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Lung cancer is one of the most common types of malignant cancer worldwide, causing a serious social and economic burden. It is classified into non-small cell lung cancer (NSCLC) and small cell lung cancer, with NSCLC accounting for 80-85% of cases. Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is highly expressed in NSCLC, playing an important role in regulating tumor growth, angiogenesis, malignant transformation, and phagocytosis. Ubiquitin-specific protease 10 (USP10) functions as a deubiquitinating enzyme to regulate substrate protein deubiquitination and reverse the ubiquitin proteasome degradation pathway. Our previous study identified an interaction between EIF4G1 and USP10; however, their regulatory mechanism remains unclear. Herein, we found that USP10 positively regulates EIF4G1 in NSCLC cells. An in vivo ubiquitination assay demonstrated deubiquitination of EIF4G1 by USP10, which reversed the ubiquitin proteasomal degradation of EIF4G1, thereby increasing its stability. Upregulation of EIF4G1 promoted cell proliferation, migration, and invasion in NSCLC cells. The current study not only reveals a novel mechanism through which USP10 positively regulates EIF4G1 in NSCLC, but also demonstrates the potential of USP10 as a therapeutic target to treat NSCLC., (© 2024. The Author(s).)

Hepatocellular carcinoma (HCC) is a highly lethal cancer, and proteomic studies have shown increased protein diversity and abundance in HCC tissues, whereas the role of protein translation has not been extensively explored in HCC. Our research focused on key molecules in the translation process to identify a potential contributor in HCC. We discovered that EIF4G2, a crucial translation initiation factor, is significantly upregulated in HCC tissues and associated with poor prognosis. This study uniquely highlights the impact of EIF4G2 deletion, which suppresses tumor growth and metastasis both in vitro and in vivo . Furthermore, polysome analysis and nascent protein synthesis assays revealed EIF4G2's role in regulating protein translation, specifically identifying PLEKHA1 as a key translational product. This represents a novel mechanistic insight into HCC malignancy. RNA immunoprecipitation (RIP) and Dual-luciferase reporter assays further revealed that EIF4G2 facilitates PLEKHA1 translation via an IRES-dependent manner. Importantly, the synergistic effects of EIF4G2 depletion and PLEKHA1 reduction in inhibiting cell migration and invasion underscore the therapeutic potential of targeting this axis. This study not only advances our understanding of translational regulation in HCC but also identifies the EIF4G2-PLEKHA1 axis as a promising therapeutic target, offering new avenues for intervention in HCC treatment.

The clinical outcomes of osteosarcoma are relatively dismal. As immunotherapy has revolutionized treatment for solid tumors, exploring novel immunotherapy-related therapeutic targets for osteosarcoma is important. In this study, we aimed to establish the connection between RNA modification and immunotherapy in osteosarcoma to identify novel therapeutic targets. An RNA modification-related signature was first developed using weight gene correlation network analysis and a machine-learning algorithm, random forest. The signature's prognostic value, drug prediction, and immune characteristics were analyzed. EIF4G2 from the signature was next identified as a critical immunotherapy determinant. EIF4G2 could also promote tumor proliferation, migration, and M2 macrophage migration by single-cell sequencing analysis and in vitro validation. Our signature and EIF4G2 are expected to provide valuable insights into the clinical management of osteosarcoma., (© 2024 Wiley Periodicals LLC.)

Background: Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the unfolded protein response transducers and effectors directly take part in the control of protein synthesis. The connection between critical functions of the unfolded protein response in cellular physiology and requirements of multiple processes for cell growth prompted us to investigate the role of the unfolded protein response in cell growth and underlying molecular mechanisms., Methods: Cardiomyocyte-specific inositol-requiring enzyme 1α (IRE1α) knockout and overexpression mouse models were generated to explore its function in vivo. Neonatal rat ventricular myocytes were isolated and cultured to evaluate the role of IRE1α in cardiomyocyte growth in vitro. Mass spectrometry was conducted to identify novel interacting proteins of IRE1α. Ribosome sequencing and polysome profiling were performed to determine the molecular basis for the function of IRE1α in translational control., Results: We show that IRE1α is required for cell growth in neonatal rat ventricular myocytes under prohypertrophy treatment and in HEK293 cells in response to serum stimulation. At the molecular level, IRE1α directly interacts with eIF4G and eIF3, 2 critical components of the translation initiation complex. We demonstrate that IRE1α facilitates the formation of the translation initiation complex around the endoplasmic reticulum and preferentially initiates the translation of transcripts with 5' terminal oligopyrimidine motifs. We then reveal that IRE1α plays an important role in determining the selectivity and translation of these transcripts. We next show that IRE1α stimulates the translation of epidermal growth factor receptor through an unannotated terminal oligopyrimidine motif in its 5' untranslated region. We further demonstrate a physiological role of IRE1α-governed protein translation by showing that IRE1α is essential for cardiomyocyte growth and cardiac functional maintenance under hemodynamic stress in vivo., Conclusions: These studies suggest a noncanonical, essential role of IRE1α in orchestrating protein synthesis, which may have important implications in cardiac hypertrophy in response to pressure overload and general cell growth under other physiological and pathological conditions., Competing Interests: None.

Background: Abnormal lipid deposition is an important driver of the progression of metabolic dysfunction-associated steatotic liver disease (MASLD). MicroRNA-411-5p (miR-411-5p) and eukaryotic translation initiation factor 4γ2 (EIF4G2) are related to abnormal lipid deposition, but the specific mechanism is unknown., Methods: A high-fat, high-cholesterol diet (HFHCD) and a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) and a high-fructose diet (HFrD) were used to establish MASLD rat and mouse models, respectively. MiR-411-5p agomir and mimic were used to upregulate the miR-411-5p in vivo and in vitro, respectively. Adeno-associated virus type 8 (AAV8) carrying EIF4G2 short hairpin RNA (shRNA) and small interfering RNA (siRNA) were used to downregulate the EIF4G2 expression in vivo and in vitro, respectively. Liver histopathological analysis, Biochemical analysis and other experiments were used to explore the functions of miR-411-5p and EIF4G2., Results: MiR-411-5p was decreased in both MASLD rats and mice, and was negatively correlated with liver triglycerides and serum alanine transaminase (ALT) and aspartate transaminase (AST) levels. Upregulation of miR-411-5p alleviated liver lipid deposition and hepatocellular steatosis. Moreover, miR-411-5p targeted and downregulated EIF4G2. Downregulation of EIF4G2 not only reduced liver triglycerides and serum ALT and AST levels in MASLD model, but also alleviated lipid deposition. Notably, upregulation of miR-411-5p and downregulation of EIF4G2 led to the reduction of forkhead box class O3 (FOXO3) and inhibited the expression of sterol regulatory-element binding protein 1 (SREBP1), acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), thereby reducing fatty acid synthesis., Conclusions: Upregulation of miR-411-5p inhibits EIF4G2 to reduce the FOXO3 expression, thereby reducing fatty acid synthesis and alleviating abnormal lipid deposition in MASLD., (© 2024. The Author(s).)

Motoneurons critically depend on precise spatial and temporal control of translation for axon growth and the establishment and maintenance of neuromuscular connections. While defects in local translation have been implicated in the pathogenesis of motoneuron disorders, little is known about the mechanisms regulating axonal protein synthesis. Here, we report that motoneurons derived from Hnrnpr knockout mice show reduced axon growth accompanied by lowered synthesis of cytoskeletal and synaptic components in axons. Mutant mice display denervated neuromuscular junctions and impaired motor behavior. In axons, hnRNP R is a component of translation initiation complexes and, through interaction with O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (Ogt), modulates O-GlcNAcylation of eIF4G. Restoring axonal O-GlcNAc levels rescued local protein synthesis and axon growth defects of hnRNP R knockout motoneurons. Together, these findings demonstrate a function of hnRNP R in controlling the local production of key factors required for axon growth and formation of neuromuscular innervations., (© 2024. The Author(s).)

Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem-loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5'-UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5'-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies., (© 2024 Whittaker and Goss; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Viruses can selectively repress the translation of mRNAs involved in the antiviral response. RNA viruses exploit the Grb10-interacting GYF (glycine-tyrosine-phenylalanine) proteins 2 (GIGYF2) and eukaryotic translation initiation factor 4E (eIF4E) homologous protein 4EHP to selectively repress the translation of transcripts such as Ifnb1 , which encodes the antiviral cytokine interferon-β (IFN-β). Herein, we reveal that GIGYF1, a paralog of GIGYF2, robustly represses cellular mRNA translation through a distinct 4EHP-independent mechanism. Upon recruitment to a target mRNA, GIGYF1 binds to subunits of eukaryotic translation initiation factor 3 (eIF3) at the eIF3-eIF4G1 interaction interface. This interaction disrupts the eIF3 binding to eIF4G1, resulting in transcript-specific translational repression. Depletion of GIGYF1 induces a robust immune response by derepressing IFN-β production. Our study highlights a unique mechanism of translational regulation by GIGYF1 that involves sequestering eIF3 and abrogating its binding to eIF4G1. This mechanism has profound implications for the host response to viral infections.

Malignant pleural mesothelioma is a rare and lethal cancer caused by exposure to asbestos. The highly inflammatory environment caused by fibers accumulation forces cells to undergo profound adaptation to gain survival advantages. Prioritizing the synthesis of essential transcripts is an efficient mechanism coordinated by multiple molecules, including long non-coding RNAs. Enhancing the knowledge about these mechanisms is an essential weapon in combating mesothelioma. Linc00941 correlates to bad prognosis in various cancers, but it is reported to partake in distinct and apparently irreconcilable processes. In this work, we report that linc00941 supports the survival and aggressiveness of mesothelioma cells by influencing protein synthesis and ribosome biogenesis. Linc00941 binds to the translation initiation factor eIF4G, promoting the selective protein synthesis of cMYC, which, in turn, enhances the expression of key genes involved in translation. We analyzed a retrospective cohort of 97 mesothelioma patients' samples from our institution, revealing that linc00941 expression strongly correlates with reduced survival probability. This discovery clarifies linc00941's role in mesothelioma and proposes a unified mechanism of action for this lncRNA involving the selective translation of essential oncogenes, reconciling the discrepancies about its function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Scanning and initiation are critical steps in translation. Here, we utilized translation complex profiling (TCP-seq) to investigate 48S organization and eIF4G1-eIF1 inhibition impact. We provide global views of scanning and leaky scanning, uncovering a central role of eIF4G1-eIF1 in their regulation. We confirm AUG context importance, with non-leaky genes featuring a Kozak context and cytosine at positions -1 and +5. Capturing 48S complexes associated with eIF1, eIF4G1, eIF3, and eIF2 through selective TCP-seq revealed that the eIF3-scanning ribosome is highly vulnerable to eIF4G1-eIF1 inhibition, and eIF1 tends to dissociate upon AUG recognition. Initiation-site footprint analysis revealed a class spanning -12 to +18/19 from the AUG, representing the entire 48S and enriched with eIF2, eIF1, and eIF4G1, indicative of early initiation. Another eIF3-dependent class extends up to +26 and exhibits reduced eIF2 and eIF4G1 association, suggesting a late/alternative initiation complex. Our analysis provides an overview of scanning, initiation, and evidence for conformational rearrangements in vivo., Competing Interests: Declaration of interests The authors declare a patent application for the i14G1-12 used in this study (YEDA-P-015-IL)., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests S.A. is an advisor on the scientific advisory board of Dewpoint Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The heat shock response is crucial for cell survival. In this issue of Molecular Cell, Desroches Altamirano et al. 1 demonstrate that a temperature-induced conformational change in the translation initiation factor eIF4G is a key mechanism regulating translation during the heat shock response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The CRISPR-Cas system, initially for DNA-level gene editing and transcription regulation, has expanded to RNA targeting with the Cas13d family, notably the RfxCas13d. This advancement allows for mRNA targeting with high specificity, particularly after catalytic inactivation, broadening the exploration of translation regulation. This study introduces a CRISPR-dCas13d-eIF4G fusion module, combining dCas13d with the eIF4G translation regulatory element, enhancing target mRNA translation levels. This module, using specially designed sgRNAs, selectively boosts protein translation in targeted tissue cells without altering transcription, leading to notable protein expression upregulation. This system is applied to a kidney stone disease model, focusing on ferroptosis-linked GPX4 gene regulation. By targeting GPX4 with sgRNAs, its protein expression is upregulated in human renal cells and mouse kidney tissue, countering ferroptosis and resisting calcium oxalate-induced cell damage, hence mitigating stone formation. This study evidences the CRISPR-dCas13d-eIF4G system's efficacy in eukaryotic cells, presenting a novel protein translation research approach and potential kidney stone disease treatment advancements., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

The cap-dependent mRNA translation is dysregulated in many kinds of cancers. The interaction between eIF4E and eIF4G through a canonical eIF4E-binding motif (CEBM) determines the efficacy of the cap-dependent mRNA translation. eIF4E-binding proteins (4E-BPs) share the CEBM and compete with eIF4G for the same binding surface of eIF4E and then inhibit the mRNA translation. 4E-BPs function as tumor repressors in nature. Hyperphosphorylation of 4E-BPs regulates the structure folding and causes the dissociation of 4E-BPs from eIF4E. However, until now, there has been no structure of the full-length 4E-BPs in complex with eIF4E. The regulation mechanism of phosphorylation is still unclear. In this work, we first investigate the interactions of human eIF4E with the CEBM and an auxiliary eIF4E-binding motif (AEBM) in eIF4G and 4E-BPs. The results unravel that the structure and interactions of the CEBM are highly conserved between eIF4G and 4E-BPs. However, the extended CEBM (ECEBM) in 4E-BPs forms a longer helix than that in eIF4G. The residue R62 in the ECEBM of 4E-BP2 forms salt bridges with E32 and E70 of eIF4E. The residue R63 of 4E-BP2 forms two special hydrogen bonds with N77 of eIF4E. Both of these interactions are missing in eIF4G. The AEBM of 4E-BPs folds into a β-sheet conformation, which protects V81 inside a hydrophobic core in 4E-BP2. In eIF4G, the AEBM exists in a random coil state. The hydrophilic residues S637 and D638 of eIF4G open the hydrophobic core for solvents. The results show that the ECEBM and AEBM may be responsible for the competing advantage of 4E-BP2. Finally, based on our previous work (J. Zeng, F. Jiang and Y. D. Wu, J. Chem. Theory Comput. , 2017, 13 , 320), the human eIF4E:4E-BP2 complex (eIF4E:BP2 P18-I88 ) including all reported phosphorylation sites is predicted. The eIF4E:BP2 P18-I88 complex is different from the existing experimental eIF4E:eIF4G complex and provides an important structure for further studying the regulation mechanism of phosphorylation in 4E-BPs.

The non-canonical translation initiation factor EIF4G2 plays essential roles in cellular stress responses via translation of selective mRNA cohorts. Currently there is limited and conflicting information regarding its involvement in cancer development and progression. Here we assessed its role in endometrial cancer (EC), in a cohort of 280 EC patients across different types, grades, and stages, and found that low EIF4G2 expression highly correlated with poor overall- and recurrence-free survival in Grade 2 EC patients, monitored over a period of up to 12 years. To establish a causative connection between low EIF4G2 expression and cancer progression, we stably knocked-down EIF4G2 in two human EC cell lines in parallel. EIF4G2 depletion resulted in increased resistance to conventional therapies and increased the prevalence of molecular markers for aggressive cell subsets, altering their transcriptional and proteomic landscapes. Prominent among the proteins with decreased abundance were Kinesin-1 motor proteins, KIF5B and KLC1, 2, 3. Multiplexed imaging of the EC patient tumor cohort showed a correlation between decreased expression of the kinesin proteins, and poor survival in patients with tumors of certain grades and stages. These findings reveal potential novel biomarkers for Grade 2 EC with ramifications for patient stratification and therapeutic interventions., (© 2024. The Author(s).)

Interaction between the cap-binding protein eIF4E and the scaffolding protein eIF4G is essential for the cap-dependent translation initiation in eukaryotes. In the Saccharomyces cerevisiae eIF4G/eIF4E complex, the intrinsically disordered eIF4E-binding domain of eIF4G folds into a bracelet-like structure upon binding to eIF4E. Aiming to unveil the molecular mechanism underlying the binding-wrapping process of eIF4G with eIF4E, we performed extensive coarse-grained molecular dynamics simulations and transition path analysis in this work. The major transition pathway revealed from our simulations showed that docking of the eIF4E-binding motif of eIF4G to the folded core of eIF4E initiates the binding process and then the disordered eIF4G wraps around the N-terminal tail of eIF4E. Additionally, we identified a minor transition pathway which indicates the involvement of topological frustration in the binding process. By manipulating the interaction strength of the wrapping contacts and the latching contacts, we further dissected factors affecting the formation of topological frustration and the binding transition kinetics. Our findings provide new clues for experimental studies on the binding mechanism of eIF4G to eIF4E in the future and exemplify the involvement of topological frustration in the binding process of intrinsically disordered proteins.

Programmable protein scaffolds are invaluable in the development of genome engineering tools. The pentatricopeptide repeat (PPR) protein is an attractive platform for RNA manipulation because of its programmable RNA-binding selectivity, which is determined by the combination of amino acid species at three specific sites in the PPR motif. Translation is a key RNA regulatory step that determines the final gene expression level and is involved in various human diseases. In this study, designer PPR protein was used to develop a translational enhancement technique by fusion with the translation initiation factor eIF4G. The results showed that the PPR-eIF4G fusion protein could activate the translation of endogenous c-Myc and p53 mRNAs and control cell fate, indicating that PPR-based translational enhancement is a versatile technique applicable to various endogenous mRNAs in mammalian cells. In addition, the translational enhancement was dependent on both the target position and presence of eIF4G, suggesting the presence of an unknown translation activation mechanism., (© 2024. The Author(s).)

In vitro-transcribed (IVT) mRNA has arisen as a rapid method for the production of nucleic acid drugs. Here, we have constructed an oncolytic IVT mRNA that utilizes human rhinovirus type 2 (HRV2) internal ribosomal entry sites (IRESs) to selectively trigger translation in cancer cells with high expression of EIF4G2 and PTBP1. The oncolytic effect was provided by a long hGSDMD c .825 T>A/c.884 A>G -F1L CT mutant mRNA sequence with mitochondrial inner membrane cardiolipin targeting toxicity that triggers mitophagy. Utilizing the permuted intron-exon (PIE) splicing circularization strategy and lipid nanoparticle (LNP) encapsulation reduced immunogenicity of the mRNA and enabled delivery to eukaryotic cells in vivo. Engineered HRV2 IRESs-GSDMD p.D275E/E295G -F1L CT circRNA-LNPs (GSDMD ENG circRNA) successfully inhibited EIF4G2 + /PTBP1 + pan-adenocarcinoma xenografts growth. Importantly, in a spontaneous tumor model with abnormal EIF4G2 and PTBP1 caused by KRAS G12D mutation, GSDMD ENG circRNA significantly prevented the occurrence of pancreatic, lung and colon adenocarcinoma, improved the survival rate and induced persistent KRAS G12D tumor antigen-specific cytotoxic T lymphocyte responses., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Circular RNAs (circRNAs) have been reported to be involved in the initiation and development of cancers. The aim of this study was to determine the role of a circRNA, circ_0020123, in the development of non-small cell lung cancer (NSCLC)., Methods: The expression of circ_0020123, microRNA-146a-5p (miR-146a-5p), and eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) mRNA was detected by quantitative real-time PCR (qPCR). Western blot was used to determine the protein levels of cyclin D1, Bax, MMP-9, and EIF4G2. Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay and colony formation assay. Flow cytometry assay was applied to determine cell cycle apoptosis. Cell migration and invasion were assessed using transwell assay. The potential relationship between miR-146a-5p and circ_0020123 or EIF4G2 was ascertained by dual-luciferase reporter assay and RIP assay. The role of circ_0020123 in vivo was explored by xenograft assay., Results: Circ_0020123 was upregulated in NSCLC, and circ_0020123 knockdown repressed proliferation, migration, and invasion of NSCLC cells. Circ_0020123 targeted miR-146a-5p, and miR-146a-5p inhibitor reversed the effects of circ_0020123 knockdown on NSCLC cells. In addition, miR-146a-5p suppressed cell proliferation, migration, and invasion by targeting EIF4G2. Moreover, the antitumor role of circ_0020123 knockdown was verified in vivo., Conclusion: Knockdown of circ_0020123 inhibited NSCLC cell progression and tumor growth by targeting the miR-146a-5p/EIF4G2 axis., (© 2023 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Tumor cells often exploit the protein translation machinery, resulting in enhanced protein expression essential for tumor growth. Since canonical translation initiation is often suppressed because of cell stress in the tumor microenvironment, non-canonical translation initiation mechanisms become particularly important for shaping the tumor proteome. EIF4G2 is a non-canonical translation initiation factor that mediates internal ribosome entry site (IRES)- and uORF-dependent initiation mechanisms, which can be used to modulate protein expression in cancer. Here, we explored the contribution of EIF4G2 to cancer by screening the COSMIC database for EIF4G2 somatic mutations in cancer patients. Functional examination of missense mutations revealed deleterious effects on EIF4G2 protein-protein interactions and, importantly, on its ability to mediate non-canonical translation initiation. Specifically, one mutation, R178Q, led to reductions in protein expression and near-complete loss of function. Two other mutations within the MIF4G domain specifically affected EIF4G2's ability to mediate IRES-dependent translation initiation but not that of target mRNAs with uORFs. These results shed light on both the structure-function of EIF4G2 and its potential tumor suppressor effects., (© 2023 Meril et al.)

Protein requirements of eukaryotic cells are ensured by proteostasis, which is mediated by tight control of TORC1 activity. Upon TORC1 inhibition, protein degradation is increased and protein synthesis is reduced through inhibition of translation initiation to maintain cell viability. Here, we show that the ribosome-associated complex (RAC)/Ssb chaperone system, composed of the HSP70 chaperone Ssb and its HSP40 co-chaperone Zuo1, is required to maintain proteostasis and cell viability under TORC1 inhibition in Saccharomyces cerevisiae. In the absence of Zuo1, translation does not decrease in response to the loss of TORC1 activity. A functional interaction between Zuo1 and Ssb is required for proper translational control and proteostasis maintenance upon TORC1 inhibition. Furthermore, we have shown that the rapid degradation of eIF4G following TORC1 inhibition is mediated by autophagy and is prevented in zuo1Δ cells, contributing to decreased survival in these conditions. We found that autophagy is defective in zuo1Δ cells, which impedes eIF4G degradation upon TORC1 inhibition. Our findings identify an essential role for RAC/Ssb in regulating translation in response to changes in TORC1 signalling., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Translation initiation factor 4G (eIF4G) is an integral component of the eIF4F complex which is key to translation initiation for most eukaryotic mRNAs. Many eIF4G isoforms have been described in diverse eukaryotic organisms but we currently have a poor understanding of their functional roles and whether they regulate translation in an mRNA specific manner. The yeast Saccharomyces cerevisiae expresses two eIF4G isoforms, eIF4G1 and eIF4G2, that have previously been considered as functionally redundant with any phenotypic differences arising due to alteration in eIF4G expression levels. Using homogenic strains that express eIF4G1 or eIF4G2 as the sole eIF4G isoforms at comparable expression levels to total eIF4G, we show that eIF4G1 is specifically required to mediate the translational response to oxidative stress. eIF4G1 binds the mRNA cap and remains associated with actively translating ribosomes during oxidative stress conditions and we use quantitative proteomics to show that eIF4G1 promotes oxidative stress-specific proteome changes. eIF4G1, but not eIF4G2, binds the Slf1 LARP protein which appears to mediate the eIF4G1-dependent translational response to oxidative stress. We show similar isoform specific roles for eIF4G in human cells suggesting convergent evolution of multiple eIF4G isoforms offers significant advantages especially where translation must continue under stress conditions., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Neurodegeneration in Fragile X-associated tremor/ataxia syndrome (FXTAS) is caused by a CGG trinucleotide repeat expansion in the 5' UTR of FMR1. Expanded CGG repeat RNAs form stable secondary structures, which in turn support repeat-associated non-AUG (RAN) translation to produce toxic peptides. The parameters that impact RAN translation initiation efficiency are not well understood. Here we used a Drosophila melanogaster model of FXTAS to evaluate the role of the eIF4G family of eukaryotic translation initiation factors (EIF4G1, EIF4GII and EIF4G2/DAP5) in modulating RAN translation and CGG repeat-associated toxicity. DAP5 knockdown robustly suppressed CGG repeat-associated toxicity and inhibited RAN translation. Furthermore, knockdown of initiation factors that preferentially associate with DAP5 (such as EIF2β, EIF3F and EIF3G) also selectively suppressed CGG repeat-induced eye degeneration. In mammalian cellular reporter assays, DAP5 knockdown exhibited modest and cell-type specific effects on RAN translation. Taken together, these data support a role for DAP5 in CGG repeat associated toxicity possibly through modulation of RAN translation., Competing Interests: Declaration of Competing Interest The authors have no financial conflicts of interest associated with the work presented in this manuscript., (Published by Elsevier Inc.)

Cancer cell plasticity enables cell survival in harsh physiological environments and fate transitions such as the epithelial-to-mesenchymal transition (EMT) that underlies invasion and metastasis. Using genome-wide transcriptomic and translatomic studies, an alternate mechanism of cap-dependent mRNA translation by the DAP5/eIF3d complex is shown to be essential for metastasis, EMT, and tumor directed angiogenesis. DAP5/eIF3d carries out selective translation of mRNAs encoding EMT transcription factors and regulators, cell migration integrins, metalloproteinases, and cell survival and angiogenesis factors. DAP5 is overexpressed in metastatic human breast cancers associated with poor metastasis-free survival. In human and murine breast cancer animal models, DAP5 is not required for primary tumor growth but is essential for EMT, cell migration, invasion, metastasis, angiogenesis, and resistance to anoikis. Thus, cancer cell mRNA translation involves two cap-dependent mRNA translation mechanisms, eIF4E/mTORC1 and DAP5/eIF3d. These findings highlight a surprising level of plasticity in mRNA translation during cancer progression and metastasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

During cellular stress conditions, particularly those seen in multiple cancers, canonical cap-dependent translation is suppressed and a subset of cellular mRNAs (e.g., those encoding FGF-9, HIF-1α, and p53, among others) is known to translate in a cap-independent manner. Human eIF4GI specifically binds to the highly structured 5'-untranslated regions (5'UTRs) of these mRNAs to promote cap-independent translation. The thermodynamics of these protein-RNA interactions have not been explored, and such information will aid in understanding the basic interactions and in potential design of therapeutic drugs. Using fluorescence quenching-based assays and site-directed mutagenesis, we determined the thermodynamic properties of three eIF4GI constructs binding to the 5'UTRs of FGF-9, HIF-1α, and p53 mRNA. These three constructs were designed to explore the importance of the eIF4E binding domain of eIF4GI, which has been shown to be important in binding and selectivity. eIF4GI 557-1599 , containing the eIF4E binding domain, had higher binding enthalpy (-21 to -14 kJ mol -1 higher), suggesting increased hydrogen bonding, whereas for eIF4GI 682-1599 lacking the eIF4E binding domain, binding was entropically favored (TΔS/ΔG of 46-85%), suggesting hydrophobic forces and/or less specific binding. A third construct where a cluster of positively charged amino acids was changed to neutral amino acids showed intermediate properties. Circular dichroism spectra confirmed the significant role of eIF4E binding domain in stable bond formation between eIF4GI and mRNAs via conformational changes. Together, these data contribute to a better understanding of the molecular forces involved in eIF4GI-mRNA recognition and elucidate properties important for the design of small molecules to mediate these interactions.

Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is highly expressed in many cancers and affects their occurrence and development. However, the effect of EIF4G1 on the prognosis, biological function and the relevant mechanism in lung squamous cell carcinoma (LSCC) is unclear. Through clinical cases, Cox's proportional hazard model and Kaplan-Meier plotter survival analysis, we find the expression levels of EIF4G1 are dependent on age and clinical stage, high expression of EIF4G1 could be used to predict the overall survival of LSCC patients. LSCC cell line NCI-H1703, NCI-H226 and SK-MES-1infected with EIF4G1 siRNA are used to detect the function of EIF4G1 with cell proliferation and tumorigenesis in vivo and vitro. The data show that EIF4G1 promotes tumor cell proliferation and the G1/S transition of cell cycle in LSCC, then the biological function of LSCC is effected by the AKT/mTOR pathway. Above all, these results have demonstrated that EIF4G1 promotes LSCC cell proliferation and may represent an indicator of prognosis in LSCC., (© 2023. The Author(s).)

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.)

Protein translation is a critical regulatory event involved in nearly all physiological and pathological processes. Eukaryotic translation initiation factors are dedicated to translation initiation, the most highly regulated stage of protein synthesis. Eukaryotic translation initiation factor 4G2 (eIF4G2, also called p97, NAT1 and DAP5), an eIF4G family member that lacks the binding sites for 5' cap binding protein eIF4E, is widely considered to be a key factor for internal ribosome entry sites (IRESs)-mediated cap-independent translation. However, recent findings demonstrate that eIF4G2 also supports many other translation initiation pathways. In this review, we summarize the role of eIF4G2 in a variety of cap-independent and -dependent translation initiation events. Additionally, we also update recent findings regarding the role of eIF4G2 in apoptosis, cell survival, cell differentiation and embryonic development. These studies reveal an emerging new picture of how eIF4G2 utilizes diverse translational mechanisms to regulate gene expression., (© 2022 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Translation is mediated by precisely orchestrated sequential interactions among translation initiation components, mRNA, and ribosomes. Biochemical, structural, and genetic techniques have revealed the fundamental mechanism that determines what occurs and when, where and in what order. Most mRNAs are circularized via the eIF4E-eIF4G-PABP interaction, which stabilizes mRNAs and enhances translation by recycling ribosomes. However, studies using single-molecule fluorescence imaging have allowed for the visualization of complex data that opposes the traditional "functional circularization" theory. Here, we briefly introduce single-molecule techniques applied to studies on mRNA circularization and describe the results of in vitro and live-cell imaging. Finally, we discuss relevant insights and questions gained from single-molecule research related to translation., (© 2023. The Author(s).)

Background: Most previous studies supported that the mammalian target of rapamycin (mTOR) is over-activated in Alzheimer's disease (AD) and exacerbates the development of AD. It is unclear whether the causal associations between the mTOR signaling-related protein and the risk for AD exist., Objective: This study aims to investigate the causal effects of the mTOR signaling targets on AD., Methods: We explored whether the risk of AD varied with genetically predicted AKT, RP-S6K, EIF4E-BP, eIF4E, eIF4A, and eIF4G circulating levels using a two-sample Mendelian randomization analysis. The summary data for targets of the mTOR signaling were acquired from published genome-wide association studies for the INTERVAL study. Genetic associations with AD were retrieved from the International Genomics of Alzheimer's Project. We utilized the inverse variance weighted as the primary approach to calculate the effect estimates., Results: The elevated levels of AKT (OR = 0.910, 95% CI=0.840-0.986, p = 0.02) and RP-S6K (OR = 0.910, 95% CI=0.840-0.986, p = 0.02) may decrease the AD risk. In contrast, the elevated eIF4E levels (OR = 1.805, 95% CI=1.002-1.174, p = 0.045) may genetically increase the AD risk. No statistical significance was identified for levels of EIF4-BP, eIF4A, and eIF4G with AD risk (p > 0.05)., Conclusion: There was a causal relationship between the mTOR signaling and the risk for AD. Activating AKT and RP-S6K, or inhibiting eIF4E may be potentially beneficial to the prevention and treatment of AD.

Initiation of translation is the first of the three obligatory steps required for protein synthesis and is carried out by a large number of protein factors called initiation factors in conjunction with ribosomes. One of the key conserved protein factors in eukaryotes that plays a role in this process is eIF4A, which has three homologues in humans with eIF4A1 being the primary factor playing a role in translation initiation. eIF4As are members of the family of DEAD-box helicases that carry out different biological functions. eIF4A1s are recruited to translation initiation complexes via association with eIF4G and have ATP binding, ATP hydrolysis, RNA binding, and unwinding activities. Plasmodium and trypanosomatids such as Leishmania and Trypanosoma are parasites that cause human disease. While mechanistically the function of eIF4A1s in eukaryotes is wellunderstood, the orthologues peIF4A1s and keIF4A1s in Plasmodium and trypanosomatids are not well-studied. Here, we have used bioinformatics tools and homology modelling/structure prediction to study the motifs and functional signatures of Plasmodium and trypanosomatid peIF4A1s/keIF4A1s. We report a high degree of sequence conservation, structural conservation, and conservation of protein-protein interaction signatures of Plasmodium and trypanosomatid peIF4A1s/keIF4A1s in comparison with human eIF4A1. Thus, in spite of the great divergence in evolution between these parasites and higher eukaryotes, there is remarkable conservation of motifs and functional signatures in Plasmodium and trypanosomatid peIF4A1s/keIF4A1s.

IRES mediated translation initiation requires a different repertoire of factors than canonical cap-dependent translation. Treatments that inhibit the canonical translation factor EIF4G1 have little or no effect on the ability of the Insr and Igf1r cellular IRESes to promote translation. Transcripts for two cellular receptors contain RNA elements that facilitate translation initiation without intact EIF4G1. Cellular IRES mechanisms may resemble viral type III IRESes allowing them to promote translate with a limited number of initiation factors allowing them to work under stress conditions when canonical translation is repressed.

Background: Breast cancer (BRCA) is one of the most common cancers in women worldwide and a leading cause of death from malignancy. This study was designed to identify a novel biomarker for prognosticating the survival of BRCA patients., Methods: The prognostic potential of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) was assessed using RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) as training cohort and validation set, respectively. The functional enrichment analysis of differentially expressed genes (DEGs) was performed. The relationship between EIF4G1 and tumor microenvironment (TME) was analyzed. Immunotherapy responses were explored by the immunophenoscores (IPS) and tumor immune dysfunction and exclusion (TIDE) score. The Connectivity Map (CMap) was used to discover potentially effective therapeutic molecules against BRCA. Immunohistochemistry (IHC) was applied to compare the protein levels of EIF4G1 in normal and cancer tissues and to verify the prognostic value of EIF4G1., Results: BRCA patients with increased expression of EIF4G1 had a shorter overall survival (OS) in all cohorts and results from IHC. EIF4G1-related genes were mainly involved in DNA replication, BRCA metastasis, and the MAPK signaling pathway. Infiltration levels of CD4 + -activated memory T cells, macrophages M0, macrophages M1, and neutrophils were higher in the EIF4G1 high-expression group than those in the EIF4G1 low-expression group. EIF4G1 was positively correlated with T cell exhaustion. Lower IPS was revealed in high EIF4G1 expression patients. Five potential groups of drugs against BRCA were identified., Conclusion: EIF4G1 might regulate the TME and affect BRCA metastasis, and it is a potential prognostic biomarker and therapeutic target for BRCA.

Translation initiation is the first step in three essential processes leading to protein synthesis. It is carried out by proteins called translation initiation factors and ribosomes on the mRNA. One of the critical translation initiation factors in eukaryotes is eIF4G which is a scaffold protein that helps assemble translation initiation complexes that carry out translation initiation which ultimately leads to polypeptide synthesis. Trypanosomatids are a large family of kinetoplastids, some of which are protozoan parasites that cause diseases in humans through transmission by vectors. While the protein translation mechanisms in eukaryotes and prokaryotes are well understood, the protein translation factors and mechanisms in trypanosomatids are poorly understood necessitating further studies. Unlike other eukaryotes, trypanosomatids contain five eIF4G orthologues with diversity in length and sequences. Here, I have used bioinformatics tools to look at trypanosomatid keIF4G orthologue sequences and report that there are similarities and considerable differences in their domains/motifs organization and signature amino acid sequences that are required for different functions as compared to human eIF4G. My analysis suggests that there is likely to be considerable diversity and complexity in trypanosomatid keIF4G functions as compared to other eukaryotes., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Eukaryotic translation is a complex process that involves the interplay of various translation factors to convert genetic information into a specific amino acid chain. According to an elegant model of eukaryotic translation initiation, the 3' poly(A) tail of an mRNA, which is occupied by poly(A)-binding proteins (PABPs), communicates with the 5'-cap bound by eIF4E to enhance translation. Although the circularization of mRNA resulting from the communication is widely understood, it has yet to be directly observed. To explore mRNA circularization in translation, we analyzed the level of colocalization of eIF4E, eIF4G, and PABP on individual mRNAs in polysomal and subpolysomal fractions using single polysome analysis. Our results show that the three tested proteins barely coexist in mRNA in either polysomal or subpolysomal fractions, implying that the closed-loop structure generated by the communication between eIF4E, eIF4G, and PAPB may be transient during translation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jong-Bong Lee reports financial support was provided by Korea Ministry of Science and ICT. Yoon Ki Kim reports financial support was provided by Korea Ministry of Science and ICT. Jong-Bong Lee reports a relationship with Korea Ministry of Science and ICT that includes: funding grants. Yoon Ki Kim reports a relationship with Korea Ministry of Science and ICT that includes: funding grants., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit general translation initiation while selectively translating essential stress regulators. Unexpectedly, in plants, pattern-triggered immunity (PTI) and response to other environmental stresses occur independently of the GCN2/eIF2α pathway. Here, we show that while PTI induces mRNA decapping to inhibit general translation, defense mRNAs with a purine-rich element ("R-motif") are selectively translated using R-motif as an internal ribosome entry site (IRES). R-motif-dependent translation is executed by poly(A)-binding proteins (PABPs) through preferential association with the PTI-activating eIFiso4G over the repressive eIF4G. Phosphorylation by PTI regulators mitogen-activated protein kinase 3 and 6 (MPK3/6) inhibits eIF4G's activity while enhancing PABP binding to the R-motif and promoting eIFiso4G-mediated defense mRNA translation, establishing a link between PTI signaling and protein synthesis. Given its prevalence in both plants and animals, the PABP/R-motif translation initiation module may have a broader role in reprogramming the stress translatome., Competing Interests: Declaration of interests X.D. is a founder of Upstream Biotechnology Inc. and a member of its scientific advisory board, as well as a scientific advisory board member of Inari Agriculture Inc. G.H.G. is a founder of Upstream Biotechnology Inc. Claims protecting IP rights to purine-rich elements are pending in U.S. patent publication US2019-0352664., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to mRNA and promote translation. Translation of ferritin IRE mRNAs is regulated by iron through iron responsive elements (IREs) and iron regulatory protein (IRP). The noncoding IRE stem-loop (30-nt) structure control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. High cellular iron concentrations promote IRE RNA binding to ribosome and initiation factors, and allow synthesis of ferritin., Methods: In vitro translation assay was performed in depleted wheat germ lysate with supplementation of initiation factors. Fluorescence spectroscopy was used to characterize eIF4F/IRE binding., Results: Eukaryotic initiation factor eIF4G increases the translation of ferritin through binding to stem loop structure of iron responsive elements mRNA in the 5'-untranslated region. Our translation experiment demonstrated that exogenous addition of eIF4G selectively enhanced the translation of ferritin IRE RNA in depleted WG lysate. However, eIF4G facilitates capped IRE RNA translation significantly higher than uncapped IRE RNA translation. Addition of iron with eIF4G to depleted WG lysate significantly enhanced translation for both IRE mRNA (capped and uncapped), confirming the contribution of eIF4G and iron as a potent enhancer of ferritin IRE mRNA translation. Fluorescence data revealed that ferritin IRE strongly interacts to eIF4G ( Kd = 63 nM), but not eIF4E. Further equilibrium studies showed that iron enhanced (~4-fold) the ferritin IRE binding to eIF4G. The equilibrium binding effects of iron on ferritin IRE RNA/eIFs interaction and the temperature dependence of this reaction were measured and compared. The Kd values for the IRE binding to eIF4G ranging from 18.2 nM to 63.0 nM as temperature elevated from 5 °C to 25 °C, while the presence of iron showed much stronger affinity over the same range of temperatures. Thermodynamic parameter revealed that IRE RNA binds to eIF4G with ΔH = -42.6 ± 3.3 kJ. mole-1, ΔS = -11.5 ± 0.4 J. mole-1K-1, and ΔG = -39.2 ± 2.7 kJ. mole-1, respectively. Furthermore, addition of iron significantly changed the values of thermodynamic parameters, favoring stable complex formation, thus favoring efficient protein synthesis. This study first time demonstrate the participation of eIF4G in ferritin IRE mRNA translation., Conclusions: eIF4G specifically interacts with ferritin IRE RNA and promotes eIF4G-dependent translation., Competing Interests: The author declares no conflict of interest., (© 2022 The Author(s). Published by IMR Press.)

Splicing of precursor mRNA is an essential process for dividing cells, and splicing defects have been linked to aging and various chronic diseases. Environmental stress has recently been shown to modify alternative splicing, and molecular mechanisms that influence stress-induced alternative splicing remain unclear. Using an in vivo RNA splicing reporter, we performed a genome-wide RNAi screen in Caenorhabditis elegans and found that protein translation suppression via silencing of the conserved eukaryotic initiation factor 4G (IFG-1/eIF4G) inhibits cadmium-induced alternative splicing. Transcriptome analysis of an ifg-1-deficient mutant revealed an overall decrease in intronic and intergenic reads and prevented cadmium-induced alternative splicing compared to the wild type. We found that the ifg-1 mutant up-regulates >80 RNA splicing regulatory genes controlled by the TGF-β transcription factor SMA-2. The extended lifespan of the ifg-1 mutant is partially reduced upon sma-2 depletion and completely nullified when core spliceosome genes including snr-1, snr-2, and uaf-2 are knocked down. Depletion of snr-1 and snr-2 also diminished the enhanced cadmium resistance of the ifg-1 mutant. Together, these data describe a molecular mechanism through which translation suppression inhibits stress-induced alternative splicing and demonstrate an essential role for RNA splicing in promoting longevity and stress resistance in a translation-compromised mutant., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

eIF4G is an important eukaryotic translation initiation factor. In this study, eIF4G1, one of the eIF4G isoforms, was shown to directly participate in biogenesis of the large (60S) ribosomal subunit in Saccharomyces cerevisiae cells. Mutation of eIF4G1 decreased the amount 60S ribosomal subunits significantly. The C-terminal fragment of eIF4G1 could complement the function in 60S biogenesis. Analyses of its purified complex with mass spectrometry indicated that eIF4G1 associated with the pre-60S form directly. Strong genetic and direct protein-protein interactions were observed between eIF4G1 and Ssf1 protein. Upon deletion of eIF4G1, Ssf1, Rrp15, Rrp14 and Mak16 were abnormally retained on the pre-60S complex. This purturbed the loading of Arx1 and eL31 at the polypeptide exit tunnel (PET) site and the transition to a Nog2 complex. Our data indicate that eIF4G1 is important in facilitating PET maturation and 27S processing correctly. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Biomolecular associations forged by specific interaction among structural scaffolds are fundamental to the control and regulation of cell processes. One such structural architecture, characterized by HEAT repeats, is involved in a multitude of cellular processes, including intracellular transport, signaling, and protein synthesis. Here, we review the multitude and versatility of HEAT domains in the regulation of mRNA translation initiation. Structural and cellular biology approaches, as well as several biophysical studies, have revealed that a number of HEAT domain-mediated interactions with a host of protein factors and RNAs coordinate translation initiation. We describe the basic structural architecture of HEAT domains and briefly introduce examples of the cellular processes they dictate, including nuclear transport by importin and RNA degradation. We then focus on proteins in the translation initiation system featuring HEAT domains, specifically the HEAT domains of eIF4G, DAP5, eIF5, and eIF2Bϵ. Comparative analysis of their remarkably versatile interactions, including protein-protein and protein-RNA recognition, reveal the functional importance of flexible regions within these HEAT domains. Here we outline how HEAT domains orchestrate fundamental aspects of translation initiation and highlight open mechanistic questions in the area., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nasopharyngeal carcinoma (NPC) is the most common primary malignancy arising from the epithelial cells of nasopharynx. CircTMTC1 is upregulated in NPC patients, but its role and molecular mechanism in NPC are unknown. Normal nasopharyngeal epithelium and tumor tissues were collected. The expression of circTMTC1, miR-495, MET/eIF4G1 pathway-related molecules were examined. Colony formation and transwell assays were used to assess cell proliferation, migration, and invasion. Cell apoptosis was analyzed by annexin V and propidium iodide (PI) staining. Gene interaction was examined by RNA immunoprecipitation (RIP) and luciferase activity assays. Subcutaneous and intravenous xenograft mouse models were established to analyze NPC growth and metastasis in vivo. CircTMTC1 was highly expressed and miR-495 was downregulated in NPC, which were associated with poor prognosis of NPC. Both circTMTC1 knockdown and miR-495 overexpression inhibited NPC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) and promoted cell apoptosis. CircTMTC1 directly targeted miR-495 to promote the expression of its downstream target gene MET. miR-495 knockdown enhanced the expression of c-Myc, Cyclin D1, and survivin and accelerated NPC cell proliferation, migration, invasion, and EMT through targeting MET and activating the MET-eIF4G1 axis. CircTMTC1 silence inhibited NPC growth and lung metastasis by targeting the miR-495-MET-eIF4G1 translational regulation axis in vivo. CircTMTC1 accelerates NPC progression through targeting miR-495 and consequently activating the MET-eIF4G1 translational regulation axis, suggesting potential therapeutic targets for NPC treatment., (© 2022. The Author(s).)

Non-small cell lung cancer (NSCLC) ranks first in the morbidity and mortality of malignant tumors in China. As reported, circular RNAs (circRNAs) are emerged in the progress of NSCLC. The study was to figure out the potential mechanism of circ-UBE2D2 in the progression of NSCLC. First, plasmid vectors intervening circ-UBE2D2, microRNA (miR)-376a-3p or Eukaryotic Translation Initiation Factor 4γ2 (EIF4G2) expression were transfected into NSCLC cells, and the expression of circ-UBE2D2, miR-376a-3p and EIF4G2 was detected by reverse transcription quantitative polymerase chain reaction or Western blot. Then, cell proliferation was detected by Cell counting kit-8 assay and plate cloning. Cell apoptosis was tested by flow cytometry. Plate scratches and Transwell were used to detect cell migration and invasion. Finally, the binding sites of circRNA UBE2D2, EIF4G2 and miR-376a-3p were verified by bioinformatics website starBase analysis and dual luciferase reporter gene assay. The results manifested the up-regulation of circ-UBE2D2 expression in NSCLC tissues and cells. Circ-UBE2D2 promoted the proliferation, migration and invasion, but repressed apoptosis of NSCLC cells. Interestingly, circ-UBE2D2 directly targeted miR-376a-3p and up-regulated miR-376a-3p restrained proliferation, migration and invasion, but accelerated apoptosis of NSCLC cells. More importantly, EIF4G2 was the target of miR-376a-3p, and overexpression of EIF4G2 reversed the effects of circ-UBE2D2 downregulation on proliferation, migration, invasion and apoptosis of NSCLC cells. These results suggest that circ-UBE2D2 promotes the proliferation, migration and invasion but restrains apoptosis of lung cancer cells by regulating miR-376a-3p/EIF4G2 axis.

Regulatory T cells (Treg cells) inhibit effector T cells and maintain immune system homeostasis. Treg cell maturation in peripheral sites requires inhibition of protein kinase mTORC1 and TGF-beta-1 (TGF-beta). While Treg cell maturation requires protein synthesis, mTORC1 inhibition downregulates it, leaving unanswered how Treg cells achieve essential mRNA translation for development and immune suppression activity. Using human CD4 + T cells differentiated in culture and genome-wide transcription and translation profiling, here we report that TGF-beta transcriptionally reprograms naive T cells to express Treg cell differentiation and immune suppression mRNAs, while mTORC1 inhibition impairs translation of T cell mRNAs but not those induced by TGF-beta. Rather than canonical mTORC1/eIF4E/eIF4G translation, Treg cell mRNAs utilize the eIF4G homolog DAP5 and initiation factor eIF3d in a non-canonical translation mechanism that requires cap-dependent binding by eIF3d directed by Treg cell mRNA 5' noncoding regions. Silencing DAP5 in isolated human naive CD4 + T cells impairs their differentiation into Treg cells. Treg cell differentiation is mediated by mTORC1 downregulation and TGF-beta transcriptional reprogramming that establishes a DAP5/eIF3d-selective mechanism of mRNA translation., (© 2021. The Author(s).)

Rice black-streaked dwarf disease, caused by rice black-streaked dwarf virus (RBSDV), is a serious constraint in Chinese rice production. Breeding disease-resistant varieties through multigene aggregation is considered an effective way to control diseases, but few disease-resistant resources have been characterized thus far. To develop novel resources for resistance to RBSDV through CRISPR/Cas9-mediated genome editing, a guide RNA sequence targeting exon 1 of eIF4G was designed and cloned into a binary vector, pHUE401. This recombinant vector was used to generate mutations in the rice cultivar Nipponbare via Agrobacterium -mediated transformation. This approach produced heritable homozygous mutations in the transgene-free T1 generation. Sequence analysis of the eIF4G target region from T1 transgenic plants identified 3 bp deletion mutants, and analysis of the predicted amino acid sequence identified one amino acid deletion in mutants that possess near full-length eIF4G. Furthermore, our data suggest that eIF4G may plays an important role in rice normal development, as there were no eIF4G knock-out homozygous mutants in T1 generation plants. When homozygous mutant lines were inoculated with RBSDV, they exhibited enhanced tolerance to virus infection, without visibly affecting plant growth and development. However, the eif4g mutant plants showed the same sensitivity to rice stripe virus (RSV) infection as wild-type plants. Notably, the wild-type and mutant N-termini of eIF4G interacted directly with RBSDV P8 in yeast and in planta. Additionally, compared to wild-type plants, the eIF4G transcript level was reduced twofold in the mutant plants. These results indicate that site-specific mutation of rice eIF4G successfully conferred partial resistance specific to RBSDV associated with less transcription of eIF4G in mutants. Therefore, this study demonstrates that the novel eIF4G alleles generated by CRISPR/Cas9 represent valuable disease-resistant resources that can be used to develop RBSDV-resistant varieties.

Estrogen receptor α (ERα) is a hormone receptor and key driver for over 70% of breast cancers that has been studied for decades as a transcription factor. Unexpectedly, we discover that ERα is a potent non-canonical RNA-binding protein. We show that ERα RNA binding function is uncoupled from its activity to bind DNA and critical for breast cancer progression. Employing genome-wide cross-linking immunoprecipitation (CLIP) sequencing and a functional CRISPRi screen, we find that ERα-associated mRNAs sustain cancer cell fitness and elicit cellular responses to stress. Mechanistically, ERα controls different steps of RNA metabolism. In particular, we demonstrate that ERα RNA binding mediates alternative splicing of XBP1 and translation of the eIF4G2 and MCL1 mRNAs, which facilitates survival upon stress conditions and sustains tamoxifen resistance of cancer cells. ERα is therefore a multifaceted RNA-binding protein, and this activity transforms our knowledge of post-transcriptional regulation underlying cancer development and drug response., Competing Interests: Declaration of interests D.R. is a shareholder of eFFECTOR Therapeutics, Inc. and is a member of its scientific advisory board., (Copyright © 2021 Elsevier Inc. All rights reserved.)

eIF4F plays diverse roles in human cancers, which complicate the development of an overarching understanding of its functional and regulatory impacts across tumor types. Typically, eIF4F drives initiation from the mRNA 5' end (cap) and is composed of eIF4G1, eIF4A1, and cap-binding eIF4E. Cap-independent initiation is possible without eIF4E, from internal ribosomal entry sites (IRESs). By analyzing large public datasets, we found that cancers selectively overexpress EIF4G1 more than EIF4E. That expression imbalance supports EIF4G1 as a prognostic indicator in patients with cancer. It also attenuates "housekeeping" pathways that are usually regulated in a tissue-specific manner via cap-dependent initiation in healthy tissues and reinforce regulation of cancer-preferred pathways in cap-independent contexts. Cap-independent initiation is mechanistically attributable to eIF4G1 hyperphosphorylation that promotes binding to eIF4A1 and reduced eIF4E availability. Collectively, these findings reveal a novel model of dysregulated eIF4F function and highlight the clinical relevance of cap-(in)dependent initiation in cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Translation reinitiation is a gene-specific translational control mechanism. It is characterized by the ability of short upstream ORFs to prevent full ribosomal recycling and allow the post-termination 40S subunit to resume traversing downstream for the next initiation event. It is well known that variable transcript-specific features of various uORFs and their prospective interactions with initiation factors lend them an unequivocal regulatory potential. Here, we investigated the proposed role of the major initiation scaffold protein eIF4G in reinitiation and its prospective interactions with uORF's cis-acting features in yeast. In analogy to the eIF3 complex, we found that eIF4G and eIF4A but not eIF4E (all constituting the eIF4F complex) are preferentially retained on ribosomes elongating and terminating on reinitiation-permissive uORFs. The loss of the eIF4G contact with eIF4A specifically increased this retention and, as a result, increased the efficiency of reinitiation on downstream initiation codons. Combining the eIF4A-binding mutation with that affecting the integrity of the eIF4G1-RNA2-binding domain eliminated this specificity and produced epistatic interaction with a mutation in one specific cis-acting feature. We conclude that similar to humans, eIF4G is retained on ribosomes elongating uORFs to control reinitiation also in yeast., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Knowledge of the genetic background of many human diseases is currently lacking from genetically undiscovered regions, including Central Asia. Kazakhstan is the first Central Asian country where the genetic studies of Parkinson's disease (PD) have been emerging since it had become a member of the International Parkinson Disease Genomics Consortium. Here we report on the results of whole-exome sequencing (WES) in 50 young-onset PD (YOPD) cases from Kazakhstan., Methodology: WES was performed on 50 unrelated individuals with YOPD from Kazakhstan. Exome data were screened for novel/ultra-rare deleterious variants in known and candidate PD genes. Copy number variants and small indels were also called., Results: Only three cases (6%) were found to be positive for known PD genes including two unrelated familial PD cases with LRRK2 p.(Arg1441Cys) and one case with a homozygous pathogenic PRKN p.(Arg84Trp) variant. Four cases had novel and ultra-rare variants of uncertain significance in LRRK2, DNAJC13, and VPS35. Novel deleterious variants were found in candidate Mendelian PD genes including CSMD1, TNR, EIF4G1, and ATP13A3. Eight cases harbored the East Asian-specific LRRK2 p.(Ala419Val) variant., Conclusions: The low diagnostic yield in our study might imply that a significant proportion of YOPD cases in Central Asia remains unresolved. Therefore, a better understanding of the genetic architecture of PD among populations of Central Asian ancestry and the pathogenicity of numerous rare variants should be further investigated. WES is a valuable technique for large-scale YOPD genetic studies in Central Asia., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Radioresistance is the predominant cause for radiotherapy failure and disease progression, resulting in increased breast cancer‑associated mortality. Using gene expression signature analysis of the Library of Integrated Network‑Based Cellular Signatures (LINCS) and Gene Expression Omnibus (GEO), the aim of the present study was to systematically identify potential candidate radiosensitizers from known drugs. The similarity of integrated gene expression signatures between irradiated eukaryotic translation initiation factor 4 γ 1 (eIF4G1)‑silenced breast cancer cells and known drugs was measured using enrichment scores (ES). Drugs with positive ES were selected as potential radiosensitizers. The radiosensitizing effects of the candidate drugs were analyzed in breast cancer cell lines (MCF‑7, MX‑1 and MDA‑MB‑231) using CCK‑8 and colony formation assays following exposure to ionizing radiation. Cell apoptosis was measured using flow cytometry. The expression levels of eIF4G1 and DNA damage response (DDR) proteins were analyzed by western blotting. Bosutinib was identified as a promising radiosensitizer, as its administration markedly reduced the dosage required both for the drug and for ionizing radiation, which may be associated with fewer treatment‑associated adverse reactions. Moreover, combined treatment of ionizing radiation and bosutinib significantly increased cell killing in all three cell lines, compared with ionizing radiation or bosutinib alone. Among the three cell lines, MX‑1 cells were identified as the most sensitive to both ionizing radiation and bosutinib. Bosutinib markedly downregulated the expression of eIF4G1 in a dose‑dependent manner and also reduced the expression of DDR proteins (including ATM, XRCC4, ATRIP, and GADD45A). Moreover, eIF4G1 was identified as a key target of bosutinib that may regulate DNA damage induced by ionizing radiation. Thus, bosutinib may serve as a potential candidate radiosensitizer for breast cancer therapy.