RESUMEN
Translation initiation is a highly regulated step needed for protein synthesis. Most cell-based mechanistic work on translation initiation has been done using non-stressed cells growing in medium with sufficient nutrients and oxygen. This has yielded our current understanding of 'canonical' translation initiation, involving recognition of the mRNA cap by eIF4E1 followed by successive recruitment of initiation factors and the ribosome. Many cells, however, such as tumor cells, are exposed to stresses such as hypoxia, low nutrients or proteotoxic stress. This leads to inactivation of mTORC1 and thereby inactivation of eIF4E1. Hence the question arises how cells translate mRNAs under such stress conditions. We study here how mRNAs are translated in an eIF4E1-independent manner by blocking eIF4E1 using a constitutively active version of eIF4E-binding protein (4E-BP). Via ribosome profiling we identify a subset of mRNAs that are still efficiently translated when eIF4E1 is inactive. We find that these mRNAs preferentially release eIF4E1 when eIF4E1 is inactive and bind instead to eIF3d via its cap-binding pocket. eIF3d then enables these mRNAs to be efficiently translated due to its cap-binding activity. In sum, our work identifies eIF3d-dependent translation as a major mechanism enabling mRNA translation in an eIF4E-independent manner.
Asunto(s)
Factor 3 de Iniciación Eucariótica , Factor 4E Eucariótico de Iniciación , Biosíntesis de Proteínas , ARN Mensajero , Ribosomas , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Ribosomas/metabolismo , Unión Proteica , Caperuzas de ARN/metabolismo , Células HEK293 , Iniciación de la Cadena Peptídica Traduccional , Proteínas de Ciclo Celular , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
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-ß (IFN-ß). 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-ß 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.
Asunto(s)
Factor 3 de Iniciación Eucariótica , Factor 4G Eucariótico de Iniciación , Unión Proteica , ARN Mensajero , Factor 4G Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Interferón beta/metabolismo , Interferón beta/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Iniciación de la Cadena Peptídica Traduccional , Animales , Biosíntesis de Proteínas , Regulación de la Expresión GénicaRESUMEN
Protein translation initiation is a conserved process involving many proteins acting in concert. The 13 subunit eukaryotic initiation factor 3 (eIF3) complex is essential for assembly of the pre-initiation complex that scans mRNA and positions ribosome at the initiation codon. We previously reported that a gain-of-function (gf) mutation affecting the G subunit of the Caenorhabditis elegans eIF3 complex, eif-3.g(gf), selectively modulates protein translation in the ventral cord cholinergic motor neurons. Here, through unbiased genetic suppressor screening, we identified that the gene lin-66 mediates eif-3.g(gf)-dependent protein translation in motor neurons. LIN-66 is composed largely of low-complexity amino acid sequences with unknown functional domains. We combined bioinformatics analysis with in vivo functional dissection and identified a cold-shock domain in LIN-66 critical for its function. In cholinergic motor neurons, LIN-66 shows a close association with EIF-3.G in the cytoplasm. The low-complexity amino acid sequences of LIN-66 modulate its subcellular pattern. As cold-shock domains function broadly in RNA regulation, we propose that LIN-66 mediates stimulus-dependent protein translation by facilitating the interaction of mRNAs with EIF-3.G.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Factor 3 de Iniciación Eucariótica , Neuronas Motoras , Biosíntesis de Proteínas , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Neuronas Motoras/metabolismo , Mutación , ARN Mensajero/metabolismo , ARN Mensajero/genética , Secuencia de Aminoácidos , Respuesta al Choque por Frío , Dominios ProteicosAsunto(s)
Chaperonina con TCP-1 , Factor 3 de Iniciación Eucariótica , Pliegue de Proteína , Chaperonina con TCP-1/metabolismo , Chaperonina con TCP-1/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/química , Humanos , Subunidades de Proteína/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/química , Unión ProteicaRESUMEN
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.
Asunto(s)
Adenocarcinoma , Neoplasias de la Vejiga Urinaria , Humanos , Masculino , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Persona de Mediana Edad , Adenocarcinoma/genética , Adenocarcinoma/patología , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Factor 3 de Iniciación Eucariótica/genética , Proteínas de Fusión Oncogénica/genéticaRESUMEN
Colorectal cancer (CRC) is among the most common gastrointestinal malignancies worldwide. eIF3a is highly expressed in a variety of cancer types, yet its role in CRC remains unclear. We introduced ectopic eIF3a expression in CRC cells to investigate its relevance to various malignant behaviors. Further, we silenced eIF3a to explore its effect on tumor growth in a nude mouse tumor xenograft model. Finally, the molecular mechanisms through which eIF3a regulates malignancy in CRC cells were explored through bioinformatics analysis combined with the use of a specific PI3K inhibitor (LY294002). eIF3a was highly expressed in the peripheral blood and cancer tissue of CRC patients. Malignancy and tumor growth were significantly inhibited by silencing eIF3a, while overexpression promoted malignant behaviors, with a positive correlation between PI3K/AKT activation and eIF3a expression. Taken together, eIF3a plays an oncogenic role in CRC by regulating PI3K/AKT signaling and is a potential biomarker for CRC diagnosis and prognostic monitoring.
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Neoplasias Colorrectales , Factor 3 de Iniciación Eucariótica , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Humanos , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Animales , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Ratones Desnudos , Línea Celular Tumoral , Proliferación Celular , Ensayos Antitumor por Modelo de Xenoinjerto , Femenino , Masculino , Regulación Neoplásica de la Expresión GénicaRESUMEN
As a key regulator of intercellular communication, exosomes are essential for tumor cells. In our study, we will explore the mechanisms of exosomes from different sources on lung cancer. We isolated CD8+T cells and cancer-associated fibroblasts (CAFs) from venous blood and tumor tissues of lung cancer patients, and isolated exosomes. MiR-2682 was high expression in CD8+T-derived exosomes, and lncRNA-FOXD3-AS1 was upregulated in CAF-derived exosomes. Online bioinformatics database analysis showed that RNA Binding Motif Protein 39 (RBM39) was identified as the target of miR-2682, and eukaryotic translation initiation factors 3B (EIF3B) was identified as the RNA binding protein of FOXD3-AS1. CD8+T-derived exosomes inhibited the growth of A549 cells and promoted apoptosis, while miR-2682 inhibits reversed these effects of CD8+T-derived exosomes. CAF-derived exosomes promoted the growth of A549 cells and inhibited apoptosis, while FOXD3-AS1 siRNA reversed the effect of CAF-derived exosomes. Mechanism studies have found that miR-2682 inhibits the growth of lung cancer cells by inhibiting the expression of RBM39. FOXD3-AS1 promoted the growth of lung cancer cells by binding to EIF3B. In vivo experiments showed that CD8+T cell-derived exosome miR-2682 inhibited lung cancer tumor formation, while CAF-derived exosome FOXD3-AS1 promoted lung cancer tumor formation. This study provides mechanistic insights into the role of miR-2682 and FOXD3-AS1 in lung cancer progression and provides new strategies for lung cancer treatment.
Asunto(s)
Exosomas , Neoplasias Pulmonares , MicroARNs , Exosomas/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Animales , Células A549 , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 3 de Iniciación Eucariótica/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Apoptosis , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Ratones , Fibroblastos Asociados al Cáncer/metabolismo , Progresión de la Enfermedad , Proliferación Celular , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratones Desnudos , Masculino , Femenino , Ratones Endogámicos BALB C , Línea Celular TumoralRESUMEN
Messenger RNA (mRNA) recruitment to the 40S ribosomal subunit is mediated by eukaryotic initiation factor 4F (eIF4F). This complex includes three subunits: eIF4E (m7G cap-binding protein), eIF4A (DEAD-box helicase), and eIF4G. Mammalian eIF4G is a scaffold that coordinates the activities of eIF4E and eIF4A and provides a bridge to connect the mRNA and 40S ribosomal subunit through its interaction with eIF3. While the roles of many eIF4G binding domains are relatively clear, the precise function of RNA binding by eIF4G remains to be elucidated. In this work, we used an eIF4G-dependent translation assay to reveal that the RNA binding domain (eIF4G-RBD; amino acids 682-720) stimulates translation. This stimulating activity is observed when eIF4G is independently tethered to an internal region of the mRNA, suggesting that the eIF4G-RBD promotes translation by a mechanism that is independent of the m7G cap and mRNA tethering. Using a kinetic helicase assay, we show that the eIF4G-RBD has a minimal effect on eIF4A helicase activity, demonstrating that the eIF4G-RBD is not required to coordinate eIF4F-dependent duplex unwinding. Unexpectedly, native gel electrophoresis and fluorescence polarization assays reveal a previously unidentified direct interaction between eIF4G and the 40S subunit. Using binding assays, our data show that this 40S subunit interaction is separate from the previously characterized interaction between eIF4G and eIF3. Thus, our work reveals how eIF4F can bind to the 40S subunit using eIF3-dependent and eIF3-independent binding domains to promote translation initiation.
Asunto(s)
Factor 4E Eucariótico de Iniciación , Biosíntesis de Proteínas , Subunidades Ribosómicas Pequeñas de Eucariotas , Humanos , Factor 3 de Iniciación Eucariótica/química , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Unión Proteica , Dominios Proteicos , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Factor 4E Eucariótico de Iniciación/metabolismoRESUMEN
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.
Asunto(s)
Neoplasias de los Conductos Biliares , Colangiocarcinoma , Factor 3 de Iniciación Eucariótica , Antígeno Nuclear de Célula en Proliferación , Ubiquitina-Proteína Ligasas , Ubiquitinación , Animales , Femenino , Humanos , Masculino , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Línea Celular Tumoral , Movimiento Celular/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Regulación Neoplásica de la Expresión Génica , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Circular RNAs (circRNAs) are associated with development and progression of multiple myeloma (MM). However, the role and mechanism of circ_0005615 in MM have not been elucidated. METHODS: Circ_0005615 was determined by GEO database. quantitative RT-PCR was performed to confirm the expression of circ_0005615 in peripheral blood of MM patients and MM cells. The roles of circ_0005615 in MM were analyzed using CCK8, transwell invasion, cell apoptosis and tumor xenograft experiments. Bioinformatics tools, RIP and RNA pull down assays were conducted to explore the downstream of circ_0005615. Furthermore, the mechanism was investigated by quantitative RT-PCR, western blot, dot blot and meRIP-PCR assays. RESULTS: Circ_0005615 was upregulated in MM. Overexpression of circ_0005615 promoted cell viability and invasion, and suppressed apoptosis in vitro, which were opposite when circ_0005615 was knockdowned. Mechanistically, EIF4A3, a RNA-binding protein (RBP), could directly bind to circ_0005615 and ALKBH5, where ALKBH5 could directly combine with MAP3K4, forming a circ_0005615- EIF4A3-ALKBH5-MAP3K4 module. Furthermore, circ_0005615 overexpression increased m6A methylation of MAP3K4 by inhibiting ALKBH5, leading to decreased MAP3K4. Further functional experiments indicated that ALKBH5 overexpression weakened the promoting roles of circ_0005615 overexpression in MAP3K4 m6A methylation and tumor progression in MM. The above functions and mechanism were also verified in vivo. CONCLUSIONS: Elevated circ_0005615 decreased MAP3K4 mediated by ALKBH5 through interacting with EIF4A3, thereby accelerating MM progression. Circ_0005615 might be a promising biomarker and target of MM.
Asunto(s)
Desmetilasa de ARN, Homólogo 5 de AlkB , Progresión de la Enfermedad , Mieloma Múltiple , ARN Circular , Humanos , ARN Circular/genética , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Mieloma Múltiple/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Ratones , Animales , Apoptosis , Regulación Neoplásica de la Expresión Génica , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Ratones Desnudos , Proliferación Celular , Ensayos Antitumor por Modelo de Xenoinjerto , Adenosina/metabolismo , Adenosina/análogos & derivados , Línea Celular Tumoral , Femenino , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Factor 4A Eucariótico de Iniciación , ARN Helicasas DEAD-boxRESUMEN
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.
Asunto(s)
Empalme Alternativo , Factor 3 de Iniciación Eucariótica , Neoplasias de Cabeza y Cuello , Carcinoma de Células Escamosas de Cabeza y Cuello , Microambiente Tumoral , Humanos , Microambiente Tumoral/inmunología , Microambiente Tumoral/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Empalme Alternativo/genética , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/inmunología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proliferación Celular/genética , Pronóstico , Apoptosis/genética , Masculino , Movimiento Celular/genética , FemeninoRESUMEN
Regulation of mRNA translation by eukaryotic initiation factors (eIFs) is crucial for cell survival. In humans, eIF3 stimulates translation of the JUN mRNA which encodes the transcription factor JUN, an oncogenic transcription factor involved in cell cycle progression, apoptosis, and cell proliferation. Previous studies revealed that eIF3 activates translation of the JUN mRNA by interacting with a stem loop in the 5' untranslated region (5' UTR) and with the 5' -7-methylguanosine cap structure. In addition to its interaction site with eIF3, the JUN 5' UTR is nearly one kilobase in length, and has a high degree of secondary structure, high GC content, and an upstream start codon (uAUG). This motivated us to explore the complexity of JUN mRNA translation regulation in human cells. Here we find that JUN translation is regulated in a sequence and structure-dependent manner in regions adjacent to the eIF3-interacting site in the JUN 5' UTR. Furthermore, we identify contributions of an additional initiation factor, eIF4A, in JUN regulation. We show that enhancing the interaction of eIF4A with JUN by using the compound Rocaglamide A (RocA) represses JUN translation. We also find that both the upstream AUG (uAUG) and the main AUG (mAUG) contribute to JUN translation and that they are conserved throughout vertebrates. Our results reveal additional layers of regulation for JUN translation and show the potential of JUN as a model transcript for understanding multiple interacting modes of translation regulation.
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Factor 3 de Iniciación Eucariótica , Biosíntesis de Proteínas , Animales , Humanos , Codón Iniciador/genética , Regiones no Traducidas 5'/genética , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/genéticaRESUMEN
Eukaryotic initiation translation factor 3 subunit h (EIF3H) plays critical roles in regulating translational initiation and predicts poor cancer prognosis, but the mechanism underlying EIF3H tumorigenesis remains to be further elucidated. Here, we report that EIF3H is overexpressed in colorectal cancer (CRC) and correlates with poor prognosis. Conditional Eif3h deletion suppresses colorectal tumorigenesis in AOM/DSS model. Mechanistically, EIF3H functions as a deubiquitinase for HAX1 and stabilizes HAX1 via antagonizing ßTrCP-mediated ubiquitination, which enhances the interaction between RAF1, MEK1 and ERK1, thereby potentiating phosphorylation of ERK1/2. In addition, activation of Wnt/ß-catenin signaling induces EIF3H expression. EIF3H/HAX1 axis promotes CRC tumorigenesis and metastasis in mouse orthotopic cancer model. Significantly, combined targeting Wnt and RAF1-ERK1/2 signaling synergistically inhibits tumor growth in EIF3H-high patient-derived xenografts. These results uncover the important roles of EIF3H in mediating CRC progression through regulating HAX1 and RAF1-ERK1/2 signaling. EIF3H represents a promising therapeutic target and prognostic marker in CRC.
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Neoplasias Colorrectales , Sistema de Señalización de MAP Quinasas , Humanos , Animales , Ratones , Fosforilación , Transformación Celular Neoplásica/genética , Carcinogénesis , Vía de Señalización Wnt , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Neoplasias Colorrectales/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Immune checkpoint inhibitors (ICIs) represent a promising therapeutic approach for esophageal squamous cell carcinoma (ESCC). However, the subpopulations of ESCC patients expected to benefit from ICIs have not been clearly defined. The anti-tumor cytotoxic activity of T cells is an important pharmacological mechanism of ICIs. In this study, the prognostic value of the genes regulating tumor cells to T cell-mediated killing (referred to as GRTTKs) in ESCC was explored by using a comprehensive bioinformatics approach. Training and validation datasets were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), respectively. A prognostic risk scoring model was developed by integrating prognostic GRTTKs from TCGA and GEO datasets using a ridge regression algorithm. Patients with ESCC were divided into high- and low-risk groups based on eight GRTTKs (EIF4H, CDK2, TCEA1, SPTLC2, TMEM209, RGP1, EIF3D, and CAPZA3) to predict overall survival in the TCGA cohort. Using Kaplan-Meier curves, receiver operating characteristic curves, and C-index analysis, the high reliability of the prognostic risk-scoring model was certified. The model scores served as independent prognostic factors, and combining clinical staging with risk scoring improved the predictive value. Patients in the high-risk group exhibited abundant immune cell infiltration, including immune checkpoint expression, antigen presentation capability, immune cycle gene expression, and high tumor inflammation signature scores. The high-risk group exhibited a greater response to immunotherapy and neoadjuvant chemotherapy than the low-risk group. Drug sensitivity analysis demonstrated lower IC50 for AZD6244 and PD.0332991 in high-risk groups and lower IC50 for cisplatin, ATRA, QS11, and vinorelbine in the low-risk group. Furthermore, the differential expression of GRTTK-related signatures including CDK2, TCEA1, and TMEM209 were verified in ESCC tissues and paracancerous tissues. Overall, the novel GRTTK-based prognostic model can serve as indicators to predict the survival status and immunotherapy response of patients with ESCC, thereby providing guidance for the development of personalized treatment strategies.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Carcinoma de Células Escamosas de Esófago/genética , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Reproducibilidad de los Resultados , Linfocitos T , Genes cdc , Pronóstico , Factor 3 de Iniciación EucarióticaRESUMEN
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.
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Factor 3 de Iniciación Eucariótica , Shigella , Animales , Ratones , Gránulos de Estrés , Citoplasma , Shigella flexneriRESUMEN
The survival of Legionella spp. as intracellular pathogens relies on the combined action of protein effectors delivered inside their eukaryotic hosts by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IVb secretion system. The specific repertoire of effector arsenals varies dramatically across over 60 known species of this genera with Legionella pneumophila responsible for most cases of Legionnaires' disease in humans encoding over 360 Dot/Icm effectors. However, a small subset of "core" effectors appears to be conserved across all Legionella species raising an intriguing question of their role in these bacteria's pathogenic strategy, which for most of these effectors remains unknown. L. pneumophila Lpg0103 effector, also known as VipF, represents one of the core effector families that features a tandem of Gcn5-related N-acetyltransferase (GNAT) domains. Here, we present the crystal structure of the Lha0223, the VipF representative from Legionella hackeliae in complex with acetyl-coenzyme A determined to 1.75 Å resolution. Our structural analysis suggested that this effector family shares a common fold with the two GNAT domains forming a deep groove occupied by residues conserved across VipF homologs. Further analysis suggested that only the C-terminal GNAT domain of VipF effectors retains the active site composition compatible with catalysis, whereas the N-terminal GNAT domain binds the ligand in a non-catalytical mode. We confirmed this by in vitro enzymatic assays which revealed VipF activity not only against generic small molecule substrates, such as chloramphenicol, but also against poly-L-lysine and histone-derived peptides. We identified the human eukaryotic translation initiation factor 3 (eIF3) complex co-precipitating with Lpg0103 and demonstrated the direct interaction between the several representatives of the VipF family, including Lpg0103 and Lha0223 with the K subunit of eIF3. According to our data, these interactions involve primarily the C-terminal tail of eIF3-K containing two lysine residues that are acetylated by VipF. VipF catalytic activity results in the suppression of eukaryotic protein translation in vitro, revealing the potential function of VipF "core" effectors in Legionella's pathogenic strategy.IMPORTANCEBy translocating effectors inside the eukaryotic host cell, bacteria can modulate host cellular processes in their favor. Legionella species, which includes the pneumonia-causing Legionella pneumophila, encode a widely diverse set of effectors with only a small subset that is conserved across this genus. Here, we demonstrate that one of these conserved effector families, represented by L. pneumophila VipF (Lpg0103), is a tandem Gcn5-related N-acetyltransferase interacting with the K subunit of human eukaryotic initiation factor 3 complex. VipF catalyzes the acetylation of lysine residues on the C-terminal tail of the K subunit, resulting in the suppression of eukaryotic translation initiation factor 3-mediated protein translation in vitro. These new data provide the first insight into the molecular function of this pathogenic factor family common across Legionellae.
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Legionella pneumophila , Legionella , Enfermedad de los Legionarios , Humanos , Acetiltransferasas/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Lisina/metabolismo , Factor 3 Procariótico de Iniciación/metabolismo , Legionella/genética , Legionella pneumophila/genética , Biosíntesis de Proteínas , Proteínas Bacterianas/metabolismoRESUMEN
In a previous study, we discovered that the level of lnc-TSPAN12 was significantly elevated in hepatocellular carcinoma (HCC) and correlated with a low survival rate. However, the function and mechanism of lnc-TSPAN12 in modulating epithelial-mesenchymal transition (EMT) and metastasis in HCC remains poorly understood. This study demonstrates that lnc-TSPAN12 positively influences migration, invasion, and EMT of HCC cells in vitro and promotes hepatic metastasis in vivo. The modification of N6-methyladenosine, driven by METTL3, is essential for the stability of lnc-TSPAN12, which may partially contribute to the upregulation of lnc-TSPAN12. Mechanistically, lnc-TSPAN12 exhibits direct interactions with EIF3I and SENP1, acting as a scaffold to enhance the SENP1-EIF3I interaction. As a result, the SUMOylation of EIF3I is inhibited, preventing its ubiquitin-mediated degradation. Ultimately, this activates the Wnt/ß-catenin signaling pathway, stimulating EMT and metastasis in HCC. Our findings shed light on the regulatory mechanism of lnc-TSPAN12 in HCC metastasis and identify the lnc-TSPAN12-EIF3I/SENP1 axis as a novel therapeutic target for HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , ARN Largo no Codificante , Tetraspaninas , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Transición Epitelial-Mesenquimal , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Metiltransferasas/genética , Metiltransferasas/metabolismo , ARN Largo no Codificante/genética , Vía de Señalización WntRESUMEN
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.
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Factor 4F Eucariótico de Iniciación , Neoplasias , Humanos , Factor 4F Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/genética , Variaciones en el Número de Copia de ADN , Factor 3 de Iniciación Eucariótica , Neoplasias/genéticaRESUMEN
An RNA structure or modified RNA sequences can provide a platform for ribosome loading and internal translation initiation. The functional significance of internal translation has recently been highlighted by the discovery that a subset of circular RNAs (circRNAs) is internally translated. However, the molecular mechanisms underlying the internal initiation of translation in circRNAs remain unclear. Here, we identify eIF3g (a subunit of eIF3 complex) as a binding partner of eIF4A3, a core component of the exon-junction complex (EJC) that is deposited onto spliced mRNAs and plays multiple roles in the regulation of gene expression. The direct interaction between eIF4A3-eIF3g serves as a molecular linker between the eIF4A3 and eIF3 complex, thereby facilitating internal ribosomal entry. Protein synthesis from in vitro-synthesized circRNA demonstrates eIF4A3-driven internal translation, which relies on the eIF4A3-eIF3g interaction. Furthermore, our transcriptome-wide analysis shows that efficient polysomal association of endogenous circRNAs requires eIF4A3. Notably, a subset of endogenous circRNAs can express a full-length intact protein, such as ß-catenin, in an eIF4A3-dependent manner. Collectively, our results expand the understanding of the protein-coding potential of the human transcriptome, including circRNAs.
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Factor 3 de Iniciación Eucariótica , Factor 4A Eucariótico de Iniciación , ARN Circular , Humanos , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Proteínas , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.