Translation Initiation Factor eIF4F Research Articles

A small natural molecule CADPE kills residual colorectal cancer cells by inhibiting key transcription factors and translation initiation factors

Residual disease is the major cause for colorectal cancer (CRC) relapse. Herein, we explore whether and how a natural molecule CADPE killed heterogenic populations in a panel of CRC cell lines with KRAS/BRAF mutations that are natively resistant to EGFR- or VEGFR-targeted therapy, without sparing persistent cells, a reservoir of the disease relapse. Results showed that CADPE killed the tumor bulk and residual cells in the panel of CRC cell lines, rapidly inactivated c-Myc, STAT3, and NF-κB, and then decreased the protein levels of key signaling molecules for CRC, such as β-catenin, Notch1, and the nodes of mTOR pathways; eukaryotic translation initiation factors (eIF4F); anti-apoptotic proteins (Bcl-xl, Mcl-1, and survivin); and stemness-supporting molecules (CD133, Bim-1, and VEGF). In terms of mechanism of action, concurrent downregulation of Mcl-1, Bcl-xl, and survivin was necessary for CADPE to kill CRC bulk cells, while additional depletion of CD133 and VEGF proteins was required for killing the residual CRC cells. Moreover, the disabled c-Myc, STAT3, NF-κB, and eIF4F were associated with the broadly decreased levels of anti-apoptosis proteins and pro-stemness proteins. Consistently, CADPE suppressed CRC tumor growth associated with robust apoptosis and depleted levels of c-Myc, STAT3, NF-κB, eIF4F, anti-apoptotic proteins, and pro-stemness proteins. Our findings showed the promise of CADPE for treating CRC and suggested a rational polytherapy that disables c-Myc, STAT3, NF-κB, and eIF4F for killing CRC residual disease.

Interaction of ferritin iron responsive element (IRE) mRNA with translation initiation factor eIF4F.

The interaction of ferritin iron responsive element (IRE) mRNA with eIF4F was examined by fluorescence and circular dichroism spectroscopy. Fluorescence quenching data indicated that eIF4F contains one high affinity binding site for ferritin IRE RNA. The Scatchard analysis revealed strong binding affinity (Ka=11.1×107M-1) and binding capacity (n=1.0) between IRE RNA and eIF4F. The binding affinity of IRE RNA for eIF4F decreased (~4-fold) as temperature increased (from 5°C to 30°C). The van't Hoff analysis revealed that IRE RNA binding to eIF4F is enthalpy-driven (ΔH=-47.1±3.4kJ/mol) and entropy-opposed (ΔS=-30.1±1.5J/mol/K). The addition of iron increased the enthalpic, while decreasing the entropic contribution towards the eIF4F•IRE RNA complex, resulting in favorable free energy (ΔG=-49.8±2.8kJ/mol). Thermodynamic values and ionic strength data suggest that the presence of iron increases hydrogen bonding and decreases hydrophobic interactions, leading to formation of a more stable complex. The interaction of IRE RNA with eIF4F at higher concentrations produced significant changes in the secondary structure of the protein, as revealed from the far-UV CD results, clearly illustrating the structural alterations resulted from formation of the eIF4F•IRE RNA complex. A Lineweaver-Burk plot showed an uncompetitive binding behavior between IRE RNA and m7G cap for the eIF4F, indicating that there are different binding sites on the eIF4F for the IRE RNA and the cap analog; molecular docking analysis further supports this notion. Our findings suggest that the eIF4F•IRE RNA complex formation is accompanied by an elevated hydrogen bonding and weakened hydrophobic interactions, leading to an overall conformational change, favored in terms of its free energy. The conformational change in the eIF4F structure, caused by the IRE RNA binding, provides a more stable platform for effective IRE translation in iron homeostasis.

Translation initiation factors GleIF4E2 and GleIF4A can interact directly with the components of the pre-initiation complex to facilitate translation initiation in Giardia lamblia

Translation initiation factor eIF4F is essential for cap-dependent translation initiation in eukaryotes. eIF4F is a trimeric complex consisting of a scaffold protein eIF4G, cap-binding protein eIF4E and DEAD-box RNA helicase eIF4A. eIF4F binds to the 5’ cap structure of the mRNA through eIF4E and facilitates the binding of the preinitiation complex (PIC) via protein-protein interactions of eIF4G with eIF3 in mammals or with eIF5 in yeast. Initiation factor eIF4A is known to unwind the secondary structures of the 5’UTRs encountered by the PIC during its initial binding to the mRNA and while scanning for the initiation codon. In Giardia, homologs for eIF4E (GleIF4E2) and eIF4A (GleIF4A) have been identified but not for eIF4G. To address how PIC is recruited to the 5’ end of mRNA in the absence of eIF4G homolog, we have used yeast two-hybrid assays to identify potential interactions of GleIF4E2 with the components of the PIC. The results show that GleIF4E2 can interact with the β subunit of the initiation factor GleIF2, a component of the PIC. ZDOCK modeling of the GleIF4E2-GleIF2β complex revealed that the dorsal side of GleIF4E2 is likely involved in binding to GleIF2β, which mimics the interaction of mammalian eIF4E with eIF4G, and with eIF4E binding proteins. These results suggest that GleIF4E2 can facilitate the recruitment of the PIC to the 5’end of the mRNA by binding directly to the components of the PIC. The role of GleIF4A in translation initiation in Giardia is not clearly understood as the short 5’ UTRs of the mRNA are unlikely to form secondary structures. Interestingly, Pateamine A, a specific inhibitor of human eIF4A, inhibited the growth of Giardia in a dose-dependent manner, suggesting that the activity of GleIF4A is probably required for translation. Using yeast two-hybrid assays, we have identified a novel interaction of GleIF4A with i subunit of the initiation factor GleIF3 (GleIF3i), another component of the PIC. These results indicate that the GleIF4A can also interact directly with the components of the PIC. ZDOCK modeling of the GleIF3i-GleIF4A complex suggests that GleIF3i could serve as a stimulator of GleIF4A activity.

Eukaryotic initiation factor (eIF) 3 mediates Barley Yellow Dwarf Viral mRNA 3'-5' UTR interactions and 40S ribosomal subunit binding to facilitate cap-independent translation.

Barley Yellow Dwarf Virus (BYDV) is a positive strand RNA virus that lacks the canonical 5′ 7-methylguanosine cap and a 3′ poly-A tail. Instead, BYDV utilizes a cruciform cap independent translation element (CITE) in its 3′UTR RNA (BYDV-like CITE or BTE) that binds eukaryotic translation initiation factor (eIF) 4F and recruits 40S ribosomal subunits in the presence of active helicase factors (eIF4A, eIF4B, eIF4F and ATP). A long-range, 5-nucleotide, base-pairing kissing loop interaction between the 3′BTE and a 5′UTR stem-loop is necessary for translation to initiate. The 40S–eIF complex does not bind to the BYDV 5′UTR, suggesting the involvement of additional factors. We identified eIF3 as a component of the 3′BTE recruited complex using affinity-tagged 3′BTE RNA pull-down assays. Fluorescence anisotropy binding and gel shift assays showed that the 3′BTE and 5′UTR RNAs can simultaneously and non-competitively bind eIF3 in the presence of active helicase factors forming a single, macromolecular complex. Further, quantitative studies showed eIF3 increased recruitment of the 40S subunit by more than 25-fold. We propose a new role for eIF3, where eIF3 bridges BYDV’s UTRs, stabilizes the long-range 5′-3′ interaction, and facilitates recruitment of the 40S–eIF complex to the 5′UTR, leading to translation initiation.

Heterogeneity and specialized functions of translation machinery: from genes to organisms.

Regulation of mRNA translation offers the opportunity to diversify the expression and abundance of proteins made from individual gene products in cells, tissues and organisms. Emerging evidence has highlighted variation in the composition and activity of several large, highly conserved translation complexes as a means to differentially control gene expression. Heterogeneity and specialized functions of individual components of the ribosome and of the translation initiation factor complexes eIF3 and eIF4F, which are required for recruitment of the ribosome to the mRNA 5' untranslated region, have been identified. In this Review, we summarize the evidence for selective mRNA translation by components of these macromolecular complexes as a means to dynamically control the translation of the proteome in time and space. We further discuss the implications of this form of gene expression regulation for a growing number of human genetic disorders associated with mutations in the translation machinery.

The MNK-eIF4E Signaling Axis Contributes to Injury-Induced Nociceptive Plasticity and the Development of Chronic Pain.

Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5' cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4ES209A ). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca2+ imaging experiments on dorsal root ganglion neurons, NGF- and IL-6-induced increases in excitability were attenuated in neurons from eIF4ES209A mice. These effects were recapitulated in Mnk1/2-/- mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4ES209A and Mnk1/2-/- mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2-eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a debilitating disease affecting approximately one in three Americans. Chronic pain is thought to be driven by changes in the excitability of peripheral nociceptive neurons, but the precise mechanisms controlling these changes are not elucidated. Emerging evidence demonstrates that mRNA translation regulation pathways are key factors in changes in nociceptor excitability. Our work demonstrates that a single phosphorylation site on the 5' cap-binding protein eIF4E is a critical mechanism for changes in nociceptor excitability that drive the development of chronic pain. We reveal a new mechanistic target for the development of a chronic pain state and propose that targeting the upstream kinase, MAPK interacting kinase 1/2, could be used as a therapeutic approach for chronic pain.

Downregulation of eIF4G by microRNA-503 enhances drug sensitivity of MCF-7/ADR cells through suppressing the expression of ABC transport proteins.

Overexpression of adenosine triphosphate-binding cassette (ABC) transport protein is emerging as a critical contributor to anticancer drug resistance. The eukaryotic translation initiation factor (eIF) 4F complex, the key modulator of mRNA translation, is regulated by the phosphoinositide 3-kinase-AKT-mammalian target of rapamycin pathway in anticancer drug-resistant tumors. The present study demonstrated the roles of ABC translation protein alterations in the acquisition of the Adriamycin (ADM)-resistant phenotype of MCF-7 human breast cells. Quantitative polymerase chain reaction and western blot analysis were applied to examine the differences in mRNA and protein levels, respectively. It was found that the expression of the ABC sub-family B member 1, ABC sub-family C member 1 and ABC sub-family G member 2 transport proteins were upregulated in MCF-7/ADR cells. An MTT assay was used to detect the cell viability, from the results MCF-7/ADR cells were less sensitive to ADM, tamoxifen (TAM) and taxol (TAX) treatment compared with MCF-7 cells. We predicted that the 3′-untranslated region of eukaryotic translation initiation factor 4-γ 1 (eIF4G) contains a potential miRNA binding site for microRNA (miR)-503 through using computational programs. These binding sites were confirmed by luciferase reporter assays. eIF4G mRNA degradation was accelerated in cells transfected with miR-503 mimics. Furthermore, it was demonstrated that eIF4G and ABC translation proteins were significantly downregulated in MCF-7/ADR cells after transfection with miR-503. It was found that miR-503 mimics could sensitize the cells to treatment with ADM, TAM and TAX. These findings demonstrated for the first time that eIF4G acted as a key factor in MCF-7/ADR cells, and may be an efficient agent for preventing and reversing multi-drug resistance in breast cancer.

Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations

IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2'-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2'-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA.

Stress Granule Induction after Brain Ischemia Is Independent of Eukaryotic Translation Initiation Factor (eIF) 2α Phosphorylation and Is Correlated with a Decrease in eIF4B and eIF4E Proteins

Stress granules (SGs) are cytoplasmic ribonucleoprotein aggregates that are directly connected with the translation initiation arrest response to cellular stresses. Translation inhibition (TI) is observed in transient brain ischemia, a condition that induces persistent TI even after reperfusion, i.e. when blood flow is restored, and causes delayed neuronal death (DND) in selective vulnerable regions. We previously described a connection between TI and DND in the hippocampal cornu ammonis 1 (CA1) in an animal model of transient brain ischemia. To link the formation of SGs to TI and DND after brain ischemia, we investigated SG induction in brain regions with differential vulnerabilities to ischemia-reperfusion (IR) in this animal model. SG formation is triggered by both eukaryotic translation initiation factor (eIF) 2α phosphorylation and eIF4F complex dysfunction. We analyzed SGs by immunofluorescence colocalization of granule-associated protein T-cell internal antigen-1 with eIF3b, eIF4E, and ribosomal protein S6 and studied eIF2 and eIF4F complex. The results showed that IR stress induced SG formation in the CA1 region after 3-day reperfusion, consistent with TI and DND in CA1. SGs were formed independently of eIF2α phosphorylation, and their appearance was correlated with a decrease in the levels of eIF4F compounds, the cap-binding protein eIF4E, and eIF4B, suggesting that remodeling of the eIF4F complex was required for SG formation. Finally, pharmacological protection of CA1 ischemic neurons with cycloheximide decreased the formation of SGs and restored eIF4E and eIF4B levels in CA1. These findings link changes in eIF4B and eIF4E to SG induction in regions vulnerable to death after IR.

Sapovirus translation requires an interaction between VPg and the cap binding protein eIF4E.

ABSTRACTSapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5′ end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction.IMPORTANCE Sapoviruses, which are members of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains noncultivable in cell culture, hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein-primed mechanism of calicivirus VPg-dependent translation initiation.

Coupling 40S ribosome recruitment to modification of a cap-binding initiation factor by eIF3 subunit e.

40S ribosomes are loaded onto capped mRNAs via the multisubunit translation initiation factors eIF3 and eIF4F. While eIF4E is the eIF4F cap recognition component, the eIF4G subunit associates with 40S-bound eIF3. How this intricate process is coordinated remains poorly understood. Here, we identify an eIF3 subunit that regulates eIF4F modification and show that eIF3e is required for inducible eIF4E phosphorylation. Significantly, recruitment of the eIF4E kinase Mnk1 (MAPK signal-integrating kinase 1) to eIF4F depended on eIF3e, and eIF3e was sufficient to promote Mnk1-binding to eIF4G. This establishes a mechanism by which 40S ribosome loading imparts a phosphorylation mark on the cap-binding eIF4F complex that regulates selective mRNA translation and is synchronized by a specific eIF3 subunit.

A new role for the cellular PABP repressor Paip2 as an innate restriction factor capable of limiting productive cytomegalovirus replication

The capacity of polyadenylate-binding protein PABPC1 (PABP1) to stimulate translation is regulated by its repressor, Paip2. Paradoxically, while PABP accumulation promotes human cytomegalovirus (HCMV) protein synthesis, we show that this is accompanied by an analogous increase in the abundance of Paip2 and EDD1, an E3 ubiquitin ligase that destabilizes Paip2. Coordinate control of PABP1, Paip2, and EDD1 required the virus-encoded UL38 mTORC1 activator and resulted in augmented Paip2 synthesis, stability, and association with PABP1. Paip2 synthesis also increased following serum stimulation of uninfected normal fibroblasts, suggesting that this coregulation may play a role in how uninfected cells respond to stress. Significantly, Paip2 accumulation was dependent on PABP accrual, as preventing PABP1 accumulation suppressed viral replication and inhibited the corresponding Paip2 increase. Furthermore, depleting Paip2 restored the ability of infected cells to assemble the translation initiation factor eIF4F, promoting viral protein synthesis and replication without increasing PABP1. This establishes a new role for the cellular PABP1 inhibitor Paip2 as an innate defense that restricts viral protein synthesis and replication. Moreover, it illustrates how a stress-induced rise in PABP1 triggered by virus infection can counter and surpass a corresponding increase in Paip2 abundance and stability.

3′ Cap-Independent Translation Enhancers of Plant Viruses

In the absence of a 5' cap, plant positive-strand RNA viruses have evolved a number of different elements in their 3' untranslated region (UTR) to attract initiation factors and/or ribosomes to their templates. These 3' cap-independent translational enhancers (3' CITEs) take different forms, such as I-shaped, Y-shaped, T-shaped, or pseudoknotted structures, or radiate multiple helices from a central hub. Common features of most 3' CITEs include the ability to bind a component of the translation initiation factor eIF4F complex and to engage in an RNA-RNA kissing-loop interaction with a hairpin loop located at the 5' end of the RNA. The two T-shaped structures can bind to ribosomes and ribosomal subunits, with one structure also able to engage in a simultaneous long-distance RNA-RNA interaction. Several of these 3' CITEs are interchangeable and there is evidence that natural recombination allows exchange of modular CITE units, which may overcome genetic resistance or extend the virus's host range.

Cation-dependent folding of 3′ cap-independent translation elements facilitates interaction of a 17-nucleotide conserved sequence with eIF4G

The 3′-untranslated regions of many plant viral RNAs contain cap-independent translation elements (CITEs) that drive translation initiation at the 5′-end of the mRNA. The barley yellow dwarf virus-like CITE (BTE) stimulates translation by binding the eIF4G subunit of translation initiation factor eIF4F with high affinity. To understand this interaction, we characterized the dynamic structural properties of the BTE, mapped the eIF4G-binding sites on the BTE and identified a region of eIF4G that is crucial for BTE binding. BTE folding involves cooperative uptake of magnesium ions and is driven primarily by charge neutralization. Footprinting experiments revealed that functional eIF4G fragments protect the highly conserved stem–loop I and a downstream bulge. The BTE forms a functional structure in the absence of protein, and the loop that base pairs the 5′-untranslated region (5′-UTR) remains solvent-accessible at high eIF4G concentrations. The region in eIF4G between the eIF4E-binding site and the MIF4G region is required for BTE binding and translation. The data support the model in which the eIF4F complex binds directly to the BTE which base pairs simultaneously to the 5′-UTR, allowing eIF4F to recruit the 40S ribosomal subunit to the 5′-end.

Yeast Eukaryotic Initiation Factor 4B (eIF4B) Enhances Complex Assembly between eIF4A and eIF4G in Vivo

Translation initiation factor eIF4F (eukaryotic initiation factor 4F), composed of eIF4E, eIF4G, and eIF4A, binds to the m(7)G cap structure of mRNA and stimulates recruitment of the 43S preinitiation complex and subsequent scanning to the initiation codon. The HEAT domain of eIF4G stabilizes the active conformation of eIF4A required for its RNA helicase activity. Mammalian eIF4B also stimulates eIF4A activity, but this function appears to be lacking in yeast, making it unclear how yeast eIF4B (yeIF4B/Tif3) stimulates translation. We identified Ts(-) mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A·eIF4G complexes in vivo, and the rescue of specific mutant eIF4A·eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.

Poly(A) tail affects equilibrium and thermodynamic behavior of tobacco etch virus mRNA with translation initiation factors eIF4F, eIF4B and PABP

We have investigated the effects of poly(A)-tail on binding of eIF4F, eIF4B and PABP with tobacco etch virus (TEV) IRES RNA. The fluorescence anisotropy data showed that the addition of poly(A) 20 increases the binding affinity of eIF4F·4B and eIF4F·PABP complexes to IRES RNA ~ 2- and 4-fold, respectively. However, the binding affinity of eIF4F with PK1 was enhanced ~ 11-fold with the addition of PABP, eIF4B, and poly(A) 20 together. Whereas, poly(A) 20 alone increases the binding affinity of eIF4F·4B·PABP with PK1 RNA about 3-fold, showing an additive effect rather than the large increase in affinity as shown for cap binding. Thermodynamic data showed that PK1 RNA binding to protein complexes in the presence of poly(A) 20 was enthalpy-driven and entropy-favorable. Poly(A) 20 decreased the entropic contribution 75% for binding of PK1 RNA to eIF4F·4B·PABP as compared to eIF4F alone, suggesting reduced hydrophobic interactions for complex formation and an overall conformational change. Overall, these results demonstrate the first direct effect of poly(A) on the equilibrium and thermodynamics of eIF4F and eIF4F·4B·PABP with IRES-RNA.

Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k(on)) and dissociation rate constant (k(off)) for eIF4F binding to IRES were 59 +/- 2.1 micro s(-1) and 12.9 +/- 0.3 s(-1), respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F approximately 3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.

Effects of poly(A)-binding protein on the interactions of translation initiation factor eIF4F and eIF4F·4B with internal ribosome entry site (IRES) of tobacco etch virus RNA

In wheat germ, the interaction between poly(A)-binding protein and eukaryotic initiation factor eIF 4G increases the affinity of eIF4E for the cap by 20-40-fold. Recent findings that wheat germ eIF4G is required for interaction with the IRES, pseudoknot 1 (PK1), of tobacco etch virus to promote cap-independent translation led us to investigate the effects of PABP on the interaction of eIF4F with PK1. The fluorescence anisotropy data showed addition of PABP to eIF4F increased the binding affinity approximately 2.0-fold for PK1 RNA as compared with eIF4F alone. Addition of both PABP and eIF4B to eIF4F enhance binding affinity to PK1 about 4-fold, showing an additive effect rather than the large increase in affinity shown for cap binding. The van't Hoff analyses showed that PK1 RNA binding to eIF4F, eIF4F.PABP, eIF4F.4B and eIF4F.4B.PABP is enthalpy-driven and entropy-favorable. PABP and eIF4B decreased the entropic contribution 65% for binding of PK1 RNA to eIF4F. The lowering of entropy for the formation of eIF4F.4B.PABP-PK1 complex suggested reduced hydrophobic interactions for complex formation. Overall, these results demonstrate the first direct effect of PABP on the interaction of eIF4F and eIF4F.4B with PK1 RNA.

Poly(A)‐Binding Protein Affects the Kinetics of Tobacco Etch Virus Pseudoknot RNA Binding to Wheat germ Translation Initiation Factor eIF4F

Previous studies indicated that the pseudoknot (PK) of tobacco etch virus (TEV) RNA is necessary to promote cap‐independent translation (Zeenko and Gallie, 2005, J.B.C; 280, 26813‐26824). We investigated the effect of poly(A)‐binding protein (PABP) on the PK1 RNA binding to eIF4F in the absence and presence of eIF4B. Equilibrium studies of PK1 RNA binding to eIF4F showed ~ 2‐fold stronger affinity in the presence of PABP. Addition of PABP and eIF4B to the eIF4F enhances binding affinity ~3.5‐fold as compared to eIF4F binding to PK1 RNA. Binding of eIF4F·PABP and eIF4F·4B·PABP to PK1 RNA is both enthalpy and entropy‐favorable. Further, kinetic analysis of eIF4F, eIF4F·PABP and eIF4F·4B·PABP with PK1 RNA and the temperature dependence of this reaction were measured and compared. The stopped‐flow fluorescence anisotropy data showed that the observed rate constant for the binding of PK1 RNA increased linearly with an increase in eIF4F, eIF4F·PABP or eIF4F·4B·PABP initiation factor concentration. The association rate constant (kon) for PK1 binding was ~ 2.8‐fold faster for eIF4F·4B·PABP than eIF4F. The dissociation rate constant (koff) was ~1.5‐fold slower for eIF4F‐PK1 in the presence of eIF4B and PABP. The activation energy of the PK1 RNA binding reaction was markedly reduced in the presence of PABP and eIF4B. The enhancement of kon, lowering of koff, and reduction in energy barrier for the formation of eIF4F·4B·PABP‐PK1 complex suggests a more stable platform for efficient translation of protein synthesis. These results demonstrate the first direct kinetic measurements of plant translation initiation factors binding to an internal ribosome entry site (IRES).

The 3' cap-independent translation element of Barley yellow dwarf virus binds eIF4F via the eIF4G subunit to initiate translation.

The 3' cap-independent translation element (BTE) of Barley yellow dwarf virus RNA confers efficient translation initiation at the 5' end via long-distance base pairing with the 5'-untranslated region (UTR). Here we provide evidence that the BTE functions by recruiting translation initiation factor eIF4F. We show that the BTE interacts specifically with the cap-binding initiation factor complexes eIF4F and eIFiso4F in a wheat germ extract (wge). In wge depleted of cap-interacting factors, addition of eIF4F (and to a lesser extent, eIFiso4F) allowed efficient translation of an uncapped reporter construct (BLucB) containing the BTE in its 3' UTR. Translation of BLucB required much lower levels of eIF4F or eIFiso4F than did a capped, nonviral mRNA. Both full-length eIF4G and the carboxy-terminal half of eIF4G lacking the eIF4E binding site stimulated translation to 70% of the level obtained with eIF4F, indicating a minor role for the cap-binding protein, eIF4E. In wge inhibited by either BTE in trans or cap analog, eIF4G alone restored translation nearly as much as eIF4F, while addition of eIF4E alone had no effect. The BTE bound eIF4G (Kd = 177 nm) and eIF4F (Kd = 37 nm) with high affinity, but very weakly to eIF4E. These interactions correlate with the ability of the factors to facilitate BTE-mediated translation. These results and previous observations are consistent with a model in which eIF4F is delivered to the 5' UTR by the BTE, and they show that eIF4G, but not eIF4E, plays a major role in this novel mechanism of cap-independent translation.