m7G-related genes-NCBP2 and EIF4E3 determine immune contexture in head and neck squamous cell carcinoma by regulating CCL4/CCL5 expression.

Aberrant N7 -methylguanosine (m7G) levels closely correlate with tumor genesis and progression. NCBP2 and EIF4E3 are two important m7G-related cap-binding genes. This study aimed to identify the relationship between the EIF4E3/NCBP2 function and immunological characteristics of head and neck squamous cell carcinoma (HNSCC). Hierarchical clustering was employed in classifying HNSCC patients into two groups based on the expressions of NCBP2 and EIF4E3. The differentially expressed genes were identified between the two groups, and GO functional enrichment was subsequently performed. Weighted gene co-expression network analysis was conducted to identify the hub genes related to EIF4E3/NCBP2 expression and immunity. The differential infiltration of immune cells and the response to immunotherapy were compared between the two groups. Single-cell sequence and trajectory analyses were performed to predict cell differentiation and display the expression of EIF4E3/NCBP2 in each state. In addition, quantitative real-time PCR, spatial transcriptome analysis, transwell assay, and western blotting were conducted to verify the biological function of EIF4E3/NCBP2. Here, group A showed a higher EIF4E3 expression and a lower NCBP2 expression, which had higher immune scores, proportion of most immune cells, immune activities, expression of immunomodulatory targets, and a better response to cancer immunotherapy. Besides, 56 hub molecules with notable immune regulation significance were identified. A risk model containing 17 hub genes and a prognostic nomogram was successfully established. Moreover, HNSCC tissues had a lower EIF4E3 expression and a higher NCBP2 expression than normal tissues. NCBP2 and EIF4E3 played a vital role in the differentiation of monocytes. Furthermore, the expression of CCL4/CCL5 can be regulated via EIF4E3 overexpression and NCBP2 knockdown. Collectively, NCBP2 and EIF4E3 can affect downstream gene expression, as well as immune contexture and response to immunotherapy, which could induce "cold-to-hot" tumor transformation in HNSCC patients.

Major splice variants and multiple polyadenylation site utilization in mRNAs encoding human translation initiation factors eIF4E1 and eIF4E3 regulate the translational regulators?

Alternative polyadenylation is an important and pervasive mechanism that generates heterogeneous 3'-termini of mRNA and is considered an important regulator of gene expression. We performed bioinformatics analyses of ESTs and the 3'-UTRs of the main transcript splice variants of the translational initiation factor eIF4E1 and its family members, eIF4E2 and eIF4E3. This systematic analysis led to the prediction of new polyadenylation signals. All identified polyadenylation sites were subsequently verified by 3'RACE of transcripts isolated from human lymphoblastic cell lines. This led to the observation that multiple simultaneous polyadenylation site utilization occurs in single cell population. Importantly, we described the use of new polyadenylation site in the eIF4E1 mRNA, which lacked any known polyadenylation signal. The proportion of eIF4E1 transcripts derived from the first two polyadenylation sites in eIF4E1 mRNA achieved 15% in a wide range of cell lines. This result demonstrates the ubiquitous presence of ARE-lacking transcripts, which escape HuR/Auf1-mediated control, the main mechanism of eIF4E1 gene expression regulation. We found many EST clones documenting the significant production of transcript variants 2-4 of eIF4E2 gene that encode proteins with C-termini that were distinct from the mainly studied prototypical isoform A. Similarly, eIF4E3 mRNAs are produced as two main variants with the same very long 3'-UTR with potential for heavy post-transcriptional regulation. We identified sparsely documented transcript variant 1 of eIF4E3 gene in human placenta. eIF4E3 truncated transcript variants were found mainly in brain. We propose to elucidate the minor splice variants of eIF4E2 and eIF4E3 in great detail because they might produce proteins with modified features that fulfill different cellular roles from their major counterparts.

Distinct recruitment of human eIF4E isoforms to processing bodies and stress granules.

BACKGROUND: Eukaryotic translation initiation factor 4E (eIF4E) plays a pivotal role in the control of cap-dependent translation initiation, modulates the fate of specific mRNAs, occurs in processing bodies (PBs) and is required for formation of stress granules (SGs). In this study, we focused on the subcellular localization of a representative compendium of eIF4E protein isoforms, particularly on the less studied members of the human eIF4E protein family, eIF4E2 and eIF4E3. RESULTS: We showed that unlike eIF4E1, its less studied isoform eIF4E3_A, encoded by human chromosome 3, localized to stress granules but not PBs upon both heat shock and arsenite stress. Furthermore, we found that eIF4E3_A interacts with human translation initiation factors eIF4G1, eIF4G3 and PABP1 in vivo and sediments into the same fractions as canonical eIF4E1 during polysome analysis in sucrose gradients. Contrary to this finding, the truncated human eIF4E3 isoform, eIF4E3_B, showed no localization to SGs and no binding to eIF4G. We also highlighted that eIF4E2 may exhibit distinct functions under different stresses as it readily localizes to P-bodies during arsenite and heat stresses, whereas it is redirected to stress granules only upon heat shock. We extended our study to a number of protein variants, arising from alternative mRNA splicing, of each of the three eIF4E isoforms. Our results surprisingly uncovered differences in the ability of eIF4E1_1 and eIF4E1_3 to form stress granules in response to cellular stresses. CONCLUSION: Our comparison of all three human eIF4E isoforms and their protein variants enriches the intriguing spectrum of roles attributed to the eukaryotic initiation translation factors of the 4E family, which exhibit a distinctive localization within different RNA granules under different stresses. The localization of eIF4E3_A to stress granules, but not to processing bodies, along with its binding to eIF4G and PABP1 suggests a role of human eIF4E3_A in translation initiation rather than its involvement in a translational repression and mRNA decay and turnover. The localization of eIF4E2 to stress granules under heat shock but not arsenite stress indicates its distinct function in cellular response to these stresses and points to the variable protein content of SGs as a consequence of different stress insults.

Delineation of the 3p14.1p13 microdeletion associated with syndromic distal limb contractures.

Distal limb contractures (DLC) represent a heterogeneous clinical and genetic condition. Overall, 20-25% of the DLC are caused by mutations in genes encoding the muscle contractile apparatus. Large interstitial deletions of the 3p have already been diagnosed by standard chromosomal analysis, but not associated with a specific phenotype. We report on four patients with syndromic DLC presenting with a de novo 3p14.1p13 microdeletion. The clinical features associated multiple contractures, feeding problems, developmental delay, and intellectual disability. Facial dysmorphism was constant with low-set posteriorly rotated ears and blepharophimosis. Review of previously reported cases with a precise mapping of the deletions, documented a 250 kb smallest region of overlap (SRO) necessary for DLC. This region contained one gene, EIF4E3, the first three exons of the FOXP1 gene, and an intronic enhancer of FOXP1 named hs1149. Sanger sequencing and locus quantification of hs1149, EIF4E3, and FOXP1 in a cohort of 11 French patients affected by DLC appeared normal. In conclusion, we delineate a new microdeletion syndrome involving the 3p14.1p13 locus and associated with DLC and severe developmental delay.

CircVDAC3 sequesters microRNA-592 and elevates EIF4E3 expression to inhibit the progression of gastric cancer.

BACKGROUND: Accumulating evidence has shown that circular RNAs (circRNAs) are involved in gastric cancer (GC) tumorigenesis. However, specific functional circRNAs in GC remain to be discovered, and their underlying mechanisms remain to be elucidated. METHODS: CircRNAs that were differentially expressed between GC tissues and controls were analyzed using a circRNA microarray dataset. The expression of circVDAC3 in GC was determined using quantitative real-time PCR (qRT-PCR), and the structural features of circVDAC3 were validated. Cell function assays and animal experiments were conducted to explore the effects of circVDAC3 on GC. Finally, bioinformatics analysis, fluorescent in situ hybridization, and dual luciferase assays were used to analyze the downstream mechanisms of circVDAC3. RESULTS: Our results showed that circVDAC3 was downregulated in GC and inhibited the proliferation and metastasis of GC cells. Mechanistically, circVDAC3 acts as a competing endogenous RNA (ceRNA) of miR-592 and deregulates the repression of EIF4E3 by miR-592. EIF4E3 is downregulated in GC and overexpression of miR-592 or knockdown of EIF4E3 in circVDAC3-overexpressing cells weakens the anticancer effect of circVDAC3. CONCLUSION: Our study provides evidence that circVDAC3 affects the growth and metastasis of GC cells via the circVDAC3/miR-592/EIF4E3 axis. Our findings offer valuable insights into the mechanisms underlying GC tumorigenesis and suggest novel therapeutic strategies.

Long non-coding RNA LINC01554 overexpression suppresses viability, migration, and invasion of liver cancer cells through regulating miR-148b-3p/EIF4E3.

Background: Long non-coding RNAs (lncRNAs) can be severed as competing endogenous RNAs (ceRNAs) to regulate target genes or mRNAs via sponging microRNAs (miRNAs). This study explored the effect of LINC01554 on liver cancer cells through the ceRNA mechanism. Methods: Five significantly down-regulated lncRNAs were selected for further verification, and then through bioinformatics, interactive miRNAs and mRNAs of lncRNAs were identified. The relationship between LINC01554, miR-148b-3p and EIF4E3 was detected by the dual luciferase reporter gene assay. Afterwards, HCCLM3 cells were transfected with pCDH-LINC01554, miR-148b-3p inhibitor and miR-148b-3p mimics. Cell viability, apoptosis, migration and invasion were measured by Cell Counting Kit-8, flow cytometer, and Transwell assays. Real-time quantitative PCR (RT-qPCR) and Western blot were used to measure the expressions of related genes and proteins. Results: LINC01554 was significantly down-regulated in the liver cancer cell lines, and was expressed in the cytoplasm of HCCLM3 cells. LINC01554 overexpression inhibited proliferation, migration, and invasion of HCCLM3 cells, and promote their apoptosis (P < 0.05). Besides, LINC01554 overexpression also significantly increased the levels of BAX, BCL2/BAX, P53, cleaved-Caspase3, TIMP3, E-cadherin and EIF4E3 (P < 0.05). Through bioinformatics and dual-luciferase reporter gene assay, LINC01554, miR-148b-3p and EIF4E3 were proved to interact with each other. Furthermore, the effects of miR-148b-3p knockdown on HCCLM3 cells were similar with those of LINC01554 overexpression, and miR-148b-3p mimics could reverse the changes of cell viability, apoptosis, migration, and invasion induced by LINC01554 overexpression. Conclusions: LINC01554 overexpression could suppress the growth and metastasis of HCCLM3 cells via miR-148b-3p/EIF4E3.

Comprehensive multi-omics analysis reveals m7G-related signature for evaluating prognosis and immunotherapy efficacy in osteosarcoma.

Background: Osteosarcoma is one of the most prevalent bone malignancies with a poor prognosis. The N7-methylguanosine (m7G) modification facilitates the modification of RNA structure and function tightly associated with cancer. Nonetheless, there is a lack of joint exploration of the relationship between m7G methylation and immune status in osteosarcoma. Methods: With the support of TARGET and GEO databases, we performed consensus clustering to characterize molecular subtypes based on m7G regulators in all osteosarcoma patients. The least absolute shrinkage and selection operator (LASSO) method, Cox regression, and receiver operating characteristic (ROC) curves were employed to construct and validate m7G-related prognostic features and derived risk scores. In addition, GSVA, ssGSEA, CIBERSORT, ESTIMATE, and gene set enrichment analysis were conducted to characterize biological pathways and immune landscapes. We explored the relationship between risk scores and drug sensitivity, immune checkpoints, and human leukocyte antigens by correlation analysis. Finally, the roles of EIF4E3 in cell function were verified through external experiments. Results: Two molecular isoforms based on regulator genes were identified, which presented significant discrepancies in terms of survival and activated pathways. Moreover, the six m7G regulators most associated with prognosis in osteosarcoma patients were identified as independent predictors for the construction of prognostic signature. The model was well stabilized and outperformed traditional clinicopathological features to reliably predict 3-year (AUC = 0.787) and 5-year (AUC = 0.790) survival in osteosarcoma cohorts. Patients with increased risk scores had a poorer prognosis, higher tumor purity, lower checkpoint gene expression, and were in an immunosuppressive microenvironment. Furthermore, enhanced expression of EIF4E3 indicated a favorable prognosis and affected the biological behavior of osteosarcoma cells. Conclusions: We identified six prognostic relevant m7G modulators that may provide valuable indicators for the estimation of overall survival and the corresponding immune landscape in patients with osteosarcoma.

Translation Initiation Factor eIF4E Positively Modulates Conidiogenesis, Appressorium Formation, Host Invasion and Stress Homeostasis in the Filamentous Fungi Magnaporthe oryzae.

Translation initiation factor eIF4E generally mediates the recognition of the 5'cap structure of mRNA during the recruitment of the ribosomes to capped mRNA. Although the eIF4E has been shown to regulate stress response in Schizosaccharomyces pombe positively, there is no direct experimental evidence for the contributions of eIF4E to both physiological and pathogenic development of filamentous fungi. We generated Magnaporthe oryzae eIF4E (MoeIF4E3) gene deletion strains using homologous recombination strategies. Phenotypic and biochemical analyses of MoeIF4E3 defective strains showed that the deletion of MoeIF4E3 triggered a significant reduction in growth and conidiogenesis. We also showed that disruption of MoeIF4E3 partially impaired conidia germination, appressorium integrity and attenuated the pathogenicity of ΔMoeif4e3 strains. In summary, this study provides experimental insights into the contributions of the eIF4E3 to the development of filamentous fungi. Additionally, these observations underscored the need for a comprehensive evaluation of the translational regulatory machinery in phytopathogenic fungi during pathogen-host interaction progression.