RESUMO
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.
Assuntos
Fator de Iniciação 3 em Eucariotos , Fator de Iniciação 4E em Eucariotos , Biossíntese de Proteínas , RNA Mensageiro , Ribossomos , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Ribossomos/metabolismo , Ligação Proteica , Capuzes de RNA/metabolismo , Células HEK293 , Iniciação Traducional da Cadeia Peptídica , Proteínas de Ciclo Celular , Proteínas Adaptadoras de Transdução de SinalRESUMO
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.
Assuntos
Fator de Iniciação 3 em Eucariotos , Fator de Iniciação Eucariótico 4G , Ligação Proteica , RNA Mensageiro , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Interferon beta/metabolismo , Interferon beta/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Iniciação Traducional da Cadeia Peptídica , Animais , Biossíntese de Proteínas , Regulação da Expressão GênicaAssuntos
Chaperonina com TCP-1 , Fator de Iniciação 3 em Eucariotos , Dobramento de Proteína , Chaperonina com TCP-1/metabolismo , Chaperonina com TCP-1/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/química , Humanos , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/química , Ligação ProteicaRESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Fator de Iniciação 3 em Eucariotos , Neurônios Motores , Biossíntese de Proteínas , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Neurônios Motores/metabolismo , Mutação , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Sequência de Aminoácidos , Resposta ao Choque Frio , Domínios ProteicosRESUMO
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.
Assuntos
Exossomos , Neoplasias Pulmonares , MicroRNAs , Exossomos/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Células A549 , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Apoptose , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Camundongos , Fibroblastos Associados a Câncer/metabolismo , Progressão da Doença , Proliferação de Células , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Camundongos Nus , Masculino , Feminino , Camundongos Endogâmicos BALB C , Linhagem Celular TumoralRESUMO
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.
Assuntos
Neoplasias Colorretais , Fator de Iniciação 3 em Eucariotos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Humanos , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Camundongos Nus , Linhagem Celular Tumoral , Proliferação de Células , Ensaios Antitumorais Modelo de Xenoenxerto , Feminino , Masculino , Regulação Neoplásica da Expressão GênicaRESUMO
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.
Assuntos
Fator de Iniciação 4E em Eucariotos , Biossíntese de Proteínas , Subunidades Ribossômicas Menores de Eucariotos , Humanos , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Ligação Proteica , Domínios Proteicos , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fator de Iniciação 4E em Eucariotos/metabolismoRESUMO
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.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Progressão da Doença , Mieloma Múltiplo , RNA Circular , Humanos , RNA Circular/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Mieloma Múltiplo/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Camundongos , Animais , Apoptose , Regulação Neoplásica da Expressão Gênica , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Camundongos Nus , Proliferação de Células , Ensaios Antitumorais Modelo de Xenoenxerto , Adenosina/metabolismo , Adenosina/análogos & derivados , Linhagem Celular Tumoral , Feminino , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Fator de Iniciação 4A em Eucariotos , RNA Helicases DEAD-boxRESUMO
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.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Fator de Iniciação 3 em Eucariotos , Antígeno Nuclear de Célula em Proliferação , Ubiquitina-Proteína Ligases , Ubiquitinação , Animais , Feminino , Humanos , Masculino , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Regulação Neoplásica da Expressão Gênica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
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.
Assuntos
Processamento Alternativo , Fator de Iniciação 3 em Eucariotos , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas de Cabeça e Pescoço , Microambiente Tumoral , Humanos , Microambiente Tumoral/imunologia , Microambiente Tumoral/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Processamento Alternativo/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/imunologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/genética , Prognóstico , Apoptose/genética , Masculino , Movimento Celular/genética , FemininoRESUMO
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.
Assuntos
Fator de Iniciação 3 em Eucariotos , Biossíntese de Proteínas , Animais , Humanos , Códon de Iniciação/genética , Regiões 5' não Traduzidas/genética , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Neoplasias Colorretais , Sistema de Sinalização das MAP Quinases , Humanos , Animais , Camundongos , Fosforilação , Transformação Celular Neoplásica/genética , Carcinogênese , Via de Sinalização Wnt , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Neoplasias Colorretais/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
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.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , RNA Longo não Codificante , Tetraspaninas , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Transição Epitelial-Mesenquimal , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Longo não Codificante/genética , Via de Sinalização WntRESUMO
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.
Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Humanos , Acetiltransferases/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Lisina/metabolismo , Fator de Iniciação 3 em Procariotos/metabolismo , Legionella/genética , Legionella pneumophila/genética , Biossíntese de Proteínas , Proteínas de Bactérias/metabolismoRESUMO
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.
Assuntos
Fator de Iniciação 3 em Eucariotos , Fator de Iniciação 4A em Eucariotos , RNA Circular , Humanos , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Proteínas , Ribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Fator de Iniciação 3 em Eucariotos , Biossíntese de Proteínas , Humanos , RNA Mensageiro/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/metabolismo , Ribossomos/genética , Sequências Hélice-Alça-HéliceRESUMO
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.
Assuntos
Neoplasias Colorretais , Fator de Iniciação 3 em Eucariotos , Regulação Neoplásica da Expressão Gênica , Fosfoglicerato Desidrogenase , Humanos , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/fisiopatologia , Metiltransferases/metabolismo , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , RNA Mensageiro/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Regulação para Cima , Técnicas de Silenciamento de Genes , Regulação Neoplásica da Expressão Gênica/genética , Animais , Camundongos , Camundongos Endogâmicos BALB C , Feminino , XenoenxertosRESUMO
Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal human malignancies, and with quite limited treatment alternatives. The proteasome is responsible for most of the protein degradation in eukaryotic cells and required for the maintenance of intracellular homeostasis. However, its potential role in HCC is largely unknown. In the current study, we identified eukaryotic translation initiation factor 3 subunit H (EIF3H), belonging to the JAB1/MPN/MOV34 (JAMM) superfamily, as a bona fide deubiquitylase of O-GlcNAc transferase (OGT) in HCC. We explored that EIF3H was positively associated with OGT in HCC and was related to the unfavorable prognosis. EIF3H could interact with, deubiquitylate, and stabilize OGT in a deubiquitylase-dependent manner. Specifically, EIF3H was associated with the GT domain of ERα via its JAB/MP domain, thus inhibiting the K48-linked ubiquitin chain on OGT. Besides, we demonstrated that the knockdown of EIF3H significantly reduced OGT protein expression, cell proliferation and invasion, and caused G1/S arrest of HCC. We also found that the deletion of EIF3H prompted ferroptosis in HCC cells. Finally, the effects of EIF3H depletion could be reversed by further OGT overexpression, implying that the OGT status is indispensable for EIF3H function in HCC carcinogenesis. In summary, our study described the oncogenic function of EIF3H and revealed an interesting post-translational mechanism between EIF3H, OGT, and ferroptosis in HCC. Targeting the EIF3H may be a promising approach in HCC. Video Abstract.
Assuntos
Carcinoma Hepatocelular , Fator de Iniciação 3 em Eucariotos , Ferroptose , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Enzimas Desubiquitinantes , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama , Fator de Iniciação 3 em Eucariotos , Fator de Iniciação Eucariótico 4G , Biossíntese de Proteínas , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/genética , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Metástase Neoplásica , RNA Mensageiro/genética , Fatores de Transcrição/genética , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismoRESUMO
eIF3, whose subunits are frequently overexpressed in cancer, regulates mRNA translation from initiation to termination, but mRNA-selective functions of individual subunits remain poorly defined. Using multiomic profiling upon acute depletion of eIF3 subunits, we observed that while eIF3a, b, e, and f markedly differed in their impact on eIF3 holo-complex formation and translation, they were each required for cancer cell proliferation and tumor growth. Remarkably, eIF3k showed the opposite pattern with depletion promoting global translation, cell proliferation, tumor growth, and stress resistance through repressing the synthesis of ribosomal proteins, especially RPS15A. Whereas ectopic expression of RPS15A mimicked the anabolic effects of eIF3k depletion, disruption of eIF3 binding to the 5'-UTR of RSP15A mRNA negated them. eIF3k and eIF3l are selectively downregulated in response to endoplasmic reticulum and oxidative stress. Supported by mathematical modeling, our data uncover eIF3k-l as a mRNA-specific module which, through controlling RPS15A translation, serves as a rheostat of ribosome content, possibly to secure spare translational capacity that can be mobilized during stress.