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 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
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
Ribosome recruitment to eukaryotic mRNAs is generally thought to occur by a scanning mechanism, whereby the 40S ribosomal subunit binds in the vicinity of the 5'cap structure of the mRNA and scans until an AUG codon is encountered in an appropriate sequence context. Study of the picornaviruses allowed the characterization of an alternative mechanism of translation initiation. Picornaviruses can initiate translation via an internal ribosome entry segment (IRES), an RNA structure that directly recruits the 40S ribosomal subunits in a cap and 5' end independent fashion. Since its discovery, the notion of IRESs has extended to a number of different virus families and cellular RNAs. This review summarizes features of both cap-dependent and IRES-dependent mechanisms of translation initiation and discusses the role of cis-acting elements, which include the 5'cap, the 5'-untranslated region (UTR) and the poly(A) tail as well as the possible roles of IRESs as part of a cellular stress response mechanism and in the virus replication cycle.
Subject(s)
Humans , Animals , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Eukaryotic Cells/cytology , Eukaryotic Cells/physiology , Eukaryotic Cells/virology , Eukaryotic Initiation Factors/analysis , Eukaryotic Initiation Factors/biosynthesis , Eukaryotic Initiation Factors/genetics , Proteins , RNA, Ribosomal/analysis , RNA, Ribosomal/biosynthesis , RNA, Ribosomal/chemical synthesisABSTRACT
The presence of antisense transcripts has been described in a wide variety of eucaryotic organisms, with a diversity of implied biological functions in development, control of cell cycles, hormonal responses, control of proliferation, structure, viral replication, and others. Reports have suggested that this type of control may operate at different levels of genic expression, whether in transcription, maturation, transport, stability, or translation of a given trascript. It is evident that the expression of antisense RNA has true importance in Eukaryotes, as already established in prokaryotic organisms. The objective of the present review is to present the advances in the mechanics of regulation, and roles already established for the expressions of antisense RNA in eukaryotic organisms.
La presencia de transcritos antisentidos ha sido descrita en una amplia variedad de organismos eucariontes, implicados en una diversidad de funciones biológicas como el desarrollo, el control del ciclo celular, respuesta hormonal, control de la proliferación, estructura, replicación viral, etc. Los informes indican que este control puede darse a diversos niveles de la expresión génica, ya sea en la transcripción, maduración, transporte, estabilidad y traducción de un determinado transcrito. Es evidente que la expresión del ARN antisentido tiene una real importancia en eucariontes, como ya ha sido establecido en organismos procariontes. Esta revisión tiene como objetivo mostrar los avances en los mecanismos de regulación y roles ya establecidos de la expresión de ARN antisentidos en organismos eucariontes.