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Messenger RNA (mRNA) recruitment to the 40S ribosomal subunit is mediated by eukaryotic initiation factor 4F (eIF4F). This complex includes three subunits: eIF4E (m7G cap-binding protein), eIF4A (DEAD-box helicase), and eIF4G. Mammalian eIF4G is a scaffold that coordinates the activities of eIF4E and eIF4A and provides a bridge to connect the mRNA and 40S ribosomal subunit through its interaction with eIF3. While the roles of many eIF4G binding domains are relatively clear, the precise function of RNA binding by eIF4G remains to be elucidated. In this work, we used an eIF4G-dependent translation assay to reveal that the RNA binding domain (eIF4G-RBD; amino acids 682-720) stimulates translation. This stimulating activity is observed when eIF4G is independently tethered to an internal region of the mRNA, suggesting that the eIF4G-RBD promotes translation by a mechanism that is independent of the m7G cap and mRNA tethering. Using a kinetic helicase assay, we show that the eIF4G-RBD has a minimal effect on eIF4A helicase activity, demonstrating that the eIF4G-RBD is not required to coordinate eIF4F-dependent duplex unwinding. Unexpectedly, native gel electrophoresis and fluorescence polarization assays reveal a previously unidentified direct interaction between eIF4G and the 40S subunit. Using binding assays, our data show that this 40S subunit interaction is separate from the previously characterized interaction between eIF4G and eIF3. Thus, our work reveals how eIF4F can bind to the 40S subunit using eIF3-dependent and eIF3-independent binding domains to promote translation initiation.

Eukaryotic protein translation has slowly gained the scientific community's attention for its advanced and powerful therapeutic potential. However, recent technical developments in studying ribosomes and global translation have revolutionized our understanding of this complex multistep process. These developments have improved and deepened the current knowledge of mRNA translation, sparking excitement and new possibilities in this field. Translation factors are crucial for maintaining protein synthesis homeostasis. Since actively proliferating cancer cells depend on protein synthesis, dysregulated protein translation is central to tumorigenesis. Translation factors and their abnormal expressions directly affect multiple oncogenes and tumor suppressors. Recently, small molecules have been used to target translation factors, resulting in translation inhibition in a gene-specific manner, opening the door for developing translation inhibitors that can lead to novel chemotherapeutic drugs for treating multiple cancer types caused by dysregulated translation machinery. This review comprehensively summarizes the involvement of translation factors in tumor progression and oncogenesis. Also, it sheds light on the evolution of translation factors as novel drug targets for developing future therapeutic drugs for treating cancer. [ABSTRACT FROM AUTHOR]

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.

Eukaryotic protein translation has slowly gained the scientific community’s attention for its advanced and powerful therapeutic potential. However, recent technical developments in studying ribosomes and global translation have revolutionized our understanding of this complex multistep process. These developments have improved and deepened the current knowledge of mRNA translation, sparking excitement and new possibilities in this field. Translation factors are crucial for maintaining protein synthesis homeostasis. Since actively proliferating cancer cells depend on protein synthesis, dysregulated protein translation is central to tumorigenesis. Translation factors and their abnormal expressions directly affect multiple oncogenes and tumor suppressors. Recently, small molecules have been used to target translation factors, resulting in translation inhibition in a gene-specific manner, opening the door for developing translation inhibitors that can lead to novel chemotherapeutic drugs for treating multiple cancer types caused by dysregulated translation machinery. This review comprehensively summarizes the involvement of translation factors in tumor progression and oncogenesis. Also, it sheds light on the evolution of translation factors as novel drug targets for developing future therapeutic drugs for treating cancer.

Abstract When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning. [ABSTRACT FROM AUTHOR]

Introduction: Annexin A2 (AnxA2) plays a critical role in cell transformation, immune response, and resistance to cancer therapy. Besides functioning as a calcium- and lipidbinding protein, AnxA2 also acts as an mRNA-binding protein, for instance, by interacting with regulatory regions of specific cytoskeleton-associated mRNAs.Methods and Results: Nanomolar concentrations of FL3, an inhibitor of the translation factor eIF4A, transiently increases the expression of AnxA2 in PC12 cells and stimulates shortterm transcription/translation of anxA2 mRNA in the rabbit reticulocyte lysate. AnxA2 regulates the translation of its cognate mRNA by a feed-back mechanism, which can partly be relieved by FL3. Results obtained using the holdup chromatographic retention assay results suggest that AnxA2 interacts transiently with eIF4E (possibly eIF4G) and PABP in an RNA-independent manner while cap pulldown experiments indicate a more stable RNA-dependent interaction. Short-term (2 h) treatment of PC12 cells with FL3 increases the amount of eIF4A in cap pulldown complexes of total lysates, but not of the cytoskeletal fraction. AnxA2 is only present in cap analogue-purified initiation complexes from the cytoskeletal fraction and not total lysates confirming that AnxA2 binds to a specific subpopulation of mRNAs.Discussion: Thus, AnxA2 interacts with PABP1 and subunits of the initiation complex eIF4F, explaining its inhibitory effect on translation by preventing the formation of the full eIF4F complex. This interaction appears to be modulated by FL3. These novel findings shed light on the regulation of translation by AnxA2 and contribute to a better understanding of the mechanism of action of eIF4A inhibitors.

Triple-negative breast cancer (TNBC) remains clinically challenging as effective targeted therapies are lacking. In addition, patient mortality mainly results from the metastasized lesions. CXCR4 has been identified to be one of the major chemokine receptors involved in breast cancer metastasis. Previously, our lab had identified LIM and SH3 Protein 1 (LASP1) to be a key mediator in CXCR4-driven invasion. To further investigate the role of LASP1 in this process, a proteomic screen was employed and identified a novel protein-protein interaction between LASP1 and components of eukaryotic initiation 4F complex (eIF4F). We hypothesized that activation of the CXCR4-LASP1-eIF4F axis may contribute to the preferential translation of oncogenic mRNAs leading to breast cancer progression and metastasis. To test this hypothesis, we first confirmed that the gene expression of CXCR4, LASP1, and eIF4A are upregulated in invasive breast cancer. Moreover, we demonstrate that LASP1 associated with eIF4A in a CXCL12-dependent manner via a proximity ligation assay. We then confirmed this finding, and the association of LASP1 with eIF4B via co-immunoprecipitation assays. Furthermore, we show that LASP1 can interact with eIF4A and eIF4B through a GST-pulldown approach. Activation of CXCR4 signaling increased the translation of oncoproteins downstream of eIF4A. Interestingly, genetic silencing of LASP1 interrupted the ability of eIF4A to translate oncogenic mRNAs into oncoproteins. This impaired ability of eIF4A was confirmed by a previously established 5'UTR luciferase reporter assay. Finally, lack of LASP1 sensitizes 231S cells to pharmacological inhibition of eIF4A by Rocaglamide A as evident through BIRC5 expression. Overall, our work identified the CXCR4-LASP1 axis to be a novel mediator in oncogenic protein translation. Thus, our axis of study represents a potential target for future TNBC therapies.

The translation initiation complex 4F (eIF4F) is a rate-limiting factor in protein synthesis. Alterations in eIF4F activity are linked to several diseases, including cancer and infectious diseases. To this end, coronaviruses require eIF4F complex activity to produce proteins essential for their life cycle. Efforts to target coronaviruses by abrogating translation have been largely limited to repurposing existing eIF4F complex inhibitors. Here, we report the results of a high throughput screen to identify small molecules that disrupt eIF4F complex formation and inhibit coronavirus RNA and protein levels. Of 338,000 small molecules screened for inhibition of the eIF4F-driven, CAP-dependent translation, we identified SBI-1232 and two structurally related analogs, SBI- 5844 and SBI-0498, that inhibit human coronavirus OC43 (HCoV-OC43; OC43) with minimal cell toxicity. Notably, gene expression changes after OC43 infection of Vero E6 or A549 cells were effectively reverted upon treatment with SBI-5844 or SBI-0498. Moreover, SBI-5844 or SBI-0498 treatment effectively impeded the eIF4F complex assembly, with concomitant inhibition of newly synthesized OC43 nucleocapsid protein and OC43 RNA and protein levels. Overall, we identify SBI-5844 and SBI-0498 as small molecules targeting the eIF4F complex that may limit coronavirus transcripts and proteins, thereby representing a basis for developing novel therapeutic modalities against coronaviruses. [ABSTRACT FROM AUTHOR]

The eIF4F translation initiation complex plays a critical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a function of eIF4F in the negative regulation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essential for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutations. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negative feedback regulator of ERK activity, requires continuous production in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutations. Treatment with small-molecule eIF4F inhibitors disrupts the negative feedback control of MAPK signaling, leading to ERK hyperactivation and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantitative analyses reveal a high spare signaling capacity in the ERK pathway, suggesting that eIF4F-dependent feedback keeps the majority of ERK molecules inactive under normal conditions. Overall, our findings highlight the crucial role of eIF4F in regulating ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negative feedback control of MAPK activity in melanomas with BRAF and NRAS activating mutations., Competing Interests: Competing interests statement:The authors declare no competing interest.

The SARS-CoV-2 infection generates up to nine different sub-genomic mRNAs (sgRNAs), in addition to the genomic RNA (gRNA). The 5′UTR of each viral mRNA shares the first 75 nucleotides (nt.) at their 5′end, called the leader, but differentiates by a variable sequence (0 to 190 nt. long) that follows the leader. As a result, each viral mRNA has its own specific 5′UTR in term of length, RNA structure, uORF and Kozak context; each one of these characteristics could affect mRNA expression. In this study, we have measured and compared translational efficiency of each of the ten viral transcripts. Our data show that most of them are very efficiently translated in all translational systems tested. Surprisingly, the gRNA 5′UTR, which is the longest and the most structured, was also the most efficient to initiate translation. This property is conserved in the 5′UTR of SARS-CoV-1 but not in MERS-CoV strain, mainly due to the regulation imposed by the uORF. Interestingly, the translation initiation mechanism on the SARS-CoV-2 gRNA 5′UTR requires the cap structure and the components of the eIF4F complex but showed no dependence in the presence of the poly(A) tail in vitro. Our data strongly suggest that translation initiation on SARS-CoV-2 mRNAs occurs via an unusual cap-dependent mechanism. [ABSTRACT FROM AUTHOR]

Currently, there is no single concept of the optimal spatial structure of mRNA during translation. It is known that many proteins, associated with the 5' end of mRNA, interact with proteins associated with the 3' end. Moreover, this interaction often affects the activity of these proteins., It is possible within the same mRNA molecule only when the mRNA forms a circular structure in which its ends are spatially close. Discovery of such proteins, in the 90s of the 20th century, made it possible to formulate the closed-loop mRNA structure hypothesis, in which it is assumed that the ends of translationally active mRNA are fixed next to each other due to the interaction of proteins and (or) RNA. However, later it was shown that a closed-loop structure is not always necessary for translation. Moreover, some authors have proposed a model according to which the translating mRNA, on the contrary, should be unfolded into a linear structure. Thus, the spatial structure of the translating mRNA does not have to be universal for all mRNA and can change dynamically, which affects its functional activity. In this review, we have summarized a variety of experimental data and concepts on the relationship between the spatial structure of mRNA and its translational activity. [ABSTRACT FROM AUTHOR]

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]

Mouse embryonic stem cells (mESCs) can self‐renew indefinitely and maintain pluripotency. Inhibition of mechanistic target of rapamycin (mTOR) by the kinase inhibitor INK128 is known to induce paused pluripotency in mESCs cultured with traditional serum/LIF medium (SL), but the underlying mechanisms remain unclear. In this study, we demonstrate that mTOR complex 1 (mTORC1) but not complex 2 (mTORC2) mediates mTOR inhibition‐induced paused pluripotency in cells grown in both SL and 2iL medium (GSK3 and MEK inhibitors and LIF). We also show that mTORC1 regulates self‐renewal in both conditions mainly through eIF4F‐mediated translation initiation that targets mRNAs of both cytosolic and mitochondrial ribosome subunits. Moreover, inhibition of mitochondrial translation is sufficient to induce paused pluripotency. Interestingly, eIF4F also regulates maintenance of pluripotency in an mTORC1‐independent but MEK/ERK‐dependent manner in SL, indicating that translation of pluripotency genes is controlled differently in SL and 2iL. Our study reveals a detailed picture of how mTOR governs self‐renewal in mESCs and uncovers a context‐dependent function of eIF4F in pluripotency regulation. Synopsis: The mTORC1‐eIF4F axis controls mouse ESC self‐renewal through translation initiation. eIF4F regulates pluripotency in mouse ESCs cultured in serum+LIF. mTORC1 but not mTORC2 controls mouse ESC self‐renewal.Translation initiation complex eIF4F mediates mTORC1's role in mouse ESC self‐renewal.eIF4F coordinates both cytosolic and mitochondrial translation in mouse ESCs.eIF4F regulates pluripotency of mESCs grown in SL in an mTORC1‐independent but MEK/ERK‐dependent manner. [ABSTRACT FROM AUTHOR]

Herpesviral mRNAs are produced and translated by cellular machinery, rendering them susceptible to the network of regulatory events that impact translation. In response, these viruses have evolved to infiltrate and hijack translational control pathways as well as to integrate specialized host translation strategies into their own repertoire. They are robust systems to dissect mechanisms of mammalian translational regulation and continue to offer insight into cis-acting mRNA features that impact assembly and activity of the translation apparatus. Here, I discuss recent advances revealing the extent to which the three herpesvirus subfamilies regulate both host and viral translation, thereby dramatically impacting the landscape of protein synthesis in infected cells.

The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5' UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5' UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome.

The SARS-CoV-2 infection generates up to nine different sub-genomic mRNAs (sgRNAs), in addition to the genomic RNA (gRNA). The 5′UTR of each viral mRNA shares the first 75 nucleotides (nt.) at their 5′end, called the leader, but differentiates by a variable sequence (0 to 190 nt. long) that follows the leader. As a result, each viral mRNA has its own specific 5′UTR in term of length, RNA structure, uORF and Kozak context; each one of these characteristics could affect mRNA expression. In this study, we have measured and compared translational efficiency of each of the ten viral transcripts. Our data show that most of them are very efficiently translated in all translational systems tested. Surprisingly, the gRNA 5′UTR, which is the longest and the most structured, was also the most efficient to initiate translation. This property is conserved in the 5′UTR of SARS-CoV-1 but not in MERS-CoV strain, mainly due to the regulation imposed by the uORF. Interestingly, the translation initiation mechanism on the SARS-CoV-2 gRNA 5′UTR requires the cap structure and the components of the eIF4F complex but showed no dependence in the presence of the poly(A) tail in vitro. Our data strongly suggest that translation initiation on SARS-CoV-2 mRNAs occurs via an unusual cap-dependent mechanism.

Mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism. In nutrient-rich environments, mTORC1 kinase activity stimulates protein synthesis to meet cellular anabolic demands. Under nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global protein synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple proteins that stimulate mTORC1 activity or subvert autophagy, but precise roles for mTORC1 in different stages of KSHV infection remain incompletely understood. Here, we report that during latent and lytic stages of KSHV infection, chemical inhibition of mTORC1 caused eukaryotic initiation factor 4F (eIF4F) disassembly and diminished global protein synthesis, which indicated that mTORC1-mediated control of translation initiation was largely intact. We observed that mTORC1 was required for synthesis of the replication and transcription activator (RTA) lytic switch protein and reactivation from latency, but once early lytic gene expression had begun, mTORC1 was not required for genome replication, late gene expression, or the release of infectious progeny. Moreover, mTORC1 control of autophagy was dysregulated during lytic replication, whereby chemical inhibition of mTORC1 prevented ULK1 phosphorylation but did not affect autophagosome formation or rates of autophagic flux. Together, these findings suggest that mTORC1 is dispensable for viral protein synthesis and viral control of autophagy during lytic infection and that KSHV undermines mTORC1- dependent cellular processes during the lytic cycle to ensure efficient viral replication. [ABSTRACT FROM AUTHOR]

Abstract Background Translation factors eIF4E and eIF4G form eIF4F, which interacts with the messenger RNA (mRNA) 5′ cap to promote ribosome recruitment and translation initiation. Variations in the association of eIF4F with individual mRNAs likely contribute to differences in translation initiation frequencies between mRNAs. As translation initiation is globally reprogrammed by environmental stresses, we were interested in determining whether eIF4F interactions with individual mRNAs are reprogrammed and how this may contribute to global environmental stress responses. Results Using a tagged-factor protein capture and RNA-sequencing (RNA-seq) approach, we have assessed how mRNA associations with eIF4E, eIF4G1 and eIF4G2 change globally in response to three defined stresses that each cause a rapid attenuation of protein synthesis: oxidative stress induced by hydrogen peroxide and nutrient stresses caused by amino acid or glucose withdrawal. We find that acute stress leads to dynamic and unexpected changes in eIF4F–mRNA interactions that are shared among each factor and across the stresses imposed. eIF4F–mRNA interactions stabilised by stress are predominantly associated with translational repression, while more actively initiating mRNAs become relatively depleted for eIF4F. Simultaneously, other mRNAs are insulated from these stress-induced changes in eIF4F association. Conclusion Dynamic eIF4F–mRNA interaction changes are part of a coordinated early translational control response shared across environmental stresses. Our data are compatible with a model where multiple mRNA closed-loop complexes form with differing stability. Hence, unexpectedly, in the absence of other stabilising factors, rapid translation initiation on mRNAs correlates with less stable eIF4F interactions.

Triple-negative breast cancer (TNBC) remains clinically challenging as effective targeted therapies are lacking. In addition, patient mortality mainly results from the metastasized lesions. CXCR4 has been identified to be one of the major chemokine receptors involved in breast cancer metastasis. Previously, our lab had identified LIM and SH3 Protein 1 (LASP1) to be a key mediator in CXCR4-driven invasion. To further investigate the role of LASP1 in this process, a proteomic screen was employed and identified a novel protein-protein interaction between LASP1 and components of eukaryotic initiation 4F complex (eIF4F). We hypothesized that activation of the CXCR4-LASP1-eIF4F axis may contribute to the preferential translation of oncogenic mRNAs leading to breast cancer progression and metastasis. To test this hypothesis, we first confirmed that the gene expression of CXCR4, LASP1, and eIF4A are upregulated in invasive breast cancer. Moreover, we demonstrate that LASP1 associated with eIF4A in a CXCL12-dependent manner via a proximity ligation assay. We then confirmed this finding, and the association of LASP1 with eIF4B via co-immunoprecipitation assays. Furthermore, we show that LASP1 can interact with eIF4A and eIF4B through a GST-pulldown approach. Activation of CXCR4 signaling increased the translation of oncoproteins downstream of eIF4A. Interestingly, genetic silencing of LASP1 interrupted the ability of eIF4A to translate oncogenic mRNAs into oncoproteins. This impaired ability of eIF4A was confirmed by a previously established 5′UTR luciferase reporter assay. Finally, lack of LASP1 sensitizes 231S cells to pharmacological inhibition of eIF4A by Rocaglamide A as evident through BIRC5 expression. Overall, our work identified the CXCR4-LASP1 axis to be a novel mediator in oncogenic protein translation. Thus, our axis of study represents a potential target for future TNBC therapies.

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]

• Eukaryotic translation initiation factors bind RNA and the ribosome to affect changes in translation. • Capped mRNAs display strong dependence on the eIF4 factors for ribosome binding. • Factor dependence of translation initiation can be kinetically determined. • Optimized purification strategies allow larger studies of apparent affinities of translation initiation factors for the preinitiation complex. Control of translation initiation plays a critical role in the regulation of gene expression in all organisms, yet the mechanics of translation initiation in eukaryotic organisms are not well understood. Confounding studies of translation are the large number and overlapping functions of many initiation factors in cells, and a lack of cap-dependence in many in vitro systems. To shed light on intricate mechanisms that are often obscured in vivo, we use a fully reconstituted translation initiation system for analyzing RNA interactions with eukaryotic translation initiation factors and complexes from the model organism Saccharomyces cerevisiae. This system exhibits strong cap dependence, and dependence on translation factors varies with mRNA 5′ UTR sequences as expected from genome-wide studies of translation. Here we provide optimized protocols for purification and analysis of the effects of labeled and unlabeled mRNA recruitment factors on both the rate and factor dependence of mRNA recruitment to the translation preinitiation complex in response to RNA sequence- and structure-changes. In addition to providing streamlined and detailed protocols, we describe a new construct for purification of higher yields of fluorescently labeled and unlabeled full-length eIF4G. [ABSTRACT FROM AUTHOR]

The translational capability of ribosomes deprived of specific non-fundamental ribosomal proteins may be altered. Physiological mechanisms are scanty and it is unclear whether free ribosomal proteins can crosstalk with the signaling machinery. RACK1 (Receptor for Activated C Kinase 1) is a highly conserved scaffold protein, located on the 40S subunit near the mRNA exit channel. RACK1 is involved in a variety of intracellular contexts, both on and off the ribosomes, acting as a receptor for proteins in signaling like PKCs. Here we show that binding of RACK1 to ribosomes is essential for full translation of capped mRNAs and efficient recruitment of eIF4E (eukaryotic Initiation Factor 4E). In vitro, supplementing the ribosome machinery with wild-type RACK1 restores the translational capability, whereas addition of a RACK1 mutant unable to bind ribosomes does not. Out of the ribosome, RACK1 has a reduced half-life. By accumulating in living cells, free RACK1 exerts an inhibitory phenotype by impairing cell cycle progression and repressing global translation. Here we present RACK1 binding to ribosomes as a crucial way to regulate translation, possibly by interacting with known signaling-involved partners on or out of the ribosome. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea (PED) is an acute, highly infectious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which seriously endangers the healthy development of the pig industry. PEDV N protein is the most abundant viral structural protein, which can be combined with viral genomic RNA to form ribonucleoprotein complexes, thereby participating in the transcription and replication of the virus. However, how PEDV hijacks the host transcription translation system to promote viral proliferation remains unclear. In this study, we found that there is an interaction between PEDV N, polyadenylate-binding protein cytoplasmic 1 (PABPC1) and eukaryotic initiation factor 4F (eIF4F) proteins through coimmunoprecipitation, GST pulldown and fluorescence microscopy experiments. PABPC1 could bind to the poly(A) tail of the mRNA, and eIF4F could bind to the 5' end cap structure of the mRNA, so the interaction of PABPC1 and eIF4F could facilitate mRNA forming a circular shape to promote translation to the proteins. To further explore the effect of N protein capture protein translation element PABPC1 and eIF4F on PEDV replication, we overexpressed PABPC1, eIF4F (containing eIF4A, eIF4E and eIF4G) separately on Vero cells and LLC-PK1 cells, and we found that the PABPC1 and eIF4F protein could promote PEDV replication. Taken together, our data suggested that PEDV N protein promoted cyclization of viral mRNA carried by N protein through binding with PABPC1 and eIF4F proteins, thus promoting viral transcription and facilitating viral replication. • PABPC1 and eIF4F interacted with the PEDV N protein. • PABPC1 or eIF4F overexpression promoted PEDV replication. • PABPC1 or eIF4F knockdown inhibited PEDV replication in both Vero cells and LLC-PK1 cells. [ABSTRACT FROM AUTHOR]

Protein concentrations evolve under greater evolutionary constraint than mRNA levels. Translation efficiency of mRNA represents the chief determinant of basal protein concentrations. This raises a fundamental question of how mRNA and protein levels are coordinated in dynamic systems responding to physiological stimuli. This report examines the contributions of mRNA abundance and translation efficiency to protein output in cells responding to oxygen stimulus. We show that changes in translation efficiencies, and not mRNA levels, represent the major mechanism governing cellular responses to [O2] perturbations. Two distinct cap-dependent protein synthesis machineries select mRNAs for translation: the normoxic eIF4F and the hypoxic eIF4FH. O2-dependent remodeling of translation efficiencies enables cells to produce adaptive translatomes from preexisting mRNA pools. Differences in mRNA expression observed under different [O2] are likely neutral, given that they occur during evolution. We propose that mRNAs contain translation efficiency determinants for their triage by the translation apparatus on [O2] stimulus.

Over the past 15 years, sequencing of the human genome and The Cancer Genome Atlas (TCGA) project have led to comprehensive lists of single-nucleotide polymorphisms (SNPs) and gene mutations across a large number of human samples. However, our ability to predict the functional impact of SNPs and mutations on gene expression is still in its infancy. Here, we provide key examples to help understand how mutations present in genes can affect translational output.

Most of the positive-strand RNA plant viruses lack the 5′-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5′- or 3′-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3′-end of viruses belonging to the family Tombusviridae and the genus Luteovirus. Seven classes of 3′-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3′-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5′-end by different mechanisms, often long-distance RNA–RNA interactions. As previously proposed and recently found in one case in nature, 3′-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3′-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3′-CITEs belonging to different classes.

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]

Most of the positive-strand RNA plant viruses lack the 5'-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5' - or 3'-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3'-end of viruses belonging to the family Tombusviridae and the genus Luteovirus. Seven classes of 3'-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3'-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5'-end by different mechanisms, often long-distance RNA--RNA interactions. As previously proposed and recently found in one case in nature, 3'- CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3'-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3'-CITEs belonging to different classes. [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.

The bipartite genomic RNAs of red clover necrotic mosaic virus (RCNMV) lack a 5′ cap and a 3′ poly(A) tail. RNA1 encodes viral replication proteins, and RNA2 encodes a movement protein (MP). These proteins are translated in a cap-independent manner. We previously identified two cis -acting RNA elements that cooperatively recruit eukaryotic translation initiation factor (eIF) complex eIF4F or eIFiso4F to RNA1. Such cis -acting RNA elements and host factors have not been identified in RNA2. Here we found that translation of RNA1 was significantly compromised in Arabidopsis thaliana carrying eif4f mutation. RNA1 replicated efficiently in eifiso4f1 mutants, suggesting vigorous translation of the replication proteins from RNA1 in the plants. In contrast, MP accumulation was decreased in eifiso4f1 mutants but not in eif4f mutants. Collectively, these results suggest that RCNMV uses different eIF complexes for translation of its bipartite genomic RNAs, which may contribute to fine-tuning viral gene expression during infection. [ABSTRACT FROM AUTHOR]

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]

Although micro RNAs and EIF4G2 are both known to play pivotal roles in cancer progression, it remains unknown whether these pathways regulate chemosensitivity in a coordinated manner. Here, we show that miR-379 expression is significantly downregulated in chemoresistant nonsmall cell lung cancer ( NSCLC) tissues and cells. Manipulation of miR-379 levels could alter the in vitro and in vivo cisplatin ( CDDP) resistance in lung cancer ( LCa) cells. Mechanistically, miR-379 potentiated LCa chemosensitivity via modulation of CDDP-induced apoptosis by directly targeting the EIF4G2 3′ UTR. Additionally, we observed an inverse correlation between miR-379 and EIF4G2 expression in LCa tissues from patients with CDDP-based chemotherapy. Together, our findings shed new light on the potential involvement of miR-379/ EIF4G2 cascade in the pathogenesis of CDDP resistance in LCa. [ABSTRACT FROM AUTHOR]

Introduction: Annexin A2 (AnxA2) plays a critical role in cell transformation, immune response, and resistance to cancer therapy. Besides functioning as a calcium- and lipidbinding protein, AnxA2 also acts as an mRNA-binding protein, for instance, by interacting with regulatory regions of specific cytoskeleton-associated mRNAs. Methods and Results: Nanomolar concentrations of FL3, an inhibitor of the translation factor eIF4A, transiently increases the expression of AnxA2 in PC12 cells and stimulates shortterm transcription/translation of anxA2 mRNA in the rabbit reticulocyte lysate. AnxA2 regulates the translation of its cognate mRNA by a feed-back mechanism, which can partly be relieved by FL3. Results obtained using the holdup chromatographic retention assay results suggest that AnxA2 interacts transiently with eIF4E (possibly eIF4G) and PABP in an RNA-independent manner while cap pulldown experiments indicate a more stable RNA-dependent interaction. Short-term (2 h) treatment of PC12 cells with FL3 increases the amount of eIF4A in cap pulldown complexes of total lysates, but not of the cytoskeletal fraction. AnxA2 is only present in cap analogue-purified initiation complexes from the cytoskeletal fraction and not total lysates confirming that AnxA2 binds to a specific subpopulation of mRNAs. Discussion: Thus, AnxA2 interacts with PABP1 and subunits of the initiation complex eIF4F, explaining its inhibitory effect on translation by preventing the formation of the full eIF4F complex. This interaction appears to be modulated by FL3. These novel findings shed light on the regulation of translation by AnxA2 and contribute to a better understanding of the mechanism of action of eIF4A inhibitors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Grindheim, Patil, Nebigil, Désaubry and Vedeler.)

Summary Eukaryotic initiation factor 4G (eIF4G) plays a central role in translation initiation through its interactions with the cap-binding protein eIF4E. This interaction is a major drug target for repressing translation and is naturally regulated by 4E-binding proteins (4E-BPs). 4E-BPs and eIF4G compete for binding to the eIF4E dorsal surface via a shared canonical 4E-binding motif, but also contain auxiliary eIF4E-binding sequences, which were assumed to contact non-overlapping eIF4E surfaces. However, it is unknown how metazoan eIF4G auxiliary sequences bind eIF4E. Here, we describe crystal structures of human and Drosophila melanogaster eIF4E-eIF4G complexes, which unexpectedly reveal that the eIF4G auxiliary sequences bind to the lateral surface of eIF4E, using a similar mode to that of 4E-BPs. Our studies provide a molecular model of the eIF4E-eIF4G complex, shed light on the competition mechanism of 4E-BPs, and enable the rational design of selective eIF4G inhibitors to dampen dysregulated translation in disease. [ABSTRACT FROM AUTHOR]

In stressed cells, a general decrease in the rate of protein synthesis occurs due to modifications in the activity of translation initiation factors. Compelling data now indicate that these changes also permit a selective post-transcriptional expression of proteins necessary for either cell survival or completion of apoptosis when cells are exposed to severe or prolonged stress. In this review, we summarize the modifications that inhibit the activity of the main canonical translation initiation factors, and the data explaining how certain mRNAs encoding proteins involved in either cell survival or apoptosis can be selectively translated.

The eukaryotic initiation factor 4G2 (eIF4G2, DAP5, Nat1, p97) was discovered in 1997. Over the past two decades, dozens of papers have presented contradictory data on eIF4G2 function. Since its identification, eIF4G2 has been assumed to participate in noncanonical translation initiation mechanisms, but recent results indicate that it can be involved in scanning as well. In particular, eIF4G2 provides leaky scanning through some upstream open reading frames (uORFs), which are typical for long 5' UTRs of mRNAs from higher eukaryotes. It is likely the protein can also help the ribosome overcome other impediments during scanning of the 5' UTRs of animal mRNAs. This may explain the need for eIF4G2 in higher eukaryotes, as many mRNAs that encode regulatory proteins have rather long and highly structured 5' UTRs. Additionally, they often bind to various proteins, which also hamper the movement of scanning ribosomes. This review discusses the suggested mechanisms of eIF4G2 action, denotes obscure or inconsistent results, and proposes ways to uncover other fundamental mechanisms in which this important protein factor may be involved in higher eukaryotes., (© 2023 Shestakova et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Fundamental studies unraveled the role of eukaryotic initiation factor (eIF) 4E in mRNA translation and its control. Under physiological conditions, regulation of translation by eIF4E is essential to cellular homeostasis. Under stress, gene flow information is parsed by eIF4E to support adaptive mechanisms that favor cell survival. Dysregulated eIF4E activity fuels tumor formation and progression and modulates response to therapy. Thus, there has been heightened interest in understanding eIF4E function in controlling gene expression as well as developing strategies to block its activity to treat disease., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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]