then exposed to spermidine, spermine, 1-methylspermidine or 1,12-dimethylspermine in the continued presence of AbeAdo. AbeAdo-induced cytostasis was overcome by the natural polyamines, spermidine and spermine. The cytostasis was also reversed by 1-methylspermidine. 1,12-Dimethylspermine had no effect on the AbeAdo-induced cytostasis of chronically treated cells, although it was active in permitting growth of cells treated with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. The initial 12-day exposure to AbeAdo elevated intracellular putrescine levels, depleted intracellular spermidine and spermine, and resulted in the accumulation of unmodified eukaryotic translation initiation factor 5A (eIF-5A). Exposure of these cells to exogenous spermidine, which is the natural substrate for deoxyhypusine synthase, resulted in a decrease in the unmodified eIF-5A content. 1-Methylspermidine, which was found to be a substrate of deoxyhypusine synthase in vitro, also decreased the levels of unmodified eIF-5A in the AbeAdo-treated cells. Although spermine is not a substrate of deoxyhypusine synthase, spermine was converted into spermidine in the L1210 cells, and spermine addition to AbeAdo-treated cells resulted in the appearance of both intracellular spermine and spermidine and in the decrease in unmodified eIF-5A. Exogenous 1,12-dimethylspermine, which was not metabolized to spermine or to 1-methylspermidine and was not a substrate of deoxyhypusine synthase in vitro, did not decrease levels of unmodified eIF-5A. The finding that AbeAdo-induced cytostasis was only reversed by polyamines and polyamine analogues that result in the formation of hypusine or an analogue in eIF-5A is consistent with the hypothesis [Byers, Wiest, Wechter and Pegg (1993) Biochem. J. 290, 115-121] that AbeAdo-induced cytostasis is due to the depletion of the hypusine-containing form of eIF-5A, which is secondary to the depletion of spermidine by inhibition of S-adenosyl-L-methionine decarboxylase.

scholarly journals Interaction in vitro between the proteinase of Tomato ringspot virus (genus Nepovirus) and the eukaryotic translation initiation factor iso4E from Arabidopsis thaliana

2002 ◽  
Vol 83 (8) ◽  
pp. 2085-2089 ◽  
Author(s):  
Simon Léonard ◽  
Joan Chisholm ◽  
Jean-François Laliberté ◽  
Hélène Sanfaçon
Keyword(s):  
Virus Genome ◽  
Translation Initiation Factor ◽  
Ringspot Virus ◽  
Initiation Factor ◽  
Virus Infectivity ◽  
Eukaryotic Translation ◽  
Tomato Ringspot Virus ◽  
Cellular Mrnas ◽  
Eukaryotic Translation Initiation

Eukaryotic initiation factor eIF(iso)4E binds to the cap structure of mRNAs leading to assembly of the translation complex. This factor also interacts with the potyvirus VPg and this interaction has been correlated with virus infectivity. In this study, we show an interaction between eIF(iso)4E and the proteinase (Pro) of a nepovirus (Tomato ringspot virus; ToRSV) in vitro. The ToRSV VPg did not interact with eIF(iso)4E although its presence on the VPg-Pro precursor increased the binding affinity of Pro for the initiation factor. A major determinant of the interaction was mapped to the first 93 residues of Pro. Formation of the complex was inhibited by addition of m7GTP (a cap analogue), suggesting that Pro-containing molecules compete with cellular mRNAs for eIF(iso)4E binding. The possible implications of this interaction for translation and/or replication of the virus genome are discussed.

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 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.

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