scholarly journals Translation Initiation Factor eIF3b Contains a Nine-Bladed β-Propeller and Interacts with the 40S Ribosomal Subunit

Structure ◽  
2014 ◽  
Vol 22 (6) ◽  
pp. 923-930 ◽  
Author(s):  
Yi Liu ◽  
Piotr Neumann ◽  
Bernhard Kuhle ◽  
Thomas Monecke ◽  
Stephanie Schell ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
40S Ribosomal Subunit

Expression of Ribosomal Protein S6 (R6SP) and Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (EIF4EBP1) Is Correlated in Acute Myelogenous Leukemia (AML) and Is Highly Prognostic, Especially in NPM1 and FLT3 Wildtype Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2369-2369
Author(s):  
Steven M. Kornblau ◽  
Chenyue W Hu ◽  
Yihua Qiu ◽  
Suk Young Yoo ◽  
Rebecca A Murray ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Npm1 Mutation ◽  
Eukaryotic Translation ◽  
Ribosomal Protein S6 ◽  
40S Ribosomal Subunit ◽  
Eukaryotic Translation Initiation

Abstract Background. Conceptually mRNA processing and ribosomal regulation should interact as both affect mRNA translation and protein production. We studied protein expression and functional relationships between proteins in AML using a custom made reverse phase protein array (RPPA), probed with 231 strictly validated antibodies. We found a relationship between expression of Ribosomal Protein S6 (HUGO name R6SP, a.k.a. S6RP) and Eukaryotic Translation Initiation Factor 4EBinding Protein 1, (HUGO name EIF4EBP1). R6SP, a 40S ribosomal subunit component, activated by phosphorylation, regulates cell growth via selective mRNA translation. EIF4EBP1 interacts with eIF4E to recruit the 40S ribosomal subunit, thereby affecting ribosomal assembly. When phosphorylated, in response to cellular signaling, it releases eIF4E allowing transcription. Methods. Our RPPA has protein from leukemia enriched cells from 511 newly diagnosed AML patients and was probed with 231 strictly validated antibodies, including antibodies against total RPS6 and forms phosphorylated on S235-236 and S240-244, and against total EIF4EBP1 and forms phosphorylated on T37 & 46, T70 and S65. Expression was compared to normal bone marrow derived CD34+ cells. Interaction networks with the other 224 proteins were generated from the RPPA data using glasso and supplemented by the literature of known interactions. Results. A heatmap of expression of the 3 R6SP and 4 PA2 forms was generated and hierarchical k-and means clustering performed (Fig A). Using the “Prototype Clustering ”method an optimal division into four clusters (Fig B) was determined. This includes an “All-Off” state (18%), a state characterized by weak activation of RPS6 alone (RP-Only, 36%) activation of only EIF4EBP1 (EIF4EBP1-Only, 26%) and a group where both were on simultaneously (Both-On). The RPS6 interactome (Fig B) showed the expected positive correlation with mTOR, and P70 (Hugo RPS6KB1) and a previously unknown, but very strong, negative correlation with transcription factor ZNF296. The EIF4EBP1 interactome showed the expected strong positive correlation with many signal transduction pathways (MAP2K1, MAPK14) and proliferation related proteins (pRB, EIF2AK, EIF2S1, FOXO3) and negative correlation with several transcription factors (GATA3, SPI1, CREB). Cluster membership was unassociated with most clinical features including cytogenetics, FLT3 , RAS and NPM1 mutation, excluding gender (more F in All-Off, more M in Both-On, p=0.01). EIF4EBP1 and Both-On had higher WBC (p=0.0001) and % marrow (p=0.0001) and blood blasts (0.0007) and lower platelet counts (p=0.025). Response rates did not differ, although fewer All-Off were primary refractory. Relapse was more common in EIF4EBP1-Only and Both-On clusters. Overall survival (OS) and remission duration (RemDur) (Fig C) of the EIF4EBP1-Only and Both-On clusters was inferior to that of the All-Off and RP-Only clusters (OS median 41 & 45 vs. 52 &63,p=0.06, RemDur 39 & 27 weeks vs. 63 & 53, p=0.008) but this was restricted to Intermediate cytogenetics cases (Fig C “IntCyto” OS 49 & 55 weeks vs. 107& 79 p=0.01, RemDur 37 & 35 weeks vs. 89 & 53 , p = 0.005) that were FLT3 mutation ((Fig C “FLT3-WT” OS p=0.006, RemDur p0.007) and NPM1 mutation negative (Fig C “NPM1-WT”, OS p=0.006, RemDur p=0.001). Conclusions. Activation of EIF4EBP1, with or without RPS6 activation is prognostically adverse in AML, particularly in intermediate cytogenetic cases with wildtype FLT3 and NPM1. This is associated with increased proliferation. Therapy directed against EIF4EBP1 activity, e.g. that block it's phosphorylation, may have utility in the ~46% of cases of AML that demonstrate high levels of EIF4EBP1 phosphorylation, especially in FLT3/NPM1 wildtype cases. Many agents that inhibit signal transduction pathways are in clinical development, analyzing them for the ability to inhibition the activation of EIF4EBP1 might identify clinically useful molecules. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev

2019 ◽  
Author(s):  
Francisco García-de-Gracia ◽  
Daniela Toro-Ascuy ◽  
Sebastián Riquelme-Barrios ◽  
Camila Pereira-Montecinos ◽  
Bárbara Rojas-Araya ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Viral Protein ◽  
Nuclear Export ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Transcript ◽  
40S Ribosomal Subunit ◽  
Cellular Mrnas ◽  
Hiv 1

ABSTRACTTranslation initiation of the human immunodeficiency virus type-1 (HIV-1) unspliced mRNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the unspliced mRNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF restricts HIV-1 replication by interfering with Gag synthesis from the unspliced mRNA. Our results indicate that CTIF associates with Rev through its N-terminal domain and is recruited onto the unspliced mRNA ribonucleoprotein complex in order to block translation. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that CTIF restricts HIV-2 but not MLV Gag synthesis indicating an inhibitory mechanism conserved in Rev-expressing human lentiviruses.

scholarly journals Translation Initiation Factor 2γ Mutant Alters Start Codon Selection Independent of Met-tRNA Binding

2008 ◽  
Vol 28 (22) ◽  
pp. 6877-6888 ◽  
Author(s):  
Pankaj V. Alone ◽  
Chune Cao ◽  
Thomas E. Dever
Keyword(s):  
Binding Affinity ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Codon Recognition ◽  
40S Ribosomal Subunit ◽  
Initiation Factor 2

ABSTRACT Selection of the AUG start codon for translation in eukaryotes is governed by codon-anticodon interactions between the initiator Met-tRNAi Met and the mRNA. Translation initiation factor 2 (eIF2) binds Met-tRNAi Met to the 40S ribosomal subunit, and previous studies identified Sui− mutations in eIF2 that enhanced initiation from a noncanonical UUG codon, presumably by impairing Met-tRNAi Met binding. Consistently, an eIF2γ-N135D GTP-binding domain mutation impairs Met-tRNAi Met binding and causes a Sui− phenotype. Intragenic A208V and A382V suppressor mutations restore Met-tRNAi Met binding affinity and cell growth; however, only A208V suppresses the Sui− phenotype associated with the eIF2γ-N135D mutation. An eIF2γ-A219T mutation impairs Met-tRNAi Met binding but unexpectedly enhances the fidelity of initiation, suppressing the Sui− phenotype associated with the eIF2γ-N135D,A382V mutant. Overexpression of eIF1, which is thought to monitor codon-anticodon interactions during translation initiation, likewise suppresses the Sui− phenotype of the eIF2γ mutants. We propose that structural alterations in eIF2γ subtly alter the conformation of Met-tRNAi Met on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNAi Met binding affinity.

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 Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (12) ◽  
pp. 7283-7294 ◽  
Author(s):  
D Kressler ◽  
J de la Cruz ◽  
M Rojo ◽  
P Linder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
18S Rrna ◽  
Amino Acid Level ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Northern Hybridization ◽  
Ribosomal Subunits ◽  
Dead Box

A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation.

Translation Initiation Factor IF2 Interacts with the 30 S Ribosomal Subunit via Two Separate Binding Sites

2006 ◽  
Vol 362 (4) ◽  
pp. 787-799 ◽  
Author(s):  
Enrico Caserta ◽  
Jerneja Tomšic ◽  
Roberto Spurio ◽  
Anna La Teana ◽  
Cynthia L. Pon ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Binding Sites ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor

scholarly journals Identification in the Ancient Protist Giardia lamblia of Two Eukaryotic Translation Initiation Factor 4E Homologues with Distinctive Functions

2005 ◽  
Vol 4 (5) ◽  
pp. 948-959 ◽  
Author(s):  
Lei Li ◽  
Ching C. Wang
Keyword(s):  
Translation Initiation ◽  
Giardia Lamblia ◽  
Binding Protein ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Affinity Column ◽  
Eukaryotic Translation Initiation Factor ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT Eukaryotic translation initiation factor 4E (eIF4E) binds to the m7GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m7GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m2,2,7GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m7GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m2,2,7GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m2,2,7GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.

scholarly journals Assembly of 48S Translation Initiation Complexesfrom Purified Components with mRNAs That Have Some Base Pairingwithin Their 5′ UntranslatedRegions

2003 ◽  
Vol 23 (24) ◽  
pp. 8925-8933 ◽  
Author(s):  
Sergei E. Dmitriev ◽  
Ilya M. Terenin ◽  
Yan E. Dunaevsky ◽  
William C. Merrick ◽  
Ivan N. Shatsky
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Beta Globin ◽  
Globin Mrna ◽  
Initiation Factors ◽  
40S Ribosomal Subunit ◽  
Cellular Mrnas ◽  
Actin Mrna

ABSTRACT The reconstitution of translation initiation complexes from purified components is a reliable approach to determine the complete set of essential canonical initiation factors and auxiliary proteins required for the 40S ribosomal subunit to locate the initiation codon on individual mRNAs. Until now, it has been successful mostly for formation of 48S translation initiation complexes with viral IRES elements. Among cap-dependent mRNAs, only globin mRNAs and transcripts with artificial 5′ leaders were amenable to this assembly. Here, with modified conditions for the reconstitution, 48S complexes have been successfully assembled with the 5′ UTR of beta-actin mRNA (84 nucleotides) and the tripartite leader of adenovirus RNAs (232 nucleotides), though the latter has been able to use only the scanning rather then the shunting model of translation initiation with canonical initiation factors. We show that initiation factor 4B is essential for mRNAs that have even a rather moderate base pairing within their 5′ UTRs (with the cumulative stability of the secondary structure within the entire 5′ UTR < −13 kcal/mol) and not essential for beta-globin mRNA. A recombinant eIF4B poorly substitutes for the native factor. The 5′ UTRs with base-paired G residues reveal a very sharp dependence on the eIF4B concentration to form the 48S complex. The data suggest that even small variations in concentration or activity of eIF4B in mammalian cells may differentially affect the translation of different classes of cap-dependent cellular mRNAs.

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