Eukaryotic Initiation Factor 4A-I Research Articles

5'-tiRNA-Gln inhibits hepatocellular carcinoma progression by repressing translation through the interaction with eukaryotic initiation factor 4A-I.

tRNA-derived small RNAs (tsRNAs) are novel non-coding RNAs that are involved in the occurrence and progression of diverse diseases. However, their exact presence and function in hepatocellular carcinoma (HCC) remain unclear. Here, differentially expressed tsRNAs in HCC were profiled. A novel tsRNA, tRNAGln-TTG derived 5'-tiRNA-Gln, is significantly downregulated, and its expression level is correlated with progression in patients. In HCC cells, 5'-tiRNA-Gln overexpression impaired the proliferation, migration, and invasion in vitro and in vivo, while 5'-tiRNA-Gln knockdown yielded opposite results. 5'-tiRNA-Gln exerted its function by binding eukaryotic initiation factor 4A-I (EIF4A1), which unwinds complex RNA secondary structures during translation initiation, causing the partial inhibition of translation. The suppressed downregulated proteins include ARAF, MEK1/2 and STAT3, causing the impaired signaling pathway related to HCC progression. Furthermore, based on the construction of a mutant 5'-tiRNA-Gln, the sequence of forming intramolecular G-quadruplex structure is crucial for 5'-tiRNA-Gln to strongly bind EIF4A1 and repress translation. Clinically, 5'-tiRNA-Gln expression level is negatively correlated with ARAF, MEK1/2, and STAT3 in HCC tissues. Collectively, these findings reveal that 5'-tiRJNA-Gln interacts with EIF4A1 to reduce related mRNA binding through the intramolecular G-quadruplex structure, and this process partially inhibits translation and HCC progression.

Integrated microarray for identifying the hub mRNAs and constructed miRNA-mRNA network in coronary in-stent restenosis.

As a major complication after percutaneous coronary intervention (PCI) in patients who suffer from coronary artery disease, in-stent restenosis (ISR) poses a significant challenge for clinical management. A miRNA-mRNA regulatory network of ISR can be constructed to better reveal the occurrence of ISR. The relevant data set from the Gene Expression Omnibus (GEO) database was downloaded, and 284 differentially expressed miRNAs (DE-miRNAs) and 849 differentially expressed mRNAs (DE-mRNAs) were identified. As predicted by online tools, 65 final functional genes (FmRNAs) were overlapping DE-mRNAs and DE-miRNAs target genes. In the biological process (BP) terms of gene ontology (GO) functional analysis, the FmRNAs were mainly enriched in the cellular response to peptide, epithelial cell proliferation, and response to peptide hormone. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the FmRNAs were mainly enriched in breast cancer, endocrine resistance, and Cushing syndrome. Jun proto-oncogene, activator protein-1 (AP-1) transcription factor subunit (JUN), insulin-like growth factor 1 receptor (IGF1R), member RAS oncogene family (RAB14), specificity protein 1 (SP1), protein tyrosine phosphatase nonreceptor type 1 (PTPN1), DDB1 and CUL4 associated factor 10 (DCAF10), retinoblastoma-binding protein 5 (RBBP5), and eukaryotic initiation factor 4A-I (EIF4A1) were hub genes in the protein-protein interaction network (PPI network). The miRNA-mRNA network containing DE-miRNAs and hub genes was built. Hsa-miR-139-5p-JUN, hsa-miR-324-5p-SP1 axis pairs were found in the miRNA-mRNA network, which could promote ISR development. The aforementioned results indicate that the miRNA-mRNA network constructed in ISR has a regulatory role in the development of ISR and may provide new approaches for clinical treatment and experimental development.

Platelet Phenotype Analysis of COVID-19 Patients Reveals Progressive Changes in the Activation of Integrin αIIbβ3, F13A1, the SARS-CoV-2 Target EIF4A1 and Annexin A5.

Background: The fatal consequences of an infection with severe acute respiratory syndrome coronavirus 2 are not only caused by severe pneumonia, but also by thrombosis. Platelets are important regulators of thrombosis, but their involvement in the pathogenesis of COVID-19 is largely unknown. The aim of this study was to determine their functional and biochemical profile in patients with COVID-19 in dependence of mortality within 5-days after hospitalization.Methods: The COVID-19-related platelet phenotype was examined by analyzing their basal activation state via integrin αIIbβ3 activation using flow cytometry and the proteome by unbiased two-dimensional differential in-gel fluorescence electrophoresis. In total we monitored 98 surviving and 12 non-surviving COVID-19 patients over 5 days of hospital stay and compared them to healthy controls (n = 12).Results: Over the observation period the level of basal αIIbβ3 activation on platelets from non-surviving COVID-19 patients decreased compared to survivors. In line with this finding, proteomic analysis revealed a decrease in the total amount of integrin αIIb (ITGA2B), a subunit of αIIbβ3, in COVID-19 patients compared to healthy controls; the decline was even more pronounced for the non-survivors. Consumption of the fibrin-stabilizing factor coagulation factor XIIIA (F13A1) was higher in platelets from COVID-19 patients and tended to be higher in non-survivors; plasma concentrations of the latter also differed significantly. Depending on COVID-19 disease status and mortality, increased amounts of annexin A5 (ANXA5), eukaryotic initiation factor 4A-I (EIF4A1), and transaldolase (TALDO1) were found in the platelet proteome and also correlated with the nasopharyngeal viral load. Dysregulation of these proteins may play a role for virus replication. ANXA5 has also been identified as an autoantigen of the antiphospholipid syndrome, which is common in COVID-19 patients. Finally, the levels of two different protein disulfide isomerases, P4HB and PDIA6, which support thrombosis, were increased in the platelets of COVID-19 patients.Conclusion: Platelets from COVID-19 patients showed significant changes in the activation phenotype, in the processing of the final coagulation factor F13A1 and the phospholipid-binding protein ANXA5 compared to healthy subjects. Additionally, these results demonstrate specific alterations in platelets during COVID-19, which are significantly linked to fatal outcome.

Identification of 4-hydroxynonenal-modified proteins in human osteoarthritic chondrocytes

The α,β-unsaturated aldehyde 4-hydroxynonenal (HNE) is formed through lipid peroxidation during oxidative stress. As a highly reactive electrophile, it is able to form adducts with various biomolecules, including proteins. These protein modifications could modulate many signaling pathways, as well as cell differentiation and proliferation, and thus could be highly important in the context of the extracellular matrix and degradation of articular cartilage. This study specifically investigated the role of HNE as a bioactive molecule in chondrocytes of osteoarthritis (OA) patients. Chondrocyte extracts of OA and non-OA patients were analyzed for HNE binding using Western blot and bottom-up LC-MS/MS analyses. HNE-modified histones, H2A and H2B, and histone deacetylase were identified using anti-HNE antibodies. Furthermore, peptide sequencing and database searching revealed 95 distinct HNE-modified proteins and their exact modification sites, with 88 protein adducts being unique to OA chondrocytes. HNE-proteins of specific interest included histone H2A, H2B and H4, collagen alpha-3(VI) chain, eukaryotic initiation factor 4A-I, and nucleolar RNA helicase 2. Comparing their MS/MS spectra to those of HNE-modified standard peptides further validated the six HNE-proteins. SignificanceHNE binding to proteins has been shown to result in multiple abnormalities of chondrocyte phenotype and function, suggesting its contribution in OA development. Considering the increased levels of HNE in OA cartilage, this reactive aldehyde could play a role in OA. This work represents a clinically-relevant in vivo study to demonstrate the pathophysiological role of HNE in human OA. Since HNE binding can alter protein conformation and function, it remains highly relevant to study the effects of this modification in OA.

Assessment of Serological Early Biomarker Candidates for Lung Adenocarcinoma by using Multiple Reaction Monitoring-Mass Spectrometry.

Plasma markers that enable diagnosis in the early stage of lung cancer is not discovered. A liquid chromatography multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay for identifying potential early marker proteins for lung adenocarcinoma is developed. LC-MRM-MS assay is used for measuring the level of 35 candidate peptides in plasma from 102 lung adenocarcinoma patients (including n = 50, 16, 24, and 12 in stage I, II, III, and IV, respectively.) and 84healthy controls. Stable isotope labeled standard peptides are synthesized to accurately measure the amount of these proteins. Seven proteins are able to distinguish stage I patients from controls. These proteins are combined in to a protein marker panel which improve the sensitivity to discriminate stage I patients from controls with cross-validated area under the curve = 0.76. Besides, it is found that low expression of eukaryotic initiation factor 4A-I and high expression of lumican show significantly poor prognosis in overall survival (p = 0.012 and 0.0074, respectively), which may be used as prognostic biomarkers for lung cancer. Proteins highlighted here may be used for early detection of lung adenocarcinoma or therapeutics development after validation in a larger cohort.

RNA Enhancement by lncRNA Promotes Translation Through Recruitment of ILF3 and EIF4A1 to the Target Mammalian mRNAs

The previously developed technology RNA enhancement (RNAe) is reported to increase specific gene expression at post-transcriptional level via a long noncoding RNA (lncRNA). The mechanism for SINEB2-dependent enhancement of translation remains not well understood. Here we present the result of experiments with the folded states of IncRNA in doubly deionized water obtained by slowcool method. These IncRNA were used in RNA pull-down assay that yielded six IncRNA-binding proteins potentially involved in RNAe. The direct interactions of IncRNA with interleukin enhancer-binding factor 3 (ILF3) and eukaryotic initiation factor 4A-I (eIF4Al) in vivo and in vitro were confirmed in RNA-binding protein affinity experiment and electrophoretic mobility shift assay (EMSA), respectively. These observations could explain RNAe phenomenon through IncRNA-dependent guiding of ILF3 protein, which, in turn, recruits polysomes or the factors for translation initiation, and attracting eIF4Al proteins accelerating the unwinding of the secondary structure at the 5'-end of mRNA during translation initiation. Therefore, the hypothetical mechanism through which IncRNAs may regulate the translation of a specific mRNA is proposed.

Eukaryotic initiation factor 4AI interacts with NS4A of Dengue virus and plays an antiviral role

Dengue virus (DENV) is a mosquito-borne flavivirus that causes the most prevalent diseases in tropical and subtropical regions. DENV utilizes host factors to facilitate its replication, while host cells intend to restrain virus replication. NS4A of DENV has been implicated to play a crucial role during viral replication. To identify more cellular proteins that are recruited by NS4A, we carried out a tandem affinity purification assay. The mass spectrometry data revealed that human eukaryotic initiation factor 4AI (eIF4AI) was one of potential NS4A-interacting partners. Co-immunoprecipitation data confirmed the interaction between NS4A and eIF4AI, and both the N-terminal ATP-binding domain and C-terminal helicase domain of eIF4AI were involved in their association. Functionally, silencing of eIF4AI by RNAi significantly increased the replication level of DENV1, DENV2 and DENV3. And knockdown of eIF4AI markedly attenuated the phosphorylation of protein kinase regulated by dsRNA-activated (PKR) and eIF2ɑ induced by DENV2 infection. Collectively, these data suggested that a potential role of NS4A in antagonizing host antiviral defense is by recruiting eIF4AI and escaping the translation inhibition mediated by PKR.

Proteomic Analysis of Hepatic Ischemia and Reperfusion Injury in Mice

Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. I/R injury induces serious hepatic dysfunction and failure. In this study, we identified proteins that were differentially expressed between sham and I/R injured livers. Animals were subjected to hepatic ischemia for 1 hr and were sacrificed at 3hr after reperfusion. Serum ALT and AST levels were significantly increased in I/R-operated animals compared to those of sham-operated animals. Ischemic hepatic lobes of I/R-operated animals showed the hepatic lesion with unclear condensation and sinusoidal congestion. Proteins from hepatic tissue were separated using two dimensional gel electrophosresis. Protein spots with a greater than 2.5-fold change in intensity were identified by mass spectrometry. Among these proteins, glutaredoxin-3, peroxiredoxin-3, glyoxalase I, spermidine synthase, dynamin-1-like protein, annexin A4, eukaryotic initiation factor 3, eukaryotic initiation factor 4A-I, 26S proteasome, proteasome alpha 1, and proteasome beta 4 levels were significantly decreased in I/R-operated animals compared to those of sham-operated animals. These proteins are related to protein synthesis, cellular growth and stabilization, anti-oxidant action. Moreover, Western blot analysis confirmed that dynamin-1-like protein levels were decreased in I/R-operated animals. Our results suggest that hepatic I/R induces the hepatic cells damage by regulation of several proteins.

Interactions between eIF4AI and its accessory factors eIF4B and eIF4H

Ribonucleoprotein complexes (RNP) remodeling by DEAD-box proteins is required at all stages of cellular RNA metabolism. These proteins are composed of a core helicase domain lacking sequence specificity; flanking protein sequences or accessory proteins target and affect the core's activity. Here we examined the interaction of eukaryotic initiation factor 4AI (eIF4AI), the founding member of the DEAD-box family, with two accessory factors, eIF4B and eIF4H. We find that eIF4AI forms a stable complex with RNA in the presence of AMPPNP and that eIF4B or eIF4H can add to this complex, also dependent on AMPPNP. For both accessory factors, the minimal stable complex with eIF4AI appears to have 1:1 protein stoichiometry. However, because eIF4B and eIF4H share a common binding site on eIF4AI, their interactions are mutually exclusive. The eIF4AI:eIF4B and eIF4AI:eIF4H complexes have the same RNase resistant footprint as does eIF4AI alone (9-10 nucleotides [nt]). In contrast, in a selective RNA binding experiment, eIF4AI in complex with either eIF4B or eIF4H preferentially bound RNAs much longer than those bound by eIF4AI alone (30-33 versus 17 nt, respectively). The differences between the RNase resistant footprints and the preferred RNA binding site sizes are discussed, and a model is proposed in which eIF4B and eIF4H contribute to RNA affinity of the complex through weak interactions not detectable in structural assays. Our findings mirror and expand on recent biochemical and structural data regarding the interaction of eIF4AI's close relative eIF4AIII with its accessory protein MLN51.

Proteome analysis of human liver carcinoma Huh7 cells harboring hepatitis C virus subgenomic replicon

Chronic infection by hepatitis C virus (HCV) is closely correlated with serious liver diseases. Although considerable progress has been made during recent years, the mechanism of replication and pathogenesis of HCV infection are still elusive. We have applied proteomic techniques in this work to globally analyze the protein expression profiles of a human liver cell lines Huh7 in absence and presence of HCV replication, aiming at elucidating the components of HCV replication and the cellular responses to HCV replication. The protein mixtures of three subcellular fractions from Huh7 and Huh7-HCV were separated by 2-DE under various pH gradients. Differentially expressed spots were identified by MALDI-TOF MS, followed by database searching. A total of 179 comparative proteins were identified unambiguously, including proteins associated with host cytoskeleton, intracellular traffic, oxidative and ER stress, proteasome degradation, translation, apoptosis, proliferation, etc. Host proteins known to interact with HCV proteins, such as HSP27, alpha-actinin, nucleolin and eukaryotic initiation factor 4A-I, were elevated in Huh7-HCV cells. Our study provides the global information of proteomic alteration of Huh7 cells in the presence of HCV replication and the clues for further understanding of the mechanism of HCV replication and pathogenesis.

Comparative proteomic analysis of all-trans-retinoic acid treatment reveals systematic posttranscriptional control mechanisms in acute promyelocytic leukemia

AbstractAll-trans-retinoic acid (ATRA) induces growth inhibition, differentiation, and apoptosis in cancer cells, including acute promyelocytic leukemia (APL). In APL, expression of promyelocytic leukemia protein retinoic acid receptor–α (PML-RARα) fusion protein, owing to the t(15; 17) reciprocal translocation, leads to a block in the promyelocytic stage of differentiation. Here, we studied molecular mechanisms involved in ATRA-induced growth inhibition and myeloid cell differentiation in APL. By employing comprehensive high-throughput proteomic methods of 2-dimensional (2-D) gel electrophoresis and amino acid–coded mass tagging coupled with electrospray ionization (ESI) mass spectrometry, we systematically identified a total of 59 differentially expressed proteins that were consistently modulated in response to ATRA treatment. The data revealed significant down-regulation of eukaryotic initiation and elongation factors, initiation factor 2 (IF2), eukaryotic initiation factor 4AI (eIF4AI), eIF4G, eIF5, eIF6, eukaryotic elongation factor 1A-1 (eEF1A-1), EF-1-δ, eEF1γ, 14-3-3ϵ, and 14-3-3ζ/δ (P< .05). The translational inhibitor DAP5/p97/NAT1 (death-associated protein 5) and PML isoform-1 were found to be up-regulated (P< .05). Additionally, the down-regulation of heterogeneous nuclear ribonucleoproteins (hnRNPs) C1/C2, UP2, K, and F; small nuclear RNPs (snRNPs) D3 and E; nucleoprotein tumor potentiating region (TPR); and protein phosphatase 2A (PP2A) were found (P< .05); these were found to function in pre-mRNA processing, splicing, and export events. Importantly, these proteomic findings were validated by Western blot analysis. Our data in comparison with previous cDNA microarray studies and our reverse transcription–polymerase chain reaction (RT-PCR) experiments demonstrate that broad networks of posttranscriptional suppressive pathways are activated during ATRA-induced growth inhibition processes in APL.

The Human Eukaryotic Initiation Factor 4AI Gene (EIF4A1) Contains Multiple Regulatory Elements That Direct High-Level Reporter Gene Expression in Mammalian Cell Lines

The gene encoding human eukaryotic initiation factor 4A (EIF4A1) is located on chromosome 17p13, 667 bp upstream from the gene encoding the macrophage endosomal protein CD68. The EIF4AI gene contains 10 intervening sequences with the 1397-bp first intron containing a CpG-rich methylation-free island. Sequences capable of enhancing gene expression reside between positions -69 and -371 and positions -504 and -1100 of the EIF4AI 5′ flanking sequence and within introns 1, 2, 3, 7, and 9. In macrophage cell lines, EIF4A1 expression vectors give sustained high-level reporter gene expression to levels 10 times higher than that obtained using the human cytomegalovirus immediate-early gene promoter/enhancer. Sequences of the human EIF4AI gene may find application in the development of new vectors for gene therapy and genetic vaccination.

Nucleotide sequence of human cDNA encoding eukaryotic initiation factor 4AI.

Nucleotide sequence of human cDNA encoding eukaryotic initiation factor 4AI.