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The canonical DEAD-box helicase, eukaryotic initiation factor (eIF) 4A, unwinds 5' UTR secondary structures to promote mRNA translation initiation. Growing evidence has indicated that other helicases, such as DHX29 and DDX3/ded1p, also function to promote the scanning of the 40S subunit on highly structured mRNAs. It is unknown how the relative contributions of eIF4A and other helicases regulate duplex unwinding on an mRNA to promote initiation. Here, we have adapted a real-time fluorescent duplex unwinding assay to monitor helicase activity precisely in the 5' UTR of a reporter mRNA that can be translated in a cell-free extract in parallel. We monitored the rate of 5' UTR-dependent duplex unwinding in the absence or presence of an eIF4A inhibitor (hippuristanol), a dominant negative eIF4A (eIF4A-R362Q), or a mutant eIF4E (eIF4E-W73L) that can bind the m7G cap but not eIF4G. Our experiments reveal that the duplex unwinding activity in the cell-free extract is roughly evenly split between eIF4A-dependent and eIF4A-independent mechanisms. Importantly, we show that the robust eIF4A-independent duplex unwinding is not sufficient for translation. We also show that the m7G cap structure, and not the poly(A) tail, is the primary mRNA modification responsible for promoting duplex unwinding in our cell-free extract system. Overall, the fluorescent duplex unwinding assay provides a precise method to investigate how eIF4A-dependent and eIF4A-independent helicase activity regulates translation initiation in cell-free extracts. We anticipate that potential small molecule inhibitors could be tested for helicase inhibition using this duplex unwinding assay.

During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.

Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS. [Display omitted] • Heat shock induces a conformational change in the eIF4A-binding domain of eIF4G • eIF4G, eIF4E, and Pab1p assemble into arrested mRNPs and condensates • eIF4A dissociates from the eIF4F complex upon heat shock • eIF4A promotes heat shock translation Using reconstitution biochemistry and experiments in yeast cells, Desroches Altamirano et al. demonstrate that a heat-induced conformational rearrangement in eIF4G dissociates eIF4A from eIF4F and promotes the assembly of translationally arrested mRNPs and condensates with eIF4E and Pab1p. eIF4A remains soluble during heat shock and promotes heat shock translation. [ABSTRACT FROM AUTHOR]

The stage at which ribosomes are recruited to messenger RNAs (mRNAs) is an elaborate and highly regulated phase of protein synthesis. Upon completion of this step, a ribosome is positioned at an appropriate initiation codon and primed to synthesize the encoded polypeptide product. In most circumstances, this step commits the ribosome to translate the mRNA. We summarize the knowledge regarding the initiation factors implicated in this activity as well as review different mechanisms by which this process is conducted. [ABSTRACT FROM AUTHOR]

eIF4A is a DEAD-box RNA-dependent ATPase thought to unwind RNA secondary structure in the 5'-untranslated regions (UTRs) of mRNAs to promote their recruitment to the eukaryotic translation pre-initiation complex (PIC). We show that eIF4A's ATPase activity is markedly stimulated in the presence of the PIC, independently of eIF4E•eIF4G, but dependent on subunits i and g of the heteromeric eIF3 complex. Surprisingly, eIF4A accelerated the rate of recruitment of all mRNAs tested, regardless of their degree of structural complexity. Structures in the 5'-UTR and 3' of the start codon synergistically inhibit mRNA recruitment in a manner relieved by eIF4A, indicating that the factor does not act solely to melt hairpins in 5'-UTRs. Our findings that eIF4A functionally interacts with the PIC and plays important roles beyond unwinding 5'-UTR structure is consistent with a recent proposal that eIF4A modulates the conformation of the 40S ribosomal subunit to promote mRNA recruitment.

Meningiomas frequently display activation of the PI3K/AKT/mTOR pathway, leading to elevated levels of phospho-eukaryotic translation initiation factor 4E binding proteins, which enhances protein synthesis; however, it is not known whether inhibition of protein translation is an effective treatment option for meningiomas. We found that human meningiomas expressed high levels of the three components of the eukaryotic initiation factor 4F (eIF4F) translation initiation complex, eIF4A, eIF4E, and eIF4G. The expression of eIF4A and eIF4E was important in sustaining the growth of NF2 -deficient benign meningioma Ben-Men-1 cells, as shRNA-mediated knockdown of these proteins strongly reduced cell proliferation. Among a series of 23 natural compounds evaluated, silvestrol, which inhibits eIF4A, was identified as being the most growth inhibitory in both primary meningioma and Ben-Men-1 cells. Silvestrol treatment of meningioma cells prominently induced G 2 /M arrest. Consistently, silvestrol significantly decreased the amounts of cyclins D1, E1, A, and B, PCNA, and Aurora A. In addition, total and phosphorylated AKT, ERK, and FAK, which have been shown to be important drivers for meningioma cell proliferation, were markedly lower in silvestrol-treated Ben-Men-1 cells. Our findings suggest that inhibiting protein translation could be a potential treatment for meningiomas. [ABSTRACT FROM AUTHOR]

Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential ‘scanning’ of the 5′ untranslated regions (UTRs). Scanning through the 5′UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5′UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the moderately structured β-globin sequences in the 5′UTR but not that of an mRNA with a poly(A) sequence as the 5′UTR. By contrast, the nonhydrolysable ATP analogue β,γ-imidoadenosine 5′-triphosphate (AMP-PNP) inhibited translation irrespective of the 5′UTR sequence, suggesting that complexes that contain ATP-binding proteins in their ATP-bound form can obstruct and/or actively block progression of ribosome recruitment and/or scanning on mRNA. Further, using primer extension inhibition to locate SSUs on mRNA (‘toeprinting’), we identify an SSU complex which inhibits primer extension approximately eight nucleotides upstream from the usual toeprinting stop generated by SSUs positioned over the start codon. This ‘−8 nt toeprint’ was seen with mRNA 5′UTRs of different length, sequence and structure potential. Importantly, the ‘−8 nt toeprint’ was strongly stimulated by the presence of the cap on the mRNA, as well as the presence of eIFs 4F, 4A/4B and ATP, implying active scanning. We assembled cell-free translation reactions with capped mRNA featuring an extended 5′UTR and used cycloheximide to arrest elongating ribosomes at the start codon. Impeding scanning through the 5′UTR in this system with elevated magnesium and AMP-PNP (similar to the toeprinting conditions), we visualised assemblies consisting of several SSUs together with one full ribosome by electron microscopy, suggesting direct detection of scanning intermediates. Collectively, our data provide additional biochemical, molecular and physical evidence to underpin the scanning model of translation initiation in eukaryotes.

Biotherapeutics have revolutionized modern medicine by providing medicines that would not have been possible with small molecules. With respect to cancer therapies, this represents the current sector of the pharmaceutical industry having the largest therapeutic impact, as exemplified by the development of recombinant antibodies and cell-based therapies. In cancer, one of the most common regulatory alterations is the perturbation of translational control. Among these, changes in eukaryotic initiation factor 4F (eIF4F) are associated with tumor initiation, progression, and drug resistance in a number of settings. This, coupled with the fact that systemic suppression of eIF4F appears well tolerated, indicates that therapeutic agents targeting eIF4F hold much therapeutic potential. Here, we discuss opportunities offered by biologicals for this purpose. [ABSTRACT FROM AUTHOR]

The formation of ribosomal 48S initiation complexes at the start AUG codon of uncapped mRNA leader sequences was studied using the methodology of primer extension inhibition (toe-printing). The experiments were performed in the system composed of purified individual components required for translation initiation. The formation of ribosomal 48S initiation complexes at the initiation codon was tested depending on the presence of the initiation factors eIF4F, eIF4A, and eIF4B. Several mRNAs containing short leader sequences lacking the extended secondary structure were studied. It was found that 48S ribosomal complexes at mRNAs with such leaders were not formed in the absence of eIF4F. In contrast, the removal of either eIF4A or eIF4B from the experimental system was found to be dispensable for the formation of the 48S complex. [ABSTRACT FROM AUTHOR]

Protein synthesis is an essential cellular process that is highly responsive to extra- and intracellular cues such as mitogens, growth signals, nutrient and energy status, and stress. Much regulation of translation is directed towards initiation, more specifically the ribosome recruitment phase which requires the eukaryotic initiation factor (eIF) 4 F complex to prepare the mRNA for the incoming 40S ribosome (and associated factors). As many commonly deregulated signaling pathways found in cancers converge to influence protein synthesis, targeting factors involved in translation initiation is a viable anti-neoplastic strategy that has demonstrated success in pre-clinical settings. Furthermore, transcripts that are particularly sensitive to fluctuations in levels of the eIF4F complex are often associated with oncogenic characteristics (eg proliferation, survival, and angiogenesis) and their translational output appears to be preferentially reduced when eIF4F is inhibited. In particular, the enzymatic subunit of the eIF4F complex, eIF4A, has been extensively explored as a druggable target with several natural products identified as potent and selective inhibitors. In this review, we discuss the cellular regulation of eIF4A activity and its potential as a therapeutic target. This article is part of a Special Issue entitled: Translation and Cancer. [ABSTRACT FROM AUTHOR]

Abstract: Eukaryotic translation initiation factor 4A (eIF4A) is required for a cap-binding complex to recognize the 5′ end of mRNA. A eIF4A gene (Px-eIF4A) was identified from Plutella xylostella. Its full cDNA (1,700bp) encoded 422 amino acids and showed high sequence similarity (59.6–96.2%) with other insect and human eIF4As. Three dimensional analysis of Px-eIF4A indicated two globular domains, both of which were predicted to participate in RNA binding and helicase activity. Both RT-PCR and Western blotting indicated that the expression of Px-eIF4A showed a constitutive pattern regardless of developmental stage or tissue. An indirect immunofluorescence assay indicated that Px-eIF4A was localized in the cytoplasm. An immunoprecipitation with Px-eIF4A against a protein extract of P. xylostella indicated that Px-eIF4A could bind eIF4G, presumably to form a functional cap-binding complex. These results suggest that Px-eIF4A is constitutively expressed in all tissues and plays a role in the formation of the translation initiation complex in P. xylostella. [ABSTRACT FROM AUTHOR]

Interfacial inhibitors exert their biological effects through co-association with two macromolecules. The pateamine A (PatA) class of molecules function by stabilizing eukaryotic initiation factor (eIF) 4A RNA helicase onto RNA, resulting in translation initiation inhibition. Here, we present the crystal structure of an eIF4A1:RNA complex bound to an analog of the marine sponge-derived natural product PatA, C5-desmethyl PatA (DMPatA). One end of this small molecule wedges itself between two RNA bases while the other end is cradled by several protein residues. Strikingly, DMPatA interacts with the eIF4A1:RNA complex in an almost identical fashion as rocaglamide A (RocA), despite being completely unrelated from a structural standpoint. The structural data rationalize the ability of PatA analogs to target a wider range of RNA substrates compared to RocA. We define the molecular basis of how DMPatA is able to clamp eIF4A1 onto RNA, imparting potent inhibitory properties to this molecule. [Display omitted] • Crystal structure of eIF4A1⋅AMPPNP⋅poly (AG) 5 ⋅C5-desmethyl PatA (DMPatA). • DMPatA acts an interfacial inhibitor, interacting with both eIF4A1 and RNA. • DMPatA clamps eIF4A1 onto both polypurine and polypyrimide RNA templates. • DMPatA and rocaglamide A (RocA) binding sites on eIF4A1 are largely overlapping. Naineni, Liang et al. solved the crystal structure of an eIF4A1⋅AMPPNP⋅r(AG) 5 ⋅DMPatA complex. DMPatA stabilizes eIF4A1/RNA complexes to inhibit translation initiation. The structure rationalizes the broad RNA targeting range of DMPatA, defines the molecular interactions with eIF4A1, and provides a template for future analog design. [ABSTRACT FROM AUTHOR]

Rocaglates are a diverse family of biologically active molecules that have gained tremendous interest in recent years due to their promising activities in pre-clinical cancer studies. As a result, this family of compounds has been significantly expanded through the development of efficient synthetic schemes. However, it is unknown whether all of the members of the rocaglate family act through similar mechanisms of action. Here, we present a comprehensive study comparing the biological activities of >200 rocaglates to better understand how the presence of different chemical entities influences their biological activities. Through this, we find that most rocaglates preferentially repress the translation of mRNAs containing purine-rich 5′ leaders, but certain rocaglates lack this bias in translation repression. We also uncover an aspect of rocaglate mechanism of action in which the pool of translationally active eIF4F is diminished due to the sequestration of the complex onto RNA. • Rocaglates produce distinct inhibitory effects on translation initiation • Rocaglates interfere with eIF4F release from the cap structure • Rocaglates exert a bystander effect on translation initiation by sequestering eIF4F Rocaglates are a diverse family of small molecules that inhibit eIF4A. Chu et al. undertake a comparative analysis of the bioactivity of >200 rocaglates and uncover nuances in their mechanisms of action. Rocaglates interfere with eIF4F release from the cap and exert a bystander effect to inhibit translation. [ABSTRACT FROM AUTHOR]

Rocaglates share a common cyclopenta b benzofuran core that inhibits eukaryotic translation initiation by modifying the behavior of the RNA helicase, eIF4A. Working as interfacial inhibitors, rocaglates stabilize the association between eIF4A and RNA, which can lead to the formation of steric barriers that block initiating ribosomes. There is significant interest in the development and expansion of rocaglate derivatives, as several members of this family have been shown to possess potent anti-neoplastic activity in vitro and in vivo. To further our understanding of rocaglate diversity and drug design, herein we explore the RNA clamping activity of >200 unique rocaglate derivatives. Through this, we report on the identification and characterization of a potent class of synthetic rocaglates called amidino-rocaglates. These compounds are among the most potent rocaglates documented to date and, taken together, this work offers important information that will guide the future design of rocaglates with improved biological properties. • A screen of >200 rocaglates for stimulation of eIF4A1:RNA clamping was prosecuted • eIF4A1 and eIF4A2 are similarly affected by all active rocaglates • Amidino-rocaglates were identified as a novel potent class of translation inhibitors • A novel lead compound, CMLD012612, was identified and characterized A screen of >200 rocaglates identified a novel, potent chemical series, amidino-rocaglates, that target eIF4A1 and eIF4A2 to potently inhibit translation and tumor cell viability, and are effective in synergizing with DNA-damaging agents in vivo against MYC-driven lymphomas. These compounds are promising anti-cancer agents. [ABSTRACT FROM AUTHOR]

Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential 'scanning' of the 5′ untranslated regions (UTRs). Scanning through the 5′UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5′UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the moderately structured β-globin sequences in the 5′UTR but not that of an mRNA with a poly(A) sequence as the 5′UTR. By contrast, the nonhydrolysable ATP analogue β,γ-imidoadenosine 5′-triphosphate (AMP-PNP) inhibited translation irrespective of the 5′UTR sequence, suggesting that complexes that contain ATP-binding proteins in their ATP-bound form can obstruct and/or actively block progression of ribosome recruitment and/or scanning on mRNA. Further, using primer extension inhibition to locate SSUs on mRNA ('toeprinting'), we identify an SSU complex which inhibits primer extension approximately eight nucleotides upstream from the usual toeprinting stop generated by SSUs positioned over the start codon. This '−8 nt toeprint' was seen with mRNA 5′UTRs of different length, sequence and structure potential. Importantly, the '−8 nt toeprint' was strongly stimulated by the presence of the cap on the mRNA, as well as the presence of eIFs 4F, 4A/4B and ATP, implying active scanning. We assembled cell-free translation reactions with capped mRNA featuring an extended 5′UTR and used cycloheximide to arrest elongating ribosomes at the start codon. Impeding scanning through the 5′UTR in this system with elevated magnesium and AMP-PNP (similar to the toeprinting conditions), we visualised assemblies consisting of several SSUs together with one full ribosome by electron microscopy, suggesting direct detection of scanning intermediates. Collectively, our data provide additional biochemical, molecular and physical evidence to underpin the scanning model of translation initiation in eukaryotes. [ABSTRACT FROM AUTHOR]