scholarly journals RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes

2020 ◽  
Vol 48 (15) ◽  
pp. 8562-8575 ◽  
Author(s):  
Francis Robert ◽  
Regina Cencic ◽  
Renying Cai ◽  
T Martin Schmeing ◽  
Jerry Pelletier
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Structural Information ◽  
Critical Role ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Eif4f Complex ◽  
Cellular Mrnas ◽  
Open Question ◽  
Reporter Mrna

Abstract Eukaryotic cellular mRNAs possess a 5′ cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4GII can associate with either eIF4A1 or eIF4A2 to recruit ribosomes to mRNA templates. In combination with eIF4E and eIF4E3, our results indicate the presence of up to eight eIF4F complexes that can operate in translation initiation.

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.

scholarly journals Specific Isoforms of Translation Initiation Factor 4GI Show Differences in Translational Activity

2006 ◽  
Vol 26 (22) ◽  
pp. 8448-8460 ◽  
Author(s):  
Mark J. Coldwell ◽  
Simon J. Morley
Keyword(s):  
Translation Initiation ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Critical Role ◽  
Translation Initiation Factor ◽  
Protein Isoforms ◽  
Initiation Factor ◽  
Functional Studies ◽  
Eif2α Phosphorylation ◽  
Translational Activity

ABSTRACT The eukaryotic initiation factor (eIF) 4GI gene locus (eIF4GI) contains three identified promoters, generating alternately spliced mRNAs, yielding a total of five eIF4GI protein isoforms. Although eIF4GI plays a critical role in mRNA recruitment to the ribosomes, little is known about the functions of the different isoforms, their partner binding capacities, or the role of the homolog, eIF4GII, in translation initiation. To directly address this, we have used short interfering RNAs (siRNAs) expressed from DNA vectors to silence the expression of eIF4GI in HeLa cells. Here we show that reduced levels of specific mRNA and eIF4GI isoforms in HeLa cells promoted aberrant morphology and a partial inhibition of translation. The latter reflected dephosphorylation of 4E-BP1 and decreased eIF4F complex levels, with no change in eIF2α phosphorylation. Expression of siRNA-resistant Myc-tagged eIF4GI isoforms has allowed us to show that the different isoforms exhibit significant differences in their ability to restore translation rates. Here we quantify the efficiency of eIF4GI promoter usage in mammalian cells and demonstrate that even though the longest isoform of eIF4GI (eIF4GIf) was relatively poorly expressed when reintroduced, it was more efficient at promoting the translation of cellular mRNAs than the more highly expressed shorter isoforms used in previous functional studies.

scholarly journals The DEAD-box RNA helicase Ded1 has a role in the translational response to TORC1 inhibition

2019 ◽  
Vol 30 (17) ◽  
pp. 2171-2184 ◽  
Author(s):  
Peyman P. Aryanpur ◽  
David M. Renner ◽  
Emily Rodela ◽  
Telsa M. Mittelmeier ◽  
Aaron Byrd ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Helicase ◽  
Initiation Factor ◽  
Untranslated Regions ◽  
Eukaryotic Initiation Factor ◽  
Functional Requirements ◽  
Dead Box ◽  
Eif4f Complex ◽  
Mrna Pool ◽  
Mutant Cells

Ded1 is a DEAD-box RNA helicase with essential roles in translation initiation. It binds to the eukaryotic initiation factor 4F (eIF4F) complex and promotes 48S preinitiation complex assembly and start-site scanning of 5′ untranslated regions of mRNAs. Most prior studies of Ded1 cellular function were conducted in steady-state conditions during nutrient-rich growth. In this work, however, we examine its role in the translational response during target of rapamycin (TOR)C1 inhibition and identify a novel function of Ded1 as a translation repressor. We show that C-terminal mutants of DED1 are defective in down-regulating translation following TORC1 inhibition using rapamycin. Furthermore, following TORC1 inhibition, eIF4G1 normally dissociates from translation complexes and is degraded, and this process is attenuated in mutant cells. Mapping of the functional requirements for Ded1 in this translational response indicates that Ded1 enzymatic activity and interaction with eIF4G1 are required, while homo-oligomerization may be dispensable. Our results are consistent with a model wherein Ded1 stalls translation and specifically removes eIF4G1 from translation preinitiation complexes, thus removing eIF4G1 from the translating mRNA pool and leading to the codegradation of both proteins. Shared features among DED1 orthologues suggest that this role is conserved and may be implicated in pathologies such as oncogenesis.

scholarly journals CELF1 is an EIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

2019 ◽  
Author(s):  
Arindam Chaudhury ◽  
Rituraj Pal ◽  
Natee Kongchan ◽  
Na Zhao ◽  
Yingmin Zhu ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Epithelial Mesenchymal Transition ◽  
Binding Protein ◽  
Mrna Translation ◽  
Scaffolding Protein ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Mesenchymal Transition

AbstractMounting evidence is revealing a granularity within gene regulation that occurs at the level of mRNA translation. Within mammalian cells, canonical cap-dependent mRNA translation is dependent upon the interaction between the m7G cap-binding protein eukaryotic initiation factor 4E (eIF4E) and the scaffolding protein eukaryotic initiation factor 4G (eIF4G), the latter of which facilitates pre-translation initiation complex assembly, mRNA circularization, and ultimately ribosomal scanning. In breast epithelial cells, we previously demonstrated that the CELF1 RNA-binding protein promotes the translation of epithelial to mesenchymal transition (EMT) effector mRNAs containing GU-rich elements (GREs) within their 3’ untranslated regions (UTRs). Here we show that within this context, CELF1 directly binds to both the eIF4E cap-binding protein and Poly(A) binding protein (PABP), promoting translation of GRE-containing mRNAs in mesenchymal cells. Disruption of this CELF1/eIF4E interaction inhibits both EMT induction and experimental metastasis. Our findings illustrate a novel way in which non-canonical mechanisms of translation initiation underlie transitional cellular states within the context of development or human disease.

scholarly journals Cap-binding protein (eukaryotic initiation factor 4E) and 4E-inactivating protein BP-1 independently regulate cap-dependent translation.

1996 ◽  
Vol 16 (10) ◽  
pp. 5450-5457 ◽  
Author(s):  
D Feigenblum ◽  
R J Schneider
Keyword(s):  
Heat Shock ◽  
Translation Initiation ◽  
Binding Protein ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Metaphase Arrest ◽  
Animal Cells ◽  
Eukaryotic Initiation Factor 4E ◽  
Dependent Protein

Cap-dependent protein synthesis in animal cells is inhibited by heat shock, serum deprivation, metaphase arrest, and infection with certain viruses such as adenovirus (Ad). At a mechanistic level, translation of capped mRNAs is inhibited by dephosphorylation of eukaryotic initiation factor 4E (eIF-4E) (cap-binding protein) and its physical sequestration with the translation repressor protein BP-1 (PHAS-I). Dephosphorylation of BP-I blocks cap-dependent translation by promoting sequestration of eIF-4E. Here we show that heat shock inhibits translation of capped mRNAs by simultaneously inducing dephosphorylation of eIF-4E and BP-1, suggesting that cells might coordinately regulate translation of capped mRNAs by impairing both the activity and the availability of eIF-4E. Like heat shock, late Ad infection is shown to induce dephosphorylation of eIF-4E. However, in contrast to heat shock, Ad also induces phosphorylation of BP-1 and release of eIF-4E. BP-1 and eIF-4E can therefore act on cap-dependent translation in either a mutually antagonistic or cooperative manner. Three sets of experiments further underscore this point: (i) rapamycin is shown to block phosphorylation of BP-1 without inhibiting dephosphorylation of eIF-4E induced by heat shock or Ad infection, (ii) eIF-4E is efficiently dephosphorylated during heat shock or Ad infection regardless of whether it is in a complex with BP-1, and (iii) BP-1 is associated with eIF-4E in vivo regardless of the state of eIF-4E phosphorylation. These and other studies establish that inhibition of cap-dependent translation does not obligatorily involve sequestration of eIF-4E by BP-1. Rather, translation is independently regulated by the phosphorylation states of eIF-4E and the 4E-binding protein, BP-1. In addition, these results demonstrate that BP-1 and eIF-4E can act either in concert or in opposition to independently regulate cap-dependent translation. We suggest that independent regulation of eIF-4E and BP-1 might finely regulate the efficiency of translation initiation or possibly control cap-dependent translation for fundamentally different purposes.

scholarly journals Calpain Mediates Eukaryotic Initiation Factor 4G Degradation during Global Brain Ischemia

1998 ◽  
Vol 18 (8) ◽  
pp. 876-881 ◽  
Author(s):  
Robert W. Neumar ◽  
Donald J. DeGracia ◽  
Lynette L. Konkoly ◽  
John I. Khoury ◽  
Blaine C. White ◽  
...  
Keyword(s):  
Brain Ischemia ◽  
Critical Role ◽  
Initiation Factor ◽  
Ischemia And Reperfusion ◽  
Eukaryotic Initiation Factor ◽  
Global Brain Ischemia ◽  
Eukaryotic Initiation Factor 4G ◽  
Global Brain ◽  
Calpain Inhibitors

Global brain ischemia and reperfusion result in the degradation of the eukaryotic initiation factor (eIF) 4G, which plays a critical role in the attachment of the mRNA to the ribosome. Because eIF-4G is a substrate of calpain, these studies were undertaken to examine whether calpain I activation during global brain ischemia contributes to the degradation of eIF-4G in vivo. Immunoblots with antibodies against calpain I and eIF-4G were prepared from rat brain postmitochondrial supernatant incubated at 37°C with and without the addition of calcium and the calpain inhibitors calpastatin or MDL-28,170. Addition of calcium alone resulted in calpain I activation (as measured by autolysis of the 80-kDa subunit) and degradation of eIF-4G; this effect was blocked by either 1 μmol/L calpastatin or 10 μmol/L MDL-28,170. In rabbits subjected to 20 minutes of cardiac arrest, immunoblots of brain postmitochondrial supernatants showed that the percentage of autolyzed calpain I increased from 1.9% ± 1.1% to 15.8% ± 5.0% and that this was accompanied by a 68% loss of eIF-4G. MDL-28,170 pretreatment (30 mg/kg) decreased ischemia-induced calpain I autolysis 40% and almost completely blocked eIF-4G degradation. We conclude that calpain I degrades eIF-4G during global brain ischemia.

scholarly journals Unlike for cellular mRNAs and other viral internal ribosome entry sites (IRESs), the eIF3 subunit e is not required for the translational activity of the HCV IRES

2020 ◽  
Vol 295 (7) ◽  
pp. 1843-1856
Author(s):  
Baptiste Panthu ◽  
Solène Denolly ◽  
Cendrine Faivre-Moskalenko ◽  
Théophile Ohlmann ◽  
François-Loïc Cosset ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Hcv Rna ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Sites ◽  
Subunit E ◽  
Cellular Mrnas ◽  
Hcv Ires

Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5′UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels.

scholarly journals PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes

2003 ◽  
Vol 84 (12) ◽  
pp. 3263-3274 ◽  
Author(s):  
Idoia Burgui ◽  
Tomás Aragón ◽  
Juan Ortín ◽  
Amelia Nieto
Keyword(s):  
Influenza Virus ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Published Data ◽  
Ns1 Protein ◽  
Viral Mrnas ◽  
Independent Manner ◽  
Cellular Mrnas

It has previously been shown that influenza virus NS1 protein enhances the translation of viral but not cellular mRNAs. This enhancement occurs by increasing the rate of translation initiation and requires the 5′UTR sequence, common to all viral mRNAs. In agreement with these findings, we show here that viral mRNAs, but not cellular mRNAs, are associated with NS1 during virus infection. We have previously reported that NS1 interacts with the translation initiation factor eIF4GI, next to its poly(A)-binding protein 1 (PABP1)-interacting domain and that NS1 and eIF4GI are associated in influenza virus-infected cells. Here we show that NS1, although capable of binding poly(A), does not compete with PABP1 for association with eIF4GI and, furthermore, that NS1 and PABP1 interact both in vivo and in vitro in an RNA-independent manner. The interaction maps between residues 365 and 535 in PABP1 and between residues 1 and 81 in NS1. These mapping studies, together with those previously reported for NS1–eIF4GI and PABP1–eIF4GI interactions, imply that the binding of all three proteins would be compatible. Collectively, these and previously published data suggest that NS1 interactions with eIF4GI and PABP1, as well as with viral mRNAs, could promote the specific recruitment of 43S complexes to the viral mRNAs.

scholarly journals Dengue Virus Utilizes a Novel Strategy for Translation Initiation When Cap-Dependent Translation Is Inhibited

2006 ◽  
Vol 80 (6) ◽  
pp. 2976-2986 ◽  
Author(s):  
Dianna Edgil ◽  
Charlotta Polacek ◽  
Eva Harris
Keyword(s):  
Dengue Virus ◽  
Translation Initiation ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Initiation Factor 4E ◽  
Cellular Translation ◽  
Cellular Mrnas ◽  
Novel Strategy

ABSTRACT Viruses have developed numerous mechanisms to usurp the host cell translation apparatus. Dengue virus (DEN) and other flaviviruses, such as West Nile and yellow fever viruses, contain a 5′ m7GpppN-capped positive-sense RNA genome with a nonpolyadenylated 3′ untranslated region (UTR) that has been presumed to undergo translation in a cap-dependent manner. However, the means by which the DEN genome is translated effectively in the presence of capped, polyadenylated cellular mRNAs is unknown. This report demonstrates that DEN replication and translation are not affected under conditions that inhibit cap-dependent translation by targeting the cap-binding protein eukaryotic initiation factor 4E, a key regulator of cellular translation. We further show that under cellular conditions in which translation factors are limiting, DEN can alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that appears not to require a functional m7G cap. This DEN noncanonical translation is not mediated by an internal ribosome entry site but requires the interaction of the DEN 5′ and 3′ UTRs for activity, suggesting a novel strategy for translation of animal viruses.

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