Deciphering EIF3D's Role in Immune Regulation and Malignant Progression: A Pan-Cancer Analysis with a Focus on Colon Adenocarcinoma.

Background: EIF3D, a key component of the eukaryotic translation initiation factor 3 (EIF3) complex, is critical in selectively translating mRNAs with atypical cap structures. Its relationship with colon adenocarcinoma (COAD) development and immune infiltration, however, remains under-explored. This study delves into EIF3D's role in COAD using bioinformatics and in vitro experimentation. Materials and Methods: We analyzed EIF3D expression levels utilizing TCGA, GTEx, CPTAC, and TISIDB databases. The TISCH database and ssGSEA method helped in assessing EIF3D's link with the tumor immune microenvironment. EIF3D expression in CRC cells was gauged via real-time PCR. Cell proliferation was assessed using CCK8 and colony formation assays, while migration capabilities were tested through Transwell assays. Flow cytometry facilitated cell cycle distribution and apoptosis analysis. ChIP-qPCR identified transcription factors regulating EIF3D, and bulk sequencing explored EIF3D's pathways in promoting COAD. Results: EIF3D upregulation is a common feature in various tumors, especially in COAD, correlating with poor prognosis in many cancer types. It showed significant associations with immune cell and cancer-associated fibroblast (CAF) infiltration across multiple tumors. Additionally, it is closely associated with molecular and immune subtypes of multiple tumors, including COAD. Single-cell analyses depicted EIF3D's distribution and proportion in CRC immune cells. In vitro findings indicated EIF3D knockdown curtailed proliferation and migration, inducing G0/G1 arrest in COAD cells. Moreover, bulk sequencing revealed EIF3D knockdown interferes with multiple cancer-related pathways, likely by curtailing cell cycle and DNA replication activities to regulate cell proliferation. Conclusion: EIF3D emerges as a potential prognostic biomarker for tumor progression and immune infiltration, particularly in COAD, potentially predicting immunotherapy efficacy. Additionally, EIF3D represents a multifaceted target implicated in COAD's malignant progression.

Design, synthesis and biological evaluation of plant-derived miliusol derivatives achieve TNBC profound regression in vivo.

Triple-negative breast cancer has become a major problem in clinical treatment due to its high heterogeneity, and Plant-derived drug discovery has been the focus of attention for novel anti-tumor therapeutics. In this study, Miliusol, a natural product isolated from the rarely reported plant Miliusa tenuistipitata, was identified as the lead compound, and 25 miliusol derivatives were designed and synthesized under antiproliferative activity guidance. The results revealed that ZMF-24 was demonstrated to have potent anti-TNBC proliferation with IC50 values of 0.22 µM and 0.44 µM in BT-549 cells and MDA-MB-231 cells respectively with low cytotoxicity to MCF10A cells, and could significantly downregulate proliferation and migration markers. Through RNAseq analysis, molecular docking and CETSA experiment, we found that ZMF-24 could inhibit Eukaryotic translation initiation factor 3 subunit D (EIF3D) that further disrupted the energy supply of TNBC by inhibiting glycolysis, induced profound TNBC apoptosis by stimulating persistent ER stress. Importantly, ZMF-24 exhibited remarkable anti-proliferation and anti-metastasis potential in nude mice xenograft TNBC model without obvious toxicity. Collectively, the findings demonstrate ZMF-24 has significant potential as a potent chemotherapy agent against triple-negative breast cancer.

Interaction of chikungunya virus glycoproteins with macrophage factors controls virion production.

Despite their role as innate sentinels, macrophages can serve as cellular reservoirs of chikungunya virus (CHIKV), a highly-pathogenic arthropod-borne alphavirus that has caused large outbreaks among human populations. Here, with the use of viral chimeras and evolutionary selection analysis, we define CHIKV glycoproteins E1 and E2 as critical for virion production in THP-1 derived human macrophages. Through proteomic analysis and functional validation, we further identify signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 subunit K (eIF3k) as E1-binding host proteins with anti-CHIKV activities. We find that E1 residue V220, which has undergone positive selection, is indispensable for CHIKV production in macrophages, as its mutation attenuates E1 interaction with the host restriction factors SPCS3 and eIF3k. Finally, we show that the antiviral activity of eIF3k is translation-independent, and that CHIKV infection promotes eIF3k translocation from the nucleus to the cytoplasm, where it associates with SPCS3. These functions of CHIKV glycoproteins late in the viral life cycle provide a new example of an intracellular evolutionary arms race with host restriction factors, as well as potential targets for therapeutic intervention.

EIF3B affects the invasion and metastasis of hepatocellular carcinoma cells via the TGFBI/MAPK/ERK pathway.

INTRODUCTION AND OBJECTIVES: To study the effect of eukaryotic initiation factor 3B (EIF3B) on the invasion and migration of hepatocellular carcinoma (HCC) and its potential mechanism. MATERIALS AND METHODS: The clinical significance of EIF3B expression was studied with The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interaction Analysis datasets. Immunohistochemical staining and western blotting were used to examine EIF3B expression in cell lines and tissues from HCC patients. The scratch assay and transwell assay were used to measure the invasion and metastasis of different HCC cell lines in vitro. The molecular mechanism of EIF3B was determined using RNA-seq and identification of dysregulated signaling pathways. Western blotting was used to verify the alterations of EIF3B signaling functioned in the promotion of HCC progression. RESULTS: Elevated expression of EIF3B in HCC correlated significantly with aggressive clinicopathologic characteristics, including advanced tumor grade and poor prognosis. Studies with cultured cells indicated that EIF3B knockdown inhibited HCC cell invasion and metastasis by depressing the epithelial-mesenchymal transition (EMT). EIF3B also activated the TGFBI/MAPK/ERK signaling pathway by increasing the levels of pMEK and pERK. CONCLUSIONS: Our results indicate that EIF3B functions as an oncogene in HCC that accelerates cell invasion, metastasis, and the EMT by stimulation of the TGFBI/MAPK/ERK signaling pathway. EIF3B is a potential target for the treatment of HCC.

FOXA1-dependent PUS1 regulates EIF3b stability in a non-enzymatic pathway mediating prostate cancer bone metastasis.

Bone metastasis is a significant contributor to the poor prognosis in prostate cancer. Recent evidence highlights the pivotal role of pseudouridine synthases in solid tumor progression, yet the specific enzyme driving prostate cancer metastasis remains unidentified. This study uncovers a novel regulatory mechanism of the FOXA1/PUS1/EIF3b signaling axis in prostate cancer bone metastasis. We identified elevated PUS1 expression in prostate cancer tissues, correlating with higher clinical grade and worse prognosis. Knockdown of PUS1 inhibited metastasis independently of its enzymatic activity, with EIF3b acting as a downstream effector, protected from ubiquitin-mediated degradation by PUS1. Overexpression of EIF3b countered the metastasis suppression due to PUS1 knockdown. Additionally, FOXA1 was shown to enhance PUS1 expression by binding to its promoter. Mogroside IV-E, a specific PUS1 inhibitor, demonstrated potent anti-metastatic effects by reducing PUS1 expression. Our findings highlight the FOXA1/PUS1/EIF3b axis as a critical mediator of prostate cancer bone metastasis and suggest that targeting this pathway could be a promising therapeutic strategy.

Proteomic Profiling of Unannotated Microproteins in Human Placenta Reveals XRCC6P1 as a Potential Negative Regulator of Translation.

Ribosome profiling and mass spectrometry have revealed thousands of previously unannotated small and alternative open reading frames (sm/alt-ORFs) that are translated into micro/alt-proteins in mammalian cells. However, their prevalence across human tissues and biological roles remains largely undefined. The placenta is an ideal model for identifying unannotated microproteins and alt-proteins due to its considerable protein diversity that is required to sustain fetal development during pregnancy. Here, we profiled unannotated microproteins and alt-proteins in human placental tissues from preeclampsia patients or healthy individuals by proteomics, identified 52 unannotated microproteins or alt-proteins, and demonstrated that five microproteins can be translated from overexpression constructs in a heterologous cell line, although several are unstable. We further demonstrated that one microprotein, XRCC6P1, associates with translation initiation factor eIF3 and negatively regulates translation when exogenously overexpressed. Thus, we revealed a hidden sm/alt-ORF-encoded proteome in the human placenta, which may advance the mechanism studies for placenta development as well as placental disorders such as preeclampsia.

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) to drive a diverse set of mechanistic steps during translation of an mRNA into the protein it encodes. And yet, 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 in dynamic exchange between 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 dynamic compositional equilibrium and show that mRNA binding by eIF3 is not driven by the full complex but instead by the eIF3a subunit within eIF3a-containing subcomplexes. Our findings provide a mechanistic model for the role of eIF3 in mRNA recruitment 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.