ABSTRACT
Eukaryotic translation initiation factors (eIFs) are crucial for initiating protein translation and ensuring the correct assembly of mRNA-ribosomal subunit complexes. In this study, we investigated the effects of deleting six eIFs in the apicomplexan parasite Toxoplasma gondii using the CRISPR-Cas9 system. We determined the subcellular localization of these eIFs using C-terminal endogenous tagging and immunofluorescence analysis. Four eIFs (RH::315150-6HA, RH::286090-6HA, RH::249370-6HA, and RH::211410-6HA) were localized in the cytoplasm, while RH::224235-6HA was localized in the apicoplast. Additionally, RH::272640-6HA was found in both the basal complex and the cytoplasm of T. gondii. Functional characterization of the six RHΔeIFs strains was conducted using plaque assay, cell invasion assay, intracellular growth assay and egress assay in vitro, and virulence assay in mice. Disruption of five eIF genes (RHΔ315150, RHΔ272640, RHΔ249370, RHΔ211410, and RHΔ224235) did not affect the ability of the T. gondii RH strain to invade, replicate, form plaques and egress in vitro, or virulence in Kunming mice (p > 0.05). However, the RHΔ286090 strain showed slightly reduced invasion efficiency and virulence (p < 0.01) compared to the other five RHΔeIFs strains and the wild-type strain. The disruption of the TGGT1_286090 gene significantly impaired the ability of tachyzoites to differentiate into bradyzoites in both type I RH and type II Pru strains. These findings reveal that the eukaryotic translation initiation factor TGGT1_286090 is crucial for T. gondii bradyzoite differentiation and may serve as a potential target for drug development and an attenuated vaccine against T. gondii.
Subject(s)
CRISPR-Cas Systems , Eukaryotic Initiation Factors , Protozoan Proteins , Toxoplasma , Toxoplasma/genetics , Toxoplasma/pathogenicity , Toxoplasma/metabolism , Toxoplasma/growth & development , Animals , Mice , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Virulence/genetics , Toxoplasmosis/parasitology , Toxoplasmosis/genetics , HumansABSTRACT
Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.
Subject(s)
Acetyl-CoA Carboxylase , Adaptor Proteins, Signal Transducing , Cell Cycle Proteins , Cell Survival , Fatty Acids , Glucose , Mechanistic Target of Rapamycin Complex 1 , Animals , Humans , Mice , Acetyl-CoA Carboxylase/metabolism , Acetyl-CoA Carboxylase/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Line, Tumor , Eukaryotic Initiation Factors/metabolism , Eukaryotic Initiation Factors/genetics , Fatty Acids/metabolism , Glucose/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 1/genetics , NADP/metabolism , Oxidative Stress , Phosphoproteins/metabolism , Phosphoproteins/genetics , Protein BiosynthesisABSTRACT
The eukaryotic translation initiation factor eIF5B is a bacterial IF2 ortholog that plays an important role in ribosome joining and stabilization of the initiator tRNA on the AUG start codon during the initiation of translation. We identified the fluorophenyl oxazole derivative 2,2-dibromo-1-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)ethanone quinolinol as an inhibitor of fungal protein synthesis using an in vitro translation assay in a fungal system. Mutants resistant to this compound were isolated in Saccharomyces cerevisiae and were demonstrated to contain amino acid substitutions in eIF5B that conferred the resistance. These results suggest that eIF5B is a target of potential antifungal compound and that mutation of eIF5B can confer resistance. Subsequent identification of 16 other mutants revealed that primary mutations clustered mainly on domain 2 of eIF5B and secondarily mainly on domain 4. Domain 2 has been implicated in the interaction with the small ribosomal subunit during initiation of translation. The tested translation inhibitor could act by weakening the functional contact between eIF5B and the ribosome complex. This data provides the basis for the development of a new family of antifungals.
Subject(s)
Antifungal Agents , Eukaryotic Initiation Factors , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/drug effects , Eukaryotic Initiation Factors/metabolism , Eukaryotic Initiation Factors/genetics , Antifungal Agents/pharmacology , Mutation , Protein Biosynthesis/drug effects , Drug Resistance, Fungal/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Protein Synthesis Inhibitors/pharmacology , Amino Acid SubstitutionABSTRACT
The phosphorylation of eukaryotic translational initiation factors has been shown to play a significant role in controlling the synthesis of protein. Viral infection, environmental stress, and growth circumstances cause phosphorylation or dephosphorylation of plant initiation factors. Our findings indicate that casein kinase 2 can phosphorylate recombinant wheat eIFiso4E and eIFiso4G generated from E. coli in vitro. For wheat eIFiso4E, Ser-207 was found to be the in vitro phosphorylation site. eIFiso4E lacks an amino acid that can be phosphorylated at the position corresponding to Ser-209, the phosphorylation site in mammalian eIF4E, yet phosphorylation of eIFiso4E has effects on VPg binding affinity that are similar to those of phosphorylation of mammalian eIF4E. The addition of VPg and phosphorylated eIFiso4F to depleted wheat germ extract (WGE) leads to enhancement of translation of both uncapped and capped viral mRNA. The addition of PABP together with eIFiso4Fp and eIF4B to depleted WGE increases both uncapped and capped mRNA translation. However, it exhibits a translational advantage specifically for uncapped mRNA, implying that the phosphorylation of eIFiso4F hinders cap binding while promoting VPg binding, thereby facilitating uncapped translation. These findings indicate TEV virus mediates VPg-dependent translation by engaging a mechanism entailing phosphorylated eIFiso4Fp and PABP. To elucidate the molecular mechanisms underlying these observed effects, we studied the impact of PABP and/or eIF4B on the binding of VPg with eIFiso4Fp. The inclusion of PABP and eIF4B with eIFiso4Fp resulted in about 2-fold increase in affinity for VPg (Kd = 24 ± 1.7 nM), as compared to the affinity of eIFiso4Fp alone (Kd = 41.0 ± 3.1 nM). The interactions between VPg and eIFiso4Fp were determined to be both enthalpically and entropically favorable, with the enthalpic contribution accounting for 76-97% of the ΔG at 25°C, indicating a substantial role of hydrogen bonding in enhancing the stability of the complex. The binding of PABP to eIFiso4Fp·4B resulted in a conformational alteration, leading to a significant enhancement in the binding affinity to VPg. These observations suggest PABP enhances the affinity between eIFiso4Fp and VPg, leading to an overall conformational change that provides a stable platform for efficient viral translation.
Subject(s)
Eukaryotic Initiation Factors , Poly(A)-Binding Proteins , Potyvirus , Protein Binding , Protein Biosynthesis , Triticum , Phosphorylation , Potyvirus/metabolism , Potyvirus/genetics , Triticum/virology , Triticum/metabolism , Triticum/genetics , Eukaryotic Initiation Factors/metabolism , Eukaryotic Initiation Factors/genetics , Poly(A)-Binding Proteins/metabolism , Plant Proteins/metabolism , Plant Proteins/genetics , Viral Proteins/metabolism , Viral Proteins/genetics , Casein Kinase II/metabolism , Casein Kinase II/geneticsABSTRACT
Argonaute (AGO) proteins are evolutionarily conserved RNA-binding proteins that control gene expression through the small RNAs they interact with. Whether AGOs have regulatory roles independent of RNAs, however, is unknown. Here, we show that AGO1 controls cell fate decisions through facilitating protein folding. We found that in mouse embryonic stem cells (mESCs), while AGO2 facilitates differentiation via the microRNA (miRNA) pathway, AGO1 controls stemness independently of its binding to small RNAs. We determined that AGO1 specifically interacts with HOP, a co-chaperone for the HSP70 and HSP90 chaperones, and enhances the folding of a set of HOP client proteins with intrinsically disordered regions. This AGO1-mediated facilitation of protein folding is important for maintaining stemness in mESCs. Our results demonstrate divergent functions between AGO1 and AGO2 in controlling cellular states and identify an RNA-independent function of AGO1 in controlling gene expression and cell fate decisions.
Subject(s)
Argonaute Proteins , Cell Differentiation , Mouse Embryonic Stem Cells , Protein Folding , Animals , Argonaute Proteins/metabolism , Argonaute Proteins/genetics , Mice , Mouse Embryonic Stem Cells/metabolism , Mouse Embryonic Stem Cells/cytology , MicroRNAs/genetics , MicroRNAs/metabolism , Eukaryotic Initiation Factors/metabolism , Eukaryotic Initiation Factors/genetics , HSP90 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Cell LineageABSTRACT
As professional secretory cells, ß-cells require adaptable mRNA translation to facilitate a rapid synthesis of proteins, including insulin, in response to changing metabolic cues. Specialized mRNA translation programs are essential drivers of cellular development and differentiation. However, in the pancreatic ß-cell, the majority of factors identified to promote growth and development function primarily at the level of transcription. Therefore, despite its importance, the regulatory role of mRNA translation in the formation and maintenance of functional ß-cells is not well defined. In this study, we have identified a translational regulatory mechanism mediated by the specialized mRNA translation factor eukaryotic initiation factor 5A (eIF5A), which facilitates the maintenance of ß-cell identity and function. The mRNA translation function of eIF5A is only active when it is posttranslationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS). We have discovered that the absence of ß-cell DHPS in mice reduces the synthesis of proteins critical to ß-cell identity and function at the stage of ß-cell maturation, leading to a rapid and reproducible onset of diabetes. Therefore, our work has revealed a gatekeeper of specialized mRNA translation that permits the ß-cell, a metabolically responsive secretory cell, to maintain the integrity of protein synthesis necessary during times of induced or increased demand.
Subject(s)
Insulin-Secreting Cells , Peptide Initiation Factors , Animals , Mice , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolism , Protein Processing, Post-Translational , Insulin-Secreting Cells/metabolism , Protein Biosynthesis , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolismABSTRACT
Cells activate stress response pathways to survive adverse conditions. Such responses involve the inhibition of global cap-dependent translation. This inhibition is a block that essential transcripts must escape via alternative methods of translation initiation, e.g., an internal ribosome entry site (IRES). IRESs have distinct structures and generally require a limited repertoire of translation factors. Cellular IRESs have been identified in many critical cellular stress response transcripts. We previously identified cellular IRESs in the murine insulin receptor (Insr) and insulin-like growth factor 1 receptor (Igf1r) transcripts and demonstrated their resistance to eukaryotic initiation factor 4F (eIF4F) inhibition. Here, we find that eIF5B preferentially promotes Insr, Igf1r, and hepatitis C virus IRES activity through a non-canonical mechanism that requires its highly charged and disordered N terminus. We find that the N-terminal region of eIF5B can drive cytoplasmic granule formation. This eIF5B granule is triggered by cellular stress and is sufficient to specifically promote IRES activity.
Subject(s)
Hepatitis C , Internal Ribosome Entry Sites , Animals , Mice , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Eukaryotic Initiation Factor-4F/metabolism , Protein BiosynthesisABSTRACT
Members of the eukaryotic translation initiation complex are co-opted in viral infection, leading to susceptibility in many crop species, including stone fruit trees (Prunus spp.). Therefore, modification of one of those eukaryotic translation initiation factors or changes in their gene expression may result in resistance. We searched the crop and wild Prunus germplasm from the Armeniaca and Amygdalus taxonomic sections for allelic variants in the eIF4E and eIFiso4E genes, to identify alleles potentially linked to resistance to Plum pox virus (PPV). Over one thousand stone fruit accessions (1397) were screened for variation in eIF4E and eIFiso4E transcript sequences which are in single copy within the diploid Prunus genome. We identified new alleles for both genes differing from haplotypes associated with PPV susceptible accessions. Overall, analyses showed that eIFiso4E is genetically more constrained since it displayed less polymorphism than eIF4E. We also demonstrated more variations at both loci in the related wild species than in crop species. As the eIFiso4E translation initiation factor was identified as indispensable for PPV infection, a selection of ten different eIFiso4E haplotypes along 13 accessions were tested by infection with PPV and eight of them displayed a range of reduced susceptibility to resistance, indicating new potential sources of resistance to sharka.
Subject(s)
Eukaryotic Initiation Factor-4E , Prunus , Alleles , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factors/genetics , Cytoplasm , Prunus/geneticsABSTRACT
During translation, a stop codon on the mRNA signals the ribosomes to terminate the process. In certain mRNAs, the termination fails due to the recoding of the canonical stop codon, and ribosomes continue translation to generate C-terminally extended protein. This process, termed stop codon readthrough (SCR), regulates several cellular functions. SCR is driven by elements/factors that act immediately downstream of the stop codon. Here, we have analysed the process of SCR using a simple mathematical model to investigate how the kinetics of translating ribosomes influences the efficiency of SCR. Surprisingly, the analysis revealed that the rate of translation inversely regulates the efficiency of SCR. We tested this prediction experimentally in mammalian AGO1 and MTCH2 mRNAs. Reduction in translation either globally by harringtonine or locally by rare codons caused an increase in the efficiency of SCR. Thus, our study has revealed a hitherto unknown mode of regulation of SCR.
Subject(s)
Codon, Terminator , Protein Biosynthesis , RNA, Messenger , Ribosomes , Codon, Terminator/genetics , Codon, Terminator/metabolism , Ribosomes/genetics , Ribosomes/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Humans , HEK293 Cells , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolismABSTRACT
Translation initiation in eukaryotes is an early step in protein synthesis, requiring multiple factors to recruit the ribosomal small subunit to the mRNA 5' untranslated region. One such protein factor is the eukaryotic translation initiation factor 4B (eIF4B), which increases the activity of the eIF4A RNA helicase, and is linked to cell survival and proliferation. We report here the protein backbone chemical shift assignments corresponding to the C-terminal 279 residues of human eIF4B. Analysis of the chemical shift values identifies one main helical region in the area previously linked to RNA binding, and confirms that the overall C-terminal region is intrinsically disordered.
Subject(s)
Eukaryotic Initiation Factors , Peptide Initiation Factors , Humans , Nuclear Magnetic Resonance, Biomolecular , Eukaryotic Initiation Factors/chemistry , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The human eukaryotic translation initiation factor 5A (EIF5A) family consists of three members, namely EIF5A1, EIF5A2, and EIF5AL1. Recent studies have shown that the expression of EIF5As is related to many human diseases, such as diabetes, viral infection, central nervous system injury, and cancer. Among them, EIF5A1 plays different functions in various cancers, possibly as a tumor-suppressor or oncogene, while EIF5A2 promotes the occurrence and development of cancer. Yet, the biological function of EIF5AL1 is not being studied so far. Interestingly, although there are only three amino acid (at residues 36, 45, and 109) differences between EIF5A1 and EIF5AL1, we demonstrate that only EIF5A1 can be hypusinated while EIF5AL1 cannot, and EIF5AL1 has a tumor-suppressor-like function by inhibiting cell proliferation and migration. We also show that EIF5AL1 protein turnover is mediated through the proteasomal pathway, and EIF5AL1 protein turnover is much faster than that of EIF5A1, which may explain their differential protein expression level in cells. By engineering single and double mutations on these three amino acids, we pinpoint which of these amino acids are critical for hypusination and protein stability. The data of this work should fill in the gaps in EIF5As research and pave the way for future studies on EIF5AL1.
Subject(s)
Lysine , Neoplasms , Humans , Amino Acids , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Lysine/metabolism , Neoplasms/metabolism , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolism , Protein Stability , Eukaryotic Translation Initiation Factor 5AABSTRACT
Aberrant splicing is typically attributed to splice-factor (SF) mutation and contributes to malignancies including acute myeloid leukemia (AML). Here, we discovered a mutation-independent means to extensively reprogram alternative splicing (AS). We showed that the dysregulated expression of eukaryotic translation initiation factor eIF4E elevated selective splice-factor production, thereby impacting multiple spliceosome complexes, including factors mutated in AML such as SF3B1 and U2AF1. These changes generated a splicing landscape that predominantly supported altered splice-site selection for ~800 transcripts in cell lines and ~4,600 transcripts in specimens from high-eIF4E AML patients otherwise harboring no known SF mutations. Nuclear RNA immunoprecipitations, export assays, polysome analyses, and mutational studies together revealed that eIF4E primarily increased SF production via its nuclear RNA export activity. By contrast, eIF4E dysregulation did not induce known SF mutations or alter spliceosome number. eIF4E interacted with the spliceosome and some pre-mRNAs, suggesting its direct involvement in specific splicing events. eIF4E induced simultaneous effects on numerous SF proteins, resulting in a much larger range of splicing alterations than in the case of mutation or dysregulation of individual SFs and providing a novel paradigm for splicing control and dysregulation.
Subject(s)
Alternative Splicing , Leukemia, Myeloid, Acute , Humans , RNA Splicing Factors/metabolism , Eukaryotic Initiation Factor-4E/metabolism , RNA Splicing , Eukaryotic Initiation Factors/genetics , Leukemia, Myeloid, Acute/genetics , MutationABSTRACT
Argonaute (AGO) proteins, through their key role in the regulation of gene expression, participate in many biological processes, including cell differentiation, proliferation, death and DNA repair. Accurate regulation of gene expression appears to be important for the proper development of complex neural circuits. Loss of AGO proteins is known to lead to early embryonic mortality in mice with various malformations, including anomalies of the central nervous system. Single-nucleotide polymorphisms (SNPs) of AGO genes can lead to deregulation of the processes in which AGO proteins are involved. The contribution of different SNPs in depression has been extensively studied. However, there are hardly any studies on the contribution of AGO genes. The aim of our research was to assess the relationship between the occurrence of depression and the presence of SNPs in genes AGO1 (rs636882) and AGO2 (rs4961280; rs2292779; rs2977490) in a Polish population. One hundred and one subjects in the study group were diagnosed with recurrent depressive disorder by a psychiatrist. The control group comprised 117 healthy subjects. Study participants performed the HDRS (Hamilton Depression Scale) test to confirm or exclude depression and assess severity. The frequency of polymorphic variants of genes AGO1 (rs636882) and AGO2 (rs4961280; rs2292779; rs2977490) was determined using TaqMan SNP genotyping assays and the TaqMan universal PCR master mix, no AmpErase UNG. The rs4961280/AGO2 polymorphism was associated with a decrease in depression occurrence in the codominant (OR = 0.51, p = 0.034), dominant (OR = 0.49, p = 0.01), and overdominant (OR = 0.58, p = 0.049) models. Based on the obtained results, we found that the studied patients demonstrated a lower risk of depression with the presence of the polymorphic variant of the rs4961280/AGO2 gene-genotype C/A and C/A-A/A.
Subject(s)
Argonaute Proteins/genetics , Depression , Eukaryotic Initiation Factors/genetics , Alleles , Animals , Case-Control Studies , Depression/genetics , Humans , Mice , Poland , Polymorphism, Single NucleotideABSTRACT
Eukaryotic translation initiation factor 4B (eIF4B) plays an important role in mRNA translation initiation, cell survival and proliferation in vitro, but the in vivo function is poorly understood. In this study, via various experimental techniques such as hematoxylin-eosin (HE) staining, flow cytometry, Western blotting, and immunohistochemistry, we investigated the role of eIF4B in mouse embryo development using an eIF4B knockout (KO) mouse model and explored the mechanism. We found that the livers, but not lungs, brain, stomach, or pancreas, derived from eIF4B KO mouse embryos displayed severe pathological changes characterized by enhanced apoptosis and necrosis. Accordingly, high expression of cleaved-caspase 3, and excessive activation of mTOR signaling as evidenced by increased expression and phosphorylation of p70S6K and enhanced phosphorylation of 4EBP1, were observed in mouse embryonic fibroblasts and fetal livers from eIF4B KO mice. These results uncover a critical role of eIF4B in mouse embryo development and provide important insights into the biological functions of eIF4B in vivo.
Subject(s)
Fibroblasts , Ribosomal Protein S6 Kinases, 70-kDa , Animals , Apoptosis/genetics , Caspase 3 , Eosine Yellowish-(YS) , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Hematoxylin , Liver/metabolism , Mice , Ribosomal Protein S6 Kinases, 70-kDa/genetics , TOR Serine-Threonine KinasesABSTRACT
BACKGROUND: Serine and arginine-rich splicing factor 9 (SRSF9) has been linked to the occurrence and progression of various cancers; however, its effects and mechanism of action hepatocellular carcinoma (HCC) have not been reported. In this study, we used a bioinformatics approach and in vitro assays to evaluate the expression of SRSF9 in HCC, its prognostic value, and its underlying regulatory mechanisms, including analyses of related pathways and the role of methylation. METHODS: Transcriptomic and DNA methylation data for 357 HCC cases and 50 paratumor tissues in The Cancer Genome Atlas database were obtained. Additionally, protein expression data for cell lines and tissue samples were obtained from the Human Protein Atlas. The CMap databased was used to predict candidate drugs targeting SRSF9. Various cell lines were used for in vitro validation. RESULTS: SRSF9 expression was significantly elevated in HCC and was negatively regulated by its methylation site cg06116271. The low expression of SRSF9 and hypermethylation of cg06116271 were both associated with a longer overall survival time. A correlation analysis revealed ten genes that were co-expressed with SRSF9; levels of CDK4, RAN, DENR, RNF34, and ANAPC5 were positively correlated and levels of RBP4, APOC1, MASP2, HP, and HPX were negatively correlated with SRSF9 expression. The knockdown of SRSF9 in vitro inhibited the proliferation and migration of HCC cells and significantly reduced the expression of proteins in the Wnt signaling pathway (DVL2 and ß-catenin) and cell cycle pathway (Cyclin D and Cyclin E). A CMap analysis identified two drugs, camptothecin and apigenin, able to target and inhibit the expression of SRSF9. CONCLUSIONS: This study expands our understanding of the molecular biological functions of SRSF9 and cg06116271 and provides candidate diagnostic and therapeutic targets for HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Serine-Arginine Splicing Factors , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/pathology , Carrier Proteins , Cell Line, Tumor , Cell Proliferation , DNA Methylation , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Factor IX/genetics , Factor IX/metabolism , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/diagnosis , Liver Neoplasms/pathology , Mannose-Binding Protein-Associated Serine Proteases/genetics , Mannose-Binding Protein-Associated Serine Proteases/metabolism , Prognosis , RNA Splicing Factors/genetics , Retinol-Binding Proteins, Plasma , Serine/genetics , Serine/metabolism , Serine-Arginine Splicing Factors/geneticsABSTRACT
Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids in the liver. Given the high prevalence of NAFLD, its evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) is of global concern. Therapies for managing NASH-driven HCC can benefit from targeting factors that play a continuous role in NAFLD evolution to HCC. Recent work has shown that postprandial liver translation exacerbates lipid accumulation through the activity of a translation factor, eukaryotic initiation factor 6 (eIF6). Here, we test the effect of eIF6 inhibition on the progression of HCC. Mice heterozygous for eIF6 express half the level of eIF6 compared to wt mice and are resistant to the formation of HCC nodules upon exposure to a high fat/high sugar diet combined with liver damage. Histology showed that nodules in eIF6 het mice were smaller with reduced proliferation compared to wt nodules. By using an in vitro model of human HCC, we confirm that eIF6 depletion reduces the growth of HCC spheroids. We also tested three pharmacological inhibitors of eIF6 activity-eIFsixty-1, eIFsixty-4, and eIFsixty-6-and all three reduced eIF6 binding to 60S ribosomes and limited the growth of HCC spheroids. Thus, inhibition of eIF6 activity is feasible and limits HCC formation.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Non-alcoholic Fatty Liver Disease , Animals , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Eukaryotic Initiation Factors/antagonists & inhibitors , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Mice , Non-alcoholic Fatty Liver Disease/drug therapy , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/metabolism , Peptide Initiation Factors/antagonists & inhibitors , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolismABSTRACT
Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamic characterization of their ensembles remain challenging, both in isolation and when they form dynamic "fuzzy" complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Förster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a non-uniform segmental flexibility around six different labeling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on nanosecond-to-microsecond timescales. Upon hyperphosphorylation, which induces folding of â¼40 residues in 4E-BP2, the quenching rates decreased at most labeling sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs significantly increased upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step toward a mechanistic understanding of this important IDP via integrative modeling.
Subject(s)
Eukaryotic Initiation Factor-4E , Intrinsically Disordered Proteins , Eukaryotic Initiation Factor-4E/chemistry , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factors/chemistry , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Intrinsically Disordered Proteins/chemistry , Phosphorylation , Protein BindingABSTRACT
CD8+ T cell proliferation and differentiation into effector and memory states are high-energy processes associated with changes in cellular metabolism. CD28-mediated costimulation of T cells activates the PI3K/AKT/mammalian target of rapamycin signaling pathway and induces eukaryotic translation initiation factor 4E-dependent translation through the derepression by 4E-BP1 and 4E-BP2. In this study, we demonstrate that 4E-BP1/2 proteins are required for optimum proliferation of mouse CD8+ T cells and the development of an antiviral effector function. We show that translation of genes encoding mitochondrial biogenesis is impaired in T cells derived from 4E-BP1/2-deficient mice. Our findings demonstrate an unanticipated role for 4E-BPs in regulating a metabolic program that is required for cell growth and biosynthesis during the early stages of CD8+ T cell expansion.
Subject(s)
Eukaryotic Initiation Factors , Phosphoproteins , Animals , CD8-Positive T-Lymphocytes/metabolism , Cell Cycle Proteins/metabolism , Cell Proliferation , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Mammals/genetics , Mice , Organelle Biogenesis , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/metabolism , Phosphorylation , Protein BiosynthesisABSTRACT
Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.
Subject(s)
Eukaryotic Initiation Factor-1 , Eukaryotic Initiation Factors , RNA, Transfer, Met , Ribosome Subunits , Cryoelectron Microscopy , Eukaryotic Initiation Factor-1/metabolism , Eukaryotic Initiation Factors/genetics , Humans , RNA, Transfer, Met/genetics , RNA, Transfer, Met/metabolism , Ribosome Subunits/chemistry , Ribosome Subunits/metabolism , Single Molecule ImagingABSTRACT
In tumourigenesis, oncogenes or dysregulated tumour suppressor genes alter the canonical translation machinery leading to a reprogramming of the translatome that, in turn, promotes the translation of selected mRNAs encoding proteins involved in proliferation and metastasis. It is therefore unsurprising that abnormal expression levels and activities of eukaryotic initiation factors (eIFs), elongation factors (eEFs) or termination factors (eRFs) are associated with poor outcome for patients with a wide range of cancers. In this review we discuss how RNA binding proteins (RBPs) within the canonical translation factor machinery are dysregulated in cancers and how targeting such proteins is leading to new therapeutic avenues.