Identification of Rare EIF3E::RSPO2 Fusion in Recurrent and Aggressive Urachal Adenocarcinoma.

INTRODUCTION: Urachal cancer (UC) is a rare genitourinary malignancy arising from the urachus, an embryonic remnant of the placental allantois. Its diagnosis remains ambiguous with late-stage cancer detection and represents a highly aggressive disease. Due to its rarity, there is no clear consensus on molecular signatures and appropriate clinical management of UC. CASE REPORT: We report a 45-year-old man with recurrent urachal adenocarcinoma (UA) treated with cystectomies, chemotherapy, and radiotherapy. The patient initially presented with hematuria and abdominal pain. Imaging revealed a nodular mass arising from the superior wall of the urinary bladder and extending to the urachus. Biopsy results suggested moderately differentiated UA with muscle layer involvement. The tumor recurred after 20 months, following which, another partial cystectomy was performed. Repeat progression was noted indicating highly aggressive disease. Targeted next-generation sequencing revealed the presence of EIF3E::RSPO2 fusion, along with BRAF and TP53 mutations, and EGFR gene amplification. This is the first case reporting the presence of this fusion in UA. Palliative medication and radiotherapy were administered to manage the disease. CONCLUSION: Current treatment modality of surgery may be effective in the early stages of recurrent UA; however, a standard chemotherapy and radiotherapy regimen is yet to be determined for advanced stages. The detection of the rare EIF3E::RSPO2 fusion warrants further studies on the significance of this variant as a possible therapeutic target for improved clinical management.

A dynamic compositional equilibrium governs mRNA recognition by eIF3.

Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that binds both ribosomes and messenger RNAs (mRNAs) in order to drive a diverse set of mechanistic steps during translation. Despite its importance, a unifying framework explaining how eIF3 performs these numerous activities is lacking. Using single-molecule light scattering microscopy, we demonstrate that Saccharomyces cerevisiae eIF3 is an equilibrium mixture of the full complex, subcomplexes, and subunits. By extending our microscopy approach to an in vitro reconstituted eIF3 and complementing it with biochemical assays, we define the subspecies comprising this equilibrium and show that, rather than being driven by the full complex, mRNA binding by eIF3 is instead driven by the eIF3a subunit within eIF3a-containing subcomplexes. Our findings provide a mechanistic model for the role of eIF3 in the mRNA recruitment step of translation initiation and establish a mechanistic framework for explaining and investigating the other activities of eIF3.

LncRNA EIF3J-DT promotes chemoresistance in oral squamous cell carcinoma.

OBJECTIVES: This study aimed to screen oral squamous cell carcinoma (OSCC) diagnostic and prognostic candidates and investigate the potential functions and mechanisms of candidates in the chemoresistance of OSCC cell lines. MATERIALS AND METHODS: Differential expression profiling of lncRNA was performed in a large cohort of OSCC patients from the Cancer Genome Atlas database to identify OSCC diagnostic and prognostic candidates. Taxol resistance in OSCC cell lines was analyzed using MTT assay. OSCC cell lines transfected with EIF3J-DT pcDNA or siRNA were used to determine its regulatory effects on apoptosis, cell cycle distribution and autophagy using flow cytometry and western blot. RESULTS: We identified EIF3J-DT as a candidate for OSCC diagnosis and prognosis. The expression level of EIF3J-DT in OSCC cell lines correlates with taxol resistance. EIF3J-DT silencing attenuated taxol resistance, and EIF3J-DT overexpression enhanced taxol resistance in OSCC cell lines. Silencing of EIF3J-DT reduced taxol resistance by inducing apoptosis, cell cycle arrest, and ATG14-mediated autophagy inhibition in OSCC cell lines. CONCLUSIONS: We found that EIF3J-DT induced chemoresistance by regulating apoptosis, cell cycle, and autophagy in OSCC cell lines, which EIF3J-DT might provide a novel therapeutic approach for OSCC as well as a diagnostic and prognostic factor.

Eukaryotic initiation factor 3a promotes the development of diffuse large B-cell lymphoma through regulating cell proliferation.

BACKGROUND: One-third of diffuse large B-cell lymphoma (DLBCL) patients suffer relapse after standard treatment. Eukaryotic initiation factor 3a (eIF3a) is a key player in the initial stage of translation, which has been widely reported to be correlated with tumorigenesis and therapeutic response. This study aimed to explore the biological role of eIF3a, evaluate its prognostic and therapeutic potential in DLBCL. METHODS: RNA-seq datasets from GEO database were utilized to detect the expression and prognostic role of eIF3a in DLBCL patients. Protein level of eIF3a was estimated by western blot and immunohistochemical. Next, DLBCL cells were transfected with lentiviral vector either eIF3a-knockdown or empty to assess the biological role of eIF3a. Then, samples were divided into 2 clusters based on eIF3a expression and differentially expressed genes (DEGs) were identified. Function enrichment and mutation analysis of DEGs were employed to detect potential biological roles. Moreover, we also applied pan-cancer and chemosensitivity analysis for deep exploration. RESULTS: eIF3a expression was found to be higher in DLBCL than healthy controls, which was associated with worse prognosis. The expression of eIF3a protein was significantly increased in DLBCL cell lines compared with peripheral blood mononuclear cells (PBMCs) from healthy donors. eIF3a knockdown inhibited the proliferation of DLBCL cells and the expression of proliferation-related proteins and increase cell apoptosis rate. Besides, 114 DEGs were identified which had a close linkage to cell cycle and tumor immune. eIF3a and DEGs mutations were found to be correlated to chemosensitivity and vital signal pathways. Pan-cancer analysis demonstrated that high eIF3a expression was associated with worse prognosis in several tumors. Moreover, eIF3a expression was found to be related to chemosensitivity of several anti-tumor drugs in DLBCL, including Vincristine and Wee1 inhibitor. CONCLUSIONS: We firstly revealed the high expression and prognostic role of eIF3a in DLBCL, and eIF3a might promote the development of DLBCL through regulating cell proliferation and apoptosis. eIF3a expression was related to immune profile and chemosensitivity in DLBCL. These results suggest that eIF3a could serve as a potential prognostic biomarker and therapeutic target in DLBCL.

Unraveling the landscapes and regulation of scanning, leaky scanning, and 48S initiation complex conformations.

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.

EIF3B stabilizes PCNA by counteracting SYVN1-mediated ubiquitination to serve as a promotor in cholangiocarcinoma.

Cholangiocarcinoma, a prevalent hepatic malignancy, exhibits a progressively rising incidence. While Eukaryotic translation initiation factor 3 subunit B (EIF3B) has been implicated in the occurrence and development of various cancers, its specific roles in cholangiocarcinoma remain unexplored. Immunohistochemical (IHC) analysis was employed to detect EIF3B/PCNA expression in cholangiocarcinoma. Cells were manipulated using short hairpin RNA (shRNA)-mediated lentiviruses or overexpression plasmids. Statistical significance was assessed using the Student's t-test and one-way ANOVA, with P < 0.05 considered statistically significant. EIF3B exhibited robust expression in cholangiocarcinoma, demonstrating a significant correlation with the pathological grade of cholangiocarcinoma patients. Furthermore, modulation of EIF3B expression, either depletion or elevation, demonstrated the ability to inhibit or enhance cholangiocarcinoma cell survival and migration in vitro. Mechanistically, we identified Proliferating Cell Nuclear Antigen (PCNA) as a downstream gene of EIF3B, driving cholangiocarcinoma. EIF3B stabilized PCNA by inhibiting PCNA ubiquitination, a process mediated by E3 ligase SYVN1. Similar to EIF3B, PCNA levels were also abundant in cholangiocarcinoma, and knocking down PCNA impeded cholangiocarcinoma development. Intriguingly, silencing PCNA attenuated the promotion induced by EIF3B overexpression. Furthermore, the elevated P21 protein level in shEIF3B RBE cells was partially attenuated after UC2288 (P21 signaling pathway inhibitor) treatment. Our findings underscored the potential of EIF3B as a therapeutic target for cholangiocarcinoma. Unraveling its functions holds promise for the development of more specific and effective targeted therapy strategies.

Circ-EIF3I Promotes Hepatocellular Carcinoma Progression Through Modulating miR-361-3p/DUSP2 Axis.

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers globally. Circular RNAs (circRNAs) have been implicated in the development of HCC. Previous studies have confirmed that circ-EIF3I plays an important role in the progress of lung cancer. Nevertheless, the biological functions of circ-EIF3I and the underlying mechanisms by which they regulate HCC progression remain unclear. In this study, the regulatory mechanism and targets were studied with bioinformatics analysis, luciferase reporting analysis, transwell migration, Cell Counting Kit-8, and 5-Ethynyl-2'-deoxyuridine analysis. In addition, in vivo tumorigenesis and metastasis assays were employed to evaluate the roles of circ-EIF3I in HCC. The result shows that the circ-EIF3I expression was increased in HCC cell line, which means that circ-EIF3I plays a role in the progression of HCC. Downregulation of circ-EIF3I suppressed HCC cells' proliferation and migration in both in vivo and in vitro experiments. Bioinformatics and luciferase report analysis confirmed that both miR-361-3p and Dual-specificity phosphatase 2 (DUSP2) were the downstream target of circ-EIF3I. The overexpression of DUSP2 or inhibition of miR-361-3p restored HCC cells' proliferation and migration ability after silence circ-EIF3I. Taken together, our study found that downregulation of circ-EIF3I suppressed the progression of HCC through miR-361-3p/DUSP2 Axis.

RNA splicing regulator EIF3D regulates the tumor microenvironment through immunogene-related alternative splicing in head and neck squamous cell carcinoma.

Study finds that eukaryotic translation initiation factor 3 subunit D (EIF3D) may play an important role in aberrant alternative splicing (AS) events in tumors. AS possesses a pivotal role in both tumour progression and the constitution of the tumour microenvironment (TME). Regrettably, our current understanding of AS remains circumscribed especially in the context of immunogene-related alternative splicing (IGAS) profiles within Head and Neck Squamous Cell Carcinoma (HNSC). In this study, we comprehensively analyzed the function and mechanism of action of EIF3D by bioinformatics analysis combined with in vitro cellular experiments, and found that high expression of EIF3D in HNSC was associated with poor prognosis of overall survival (OS) and progression-free survival (PFS). The EIF3D low expression group had a higher degree of immune infiltration and better efficacy against PD1 and CTLA4 immunotherapy compared to the EIF3D high expression group. TCGA SpliceSeq analysis illustrated that EIF3D influenced differentially spliced alternative splicing (DSAS) events involving 105 differentially expressed immunogenes (DEIGs). We observed an induction of apoptosis and a suppression of cell proliferation, migration, and invasion in EIF3D knock-down FaDu cells. RNA-seq analysis unveiled that 531 genes exhibited differential expression following EIF3D knockdown in FaDu cells. These include 52 DEIGs. Furthermore, EIF3D knockdown influenced the patterns of 1923 alternative splicing events (ASEs), encompassing 129 IGASs. This study identified an RNA splicing regulator and revealed its regulatory role in IGAS and the TME of HNSC, suggesting that EIF3D may be a potential target for predicting HNSC prognosis and immunotherapeutic response.

Shigella induces stress granule formation by ADP-riboxanation of the eIF3 complex.

Under stress conditions, translationally stalled mRNA and associated proteins undergo liquid-liquid phase separation and condense into cytoplasmic foci called stress granules (SGs). Many viruses hijack SGs for their pathogenesis; however, whether pathogenic bacteria also exploit this pathway remains unknown. Here, we report that members of the OspC family of Shigella flexneri induce SG formation in infected cells. Mechanistically, the OspC effectors target multiple subunits of the host translation initiation factor 3 complex by ADP-riboxanation. The modification of eIF3 leads to translational arrest and thus the formation of SGs. Furthermore, OspC-mediated SGs are beneficial for S. flexneri replication within infected host cells, and bacterial strains unable to induce SGs are attenuated for virulence in a murine model of infection. Our findings reveal a mechanism by which bacterial pathogens induce SG assembly by inactivating host translational machinery and promote bacterial proliferation in host cells.

Protein complexes from mouse and chick brain that interact with phospho-KXGS motif tau/microtubule associated protein antibody.

Mouse monoclonal 12E8 antibody, which recognises conserved serine phosphorylated KXGS motifs in the microtubule binding domains of tau/tau-like microtubule associated proteins (MAPs), shows elevated binding in brain during normal embryonic development (mammals and birds) and at the early stages of human Alzheimer's disease (AD). It also labels ADF/cofilin-actin rods that form in neurites during exposure to stressors. We aimed to identify direct and indirect 12E8 binding proteins in postnatal mouse brain and embryonic chick brain by immunoprecipitation (IP), mass spectrometry and immunofluorescence. Tau and/or MAP2 were major direct 12E8-binding proteins detected in all IPs, and actin and/or tubulin were co-immunoprecipitated in most samples. Additional proteins were different in mouse versus chick brain IP. In mouse brain IPs, FSD1l and intermediate filament proteins - vimentin, α-internexin, neurofilament polypeptides - were prominent. Immunofluorescence and immunoblot using recombinant intermediate filament subunits, suggests an indirect interaction of these proteins with the 12E8 antibody. In chick brain IPs, subunits of eukaryotic translation initiation factor 3 (EIF3) were found, but no direct interaction between 12E8 and recombinant Eif3e protein was detected. Fluorescence microscopy in primary cultured chick neurons showed evidence of co-localisation of Eif3e and tubulin labelling, consistent with previous data demonstrating cytoskeletal organisation of the translation apparatus. Neither total tau or MAP2 immunolabelling accumulated at ADF/cofilin-actin rods generated in primary cultured chick neurons, and we were unable to narrow down the major antigen recognised by 12E8 antibody on ADF/cofilin-actin rods.

A novel EIF3C-related CD8+ T-cell signature in predicting prognosis and immunotherapy response of nasopharyngeal carcinoma.

PURPOSE: At present, dysfunctional CD8+ T-cells in the nasopharyngeal carcinoma (NPC) tumor immune microenvironment (TIME) have caused unsatisfactory immunotherapeutic effects, such as a low response rate of anti-PD-L1 therapy. Therefore, there is an urgent need to identify reliable markers capable of accurately predicting immunotherapy efficacy. METHODS: Utilizing various algorithms for immune-infiltration evaluation, we explored the role of EIF3C in the TIME. We next found the influence of EIF3C expression on NPC based on functional analyses and RNA sequencing. By performing correlation and univariate Cox analyses of CD8+ Tcell markers from scRNA-seq data, we identified four signatures, which were then used in conjunction with the lasso algorithm to determine corresponding coefficients in the resulting EIF3C-related CD8+ T-cell signature (ETS). We subsequently evaluated the prognostic value of ETS using univariate and multivariate Cox regression analyses, Kaplan-Meier curves, and the area under the receiver operating characteristic curve (AUROC). RESULTS: Our results demonstrate a significant relationship between low expression of EIF3C and high levels of CD8+ T-cell infiltration in the TIME, as well as a correlation between EIF3C expression and progression of NPC. Based on the expression levels of four EIF3C-related CD8+ T-cell marker genes, we constructed the ETS predictive model for NPC prognosis, which demonstrated success in validation. Notably, our model can also serve as an accurate indicator for detecting immunotherapy response. CONCLUSION: Our findings suggest that EIF3C plays a significant role in NPC progression and immune modulation, particularly in CD8+ T-cell infiltration. Furthermore, the ETS model holds promise as both a prognostic predictor for NPC patients and a tool for adjusting individualized immunotherapy strategies.

METTL16 promotes liver cancer stem cell self-renewal via controlling ribosome biogenesis and mRNA translation.

BACKGROUND: While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N6-methyladenosine (m6A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. METHODS: Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. RESULTS: METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a (eIF3a) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). CONCLUSIONS: Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.

Drosophila eIF3f1 mediates host immune defense by targeting dTak1.

Eukaryotic translation initiation factors have long been recognized for their critical roles in governing the translation of coding RNAs into peptides/proteins. However, whether they harbor functional activities at the post-translational level remains poorly understood. Here, we demonstrate that eIF3f1 (eukaryotic translation initiation factor 3 subunit f1), which encodes an archetypal deubiquitinase, is essential for the antimicrobial innate immune defense of Drosophila melanogaster. Our in vitro and in vivo evidence indicate that the immunological function of eIF3f1 is dependent on the N-terminal JAMM (JAB1/MPN/Mov34 metalloenzymes) domain. Mechanistically, eIF3f1 physically associates with dTak1 (Drosophila TGF-beta activating kinase 1), a key regulator of the IMD (immune deficiency) signaling pathway, and mediates the turnover of dTak1 by specifically restricting its K48-linked ubiquitination. Collectively, these results provide compelling insight into a noncanonical molecular function of a translation initiation factor that controls the post-translational modification of a target protein.

Hsa_circ_0092856 Promoted the Proliferation, Migration, and Invasion of NSCLC Cells by Up-Regulating the Expression of eIF3a.

Circular RNA (circRNA) plays a very important regulatory role in a variety of human malignancies such as non-small-cell lung cancer (NSCLC). In the current study, we explored the role of hsa_circ_0092856 in the progression of NSCLC. We screened CircRNA from the eIF3a gene in the Circbase database. The biological functions of hsa_circ_0092856 in NSCLC were analyzed via qRT-PCR, a CCK-8 assay, a plate cloning experiment, scratch testing, a transwell chamber experiment, an RNA nuclear mass separation experiment, an RIP experiment, and a Western blot test. The results showed that hsa_circ_0092856 was highly expressed in NSCLC cells, and the knockdown of hsa_circ_0092856 could inhibit the proliferation, migration, and invasion of NSCLC cells. The overexpression of hsa_circ_0092856 has the opposite effect. The expression of eIF3a also changed with the change in hsa_circ_0092856. These results suggest that hsa_circ_0092856 may play a key role in the progression of NSCLC by regulating the expression of eIF3a.

YTHDF2 Is a Therapeutic Target for HCC by Suppressing Immune Evasion and Angiogenesis Through ETV5/PD-L1/VEGFA Axis.

N6-methyladenosine (m6A) modification orchestrates cancer formation and progression by affecting the tumor microenvironment (TME). For hepatocellular carcinoma (HCC), immune evasion and angiogenesis are characteristic features of its TME. The role of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), as an m6A reader, in regulating HCC TME are not fully understood. Herein, it is discovered that trimethylated histone H3 lysine 4 and H3 lysine 27 acetylation modification in the promoter region of YTHDF2 enhanced its expression in HCC, and upregulated YTHDF2 in HCC predicted a worse prognosis. Animal experiments demonstrated that Ythdf2 depletion inhibited spontaneous HCC formation, while its overexpression promoted xenografted HCC progression. Mechanistically, YTHDF2 recognized the m6A modification in the 5'-untranslational region of ETS variant transcription factor 5 (ETV5) mRNA and recruited eukaryotic translation initiation factor 3 subunit B to facilitate its translation. Elevated ETV5 expression induced the transcription of programmed death ligand-1 and vascular endothelial growth factor A, thereby promoting HCC immune evasion and angiogenesis. Targeting YTHDF2 via small interference RNA-containing aptamer/liposomes successfully both inhibited HCC immune evasion and angiogenesis. Together, this findings reveal the potential application of YTHDF2 in HCC prognosis and targeted treatment.

Computational inference of eIF4F complex function and structure in human cancers.

The canonical eukaryotic initiation factor 4F (eIF4F) complex, composed of eIF4G1, eIF4A1, and the cap-binding protein eIF4E, plays a crucial role in cap-dependent translation initiation in eukaryotic cells. An alternative cap-independent initiation can occur, involving only eIF4G1 and eIF4A1 through internal ribosome entry sites (IRESs). This mechanism is considered complementary to cap-dependent initiation, particularly in tumors under stress conditions. However, the selection and molecular mechanism of specific translation initiation remains poorly understood in human cancers. Thus, we analyzed gene copy number variations (CNVs) in TCGA tumor samples and found frequent amplification of genes involved in translation initiation. Copy number gains in EIF4G1 and EIF3E frequently co-occur across human cancers. Additionally, EIF4G1 expression strongly correlates with genes from cancer cell survival pathways including cell cycle and lipogenesis, in tumors with EIF4G1 amplification or duplication. Furthermore, we revealed that eIF4G1 and eIF4A1 protein levels strongly co-regulate with ribosomal subunits, eIF2, and eIF3 complexes, while eIF4E co-regulates with 4E-BP1, ubiquitination, and ESCRT proteins. Utilizing Alphafold predictions, we modeled the eIF4F structure with and without eIF4E binding. For cap-dependent initiation, our modeling reveals extensive interactions between the N-terminal eIF4E-binding domain of eIF4G1 and eIF4E. Furthermore, the eIF4G1 HEAT-2 domain positions eIF4E near the eIF4A1 N-terminal domain (NTD), resulting in the collaborative enclosure of the RNA binding cavity within eIF4A1. In contrast, during cap-independent initiation, the HEAT-2 domain directly binds the eIF4A1-NTD, leading to a stronger interaction between eIF4G1 and eIF4A1, thus closing the mRNA binding cavity without the involvement of eIF4E.

In vitro reconstitution of SARS-CoV-2 Nsp1-induced mRNA cleavage reveals the key roles of the N-terminal domain of Nsp1 and the RRM domain of eIF3g.

SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on ß-globin, EMCV IRES, and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g's RRM domain. The cleavage site was located in the coding region 18 nt downstream from the mRNA entrance, indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1's N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g's RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of the Nsp1 NTD and eIF3g's RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.

eIF3d controls the persistent integrated stress response.

Cells respond to intrinsic and extrinsic stresses by reducing global protein synthesis and activating gene programs necessary for survival. Here, we show that the integrated stress response (ISR) is driven by the non-canonical cap-binding protein eIF3d that acts as a critical effector to control core stress response orchestrators, the translation factor eIF2α and the transcription factor ATF4. We find that during persistent stress, eIF3d activates the translation of the kinase GCN2, inducing eIF2α phosphorylation and inhibiting general protein synthesis. In parallel, eIF3d upregulates the m6A demethylase ALKBH5 to drive 5' UTR-specific demethylation of stress response genes, including ATF4. Ultimately, this cascade converges on ATF4 expression by increasing mRNA engagement of translation machinery and enhancing ribosome bypass of upstream open reading frames (uORFs). Our results reveal that eIF3d acts in a life-or-death decision point during chronic stress and uncover a synergistic signaling mechanism in which translational cascades complement transcriptional amplification to control essential cellular processes.

eIF3f Mediates SGOC Pathway Reprogramming by Enhancing Deubiquitinating Activity in Colorectal Cancer.

Numerous studies have demonstrated that individual proteins can moonlight. Eukaryotic Initiation translation factor 3, f subunit (eIF3f) is involved in critical biological functions; however, its role independent of protein translation in regulating colorectal cancer (CRC) is not characterized. Here, it is demonstrated that eIF3f is upregulated in CRC tumor tissues and that both Wnt and EGF signaling pathways are participating in eIF3f's oncogenic impact on targeting phosphoglycerate dehydrogenase (PHGDH) during CRC development. Mechanistically, EGF blocks FBXW7ß-mediated PHGDH ubiquitination through GSK3ß deactivation, and eIF3f antagonizes FBXW7ß-mediated PHGDH ubiquitination through its deubiquitinating activity. Additionally, Wnt signals transcriptionally activate the expression of eIF3f, which also exerts its deubiquitinating activity toward MYC, thereby increasing MYC-mediated PHGDH transcription. Thereby, both impacts allow eIF3f to elevate the expression of PHGDH, enhancing Serine-Glycine-One-Carbon (SGOC) signaling pathway to facilitate CRC development. In summary, the study uncovers the intrinsic role and underlying molecular mechanism of eIF3f in SGOC signaling, providing novel insight into the strategies to target eIF3f-PHGDH axis in CRC.

eIF3i promotes colorectal cancer cell survival via augmenting PHGDH translation.

Translational regulation is one of the decisive steps in gene expression, and its dysregulation is closely related to tumorigenesis. Eukaryotic translation initiation factor 3 subunit i (eIF3i) promotes tumor growth by selectively regulating gene translation, but the underlying mechanisms are largely unknown. Here, we show that eIF3i is significantly increased in colorectal cancer (CRC) and reinforces the proliferation of CRC cells. Using ribosome profiling and proteomics analysis, several genes regulated by eIF3i at the translation level were identified, including D-3-phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in the de novo serine synthesis pathway that participates in metabolic reprogramming of tumor cells. PHGDH knockdown significantly represses CRC cell proliferation and partially attenuates the excessive growth induced by eIF3i overexpression. Mechanistically, METTL3-mediated N6-methyladenosine modification on PHGDH mRNA promotes its binding with eIF3i, ultimately leading to a higher translational rate. In addition, knocking down eIF3i and PHGDH impedes tumor growth in vivo. Collectively, this study not only uncovered a novel regulatory mechanism for PHGDH translation but also demonstrated that eIF3i is a critical metabolic regulator in human cancer.