RESUMO
Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.
Assuntos
Fator de Iniciação 4F em Eucariotos , Fator de Iniciação Eucariótico 4G , Resposta ao Choque Térmico , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , RNA Mensageiro , Ribonucleoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Resposta ao Choque Térmico/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Regulação Fúngica da Expressão Gênica , Ligação Proteica , RNA Fúngico/metabolismo , RNA Fúngico/genéticaRESUMO
In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes, such as GCN4 in yeast. Complementing the ISR is the target of rapamycin (TOR) pathway, which regulates eIF4E function. Here, we probe translational control in the absence of eIF4E in Saccharomyces cerevisiae. Intriguingly, we find that loss of eIF4E leads to de-repression of GCN4 translation. In addition, we find that de-repression of GCN4 translation is accompanied by neither eIF2α phosphorylation nor reduction in initiator ternary complex (TC). Our data suggest that when eIF4E levels are depleted, GCN4 translation is de-repressed via a unique mechanism that may involve faster scanning by the small ribosome subunit due to increased local concentration of eIF4A. Overall, our findings suggest that relative levels of eIF4F components are key to ribosome dynamics and may play important roles in translational control of gene expression.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Estresse Fisiológico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fosforilação , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , Biossíntese de Proteínas , Regulação Fúngica da Expressão Gênica , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Transdução de Sinais , Ribossomos/metabolismo , Ribossomos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genéticaRESUMO
The canonical eukaryotic initiation factor 4F (eIF4F) complex, composed of eIF4G1, eIF4A1, and the cap-binding protein eIF4E, plays a crucial role in cap-dependent translation initiation in eukaryotic cells. An alternative cap-independent initiation can occur, involving only eIF4G1 and eIF4A1 through internal ribosome entry sites (IRESs). This mechanism is considered complementary to cap-dependent initiation, particularly in tumors under stress conditions. However, the selection and molecular mechanism of specific translation initiation remains poorly understood in human cancers. Thus, we analyzed gene copy number variations (CNVs) in TCGA tumor samples and found frequent amplification of genes involved in translation initiation. Copy number gains in EIF4G1 and EIF3E frequently co-occur across human cancers. Additionally, EIF4G1 expression strongly correlates with genes from cancer cell survival pathways including cell cycle and lipogenesis, in tumors with EIF4G1 amplification or duplication. Furthermore, we revealed that eIF4G1 and eIF4A1 protein levels strongly co-regulate with ribosomal subunits, eIF2, and eIF3 complexes, while eIF4E co-regulates with 4E-BP1, ubiquitination, and ESCRT proteins. Utilizing Alphafold predictions, we modeled the eIF4F structure with and without eIF4E binding. For cap-dependent initiation, our modeling reveals extensive interactions between the N-terminal eIF4E-binding domain of eIF4G1 and eIF4E. Furthermore, the eIF4G1 HEAT-2 domain positions eIF4E near the eIF4A1 N-terminal domain (NTD), resulting in the collaborative enclosure of the RNA binding cavity within eIF4A1. In contrast, during cap-independent initiation, the HEAT-2 domain directly binds the eIF4A1-NTD, leading to a stronger interaction between eIF4G1 and eIF4A1, thus closing the mRNA binding cavity without the involvement of eIF4E.
Assuntos
Fator de Iniciação 4F em Eucariotos , Neoplasias , Humanos , Fator de Iniciação 4F em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/genética , Variações do Número de Cópias de DNA , Fator de Iniciação 3 em Eucariotos , Neoplasias/genéticaRESUMO
Eukaryotic translation initiation involves recruitment of the 43S pre-initiation complex to the 5' end of mRNA by the cap-binding complex eIF4F, forming the 48S translation initiation complex (48S), which then scans along the mRNA until the start codon is recognized. We have previously shown that eIF4F binds near the mRNA exit channel of the 43S, leaving open the question of how mRNA secondary structure is removed as it enters the mRNA channel on the other side of the 40S subunit. Here we report the structure of a human 48S that shows that, in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound at the mRNA entry site, which could unwind RNA secondary structures as they enter the 48S. The structure also reveals conserved interactions between eIF4F and the 43S, probaby explaining how eIF4F can promote mRNA recruitment in all eukaryotes.
Assuntos
Fator de Iniciação 4F em Eucariotos , Iniciação Traducional da Cadeia Peptídica , Humanos , Fator de Iniciação 4F em Eucariotos/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , RNA Mensageiro/metabolismo , Códon de Iniciação/metabolismo , Ribossomos/metabolismo , DNA Helicases/metabolismo , Biossíntese de Proteínas , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismoRESUMO
The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and latent cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5'-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogeneous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence. IMPORTANCE: Toxoplasma gondii is an opportunistic pathogen important to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation is required. Here we show that the mRNA cap-binding protein, eIF4E1, regulates the encystation process. Encysted parasites reduce eIF4E1 levels, and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drives Toxoplasma encystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.