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.

Endogenous and exogenous chemical agents are known to compromise the integrity of RNA and cause ribosome stalling and collisions. Recent studies have shown that collided ribosomes serve as sensors for multiple processes, including ribosome quality control (RQC) and the integrated stress response (ISR). Since RQC and the ISR have distinct downstream consequences, it is of great importance that organisms activate the appropriate process. We previously showed that RQC is robustly activated in response to collisions and suppresses the ISR activation. However, the molecular mechanics behind this apparent competition were not immediately clear. Here we show that Hel2 does not physically compete with factors of the ISR, but instead its ribosomal-protein ubiquitination activity, and downstream resolution of collided ribosomes, is responsible for suppressing the ISR. Introducing a mutation in the RING domain of Hel2-which inhibits its ubiquitination activity and downstream RQC but imparts higher affinity of the factor for collided ribosomes-resulted in increased activation of the ISR upon MMS-induced alkylation stress. Similarly, mutating Hel2\'s lysine targets in uS10, which is responsible for RQC activation, resulted in increased Gcn4 target induction. Remarkably, the entire process of RQC appears to be limited by the action of Hel2, as the overexpression of this one factor dramatically suppressed the activation of the ISR. Collectively, our data suggest that cells evolved Hel2 to bind collided ribosomes with a relatively high affinity but kept its concentration relatively low, ensuring that it gets exhausted under stress conditions that cannot be resolved by quality control processes.

We have shown that multiple tRNA synthetase inhibitors can increase lifespan in both the nematode C. elegans and the budding yeast S. cerevisiae by acting through the conserved transcription factor Gcn4 (yeast)/ATF-4 (worms). To further understand the biology downstream from this conserved transcription factor in the yeast model system, we looked at two different yeast models known to have upregulated Gcn4 and GCN4-dependent increased replicative lifespan. These two models were rpl31aΔ yeast and yeast treated with the tRNA synthetase inhibitor borrelidin. We used both proteomic and RNAseq analysis of a block experimental design that included both of these models to identify GCN4-dependent changes in these two long-lived strains of yeast. Proteomic analysis of these yeast indicate that the long-lived yeast have increased abundances of proteins involved in amino acid biosynthesis. The RNAseq of these same yeast uncovered further regulation of protein degradation, identifying the differential expression of genes associated with autophagy and the ubiquitin-proteasome system (UPS). The data presented here further underscore the important role that GCN4 plays in the maintenance of protein homeostasis, which itself is an important hallmark of aging. In particular, the changes in autophagy and UPS-related gene expression that we have observed could also have wide-ranging implications for the understanding and treatment of diseases of aging that are associated with protein aggregation.

BACKGROUND: Edible mushrooms are delicious in flavour and rich in high-quality protein and amino acids required by humans. A transcription factor, general control nonderepressible 4 (GCN4), can regulate the expression of genes involved in amino acid metabolism in yeast and mammals. A previous study revealed that GCN4 plays a pivotal role in nitrogen utilization and growth in Ganoderma lucidum. However, its regulation is nearly unknown in mushrooms.
RESULTS: In this study, we found that the amino acid contents reached 120.51 mg per gram of mycelia in the WT strain under 60 mM asparagine (Asn) conditions, but decreased by 62.96% under 3 mM Asn conditions. Second, silencing of gcn4 resulted in a 54.2% decrease in amino acid contents under 60 mM Asn, especially for the essential and monosodium glutamate-like flavour amino acids. However, these effects were more pronounced under 3 mM Asn. Third, silencing of gcn4 markedly inhibited the expression of amino acid biosynthesis and transport genes. In addition, GCN4 enhanced the tricarboxylic acid cycle (TCA) and glycolytic pathway and inhibited the activity of target of rapamycin complex 1 (TORC1), thus being beneficial for maintaining amino acid homeostasis.
CONCLUSIONS: This study confirmed that GCN4 contributes to maintaining the amino acid contents in mushrooms under low concentrations of nitrogen. In conclusion, our study provides a research basis for GCN4 to regulate amino acid synthesis and improve the nutrient contents of edible mushrooms.

We have recently shown that multiple tRNA synthetase inhibitors can greatly increase lifespan in multiple models by acting through the conserved transcription factor ATF4. Here, we show that these compounds, and several others of the same class, can greatly upregulate mammalian ATF4 in cells in vitro, in a dose dependent manner. Further, RNASeq analysis of these cells pointed toward changes in protein turnover. In subsequent experiments here we show that multiple tRNA synthetase inhibitors can greatly upregulate activity of the ubiquitin proteasome system (UPS) in cells in an ATF4-dependent manner. The UPS plays an important role in the turnover of many damaged or dysfunctional proteins in an organism. Increasing UPS activity has been shown to enhance the survival of Huntington\'s disease cell models, but there are few known pharmacological enhancers of the UPS. Additionally, we see separate ATF4 dependent upregulation of macroautophagy upon treatment with tRNA synthetase inhibitors. Protein degradation is an essential cellular process linked to many important human diseases of aging such as Alzheimer\'s disease and Huntington\'s disease. These drugs\' ability to enhance proteostasis more broadly could have wide-ranging implications in the treatment of important age-related neurodegenerative diseases.

Candida albicans is a commensal of human mucosae, but also one of the most common fungal pathogens of humans. Systemic infections caused by this fungus, mostly affecting immunocompromised patients, are associated to fatality rates as high as 50% despite the available treatments. In order to improve this situation, it is necessary to fully understand how C. albicans is able to cause disease and how it copes with the host defenses. Our previous studies have revealed the importance of the C. albicans gene MBF1 in virulence and ability to colonize internal organs of mammalian and insect hosts. MBF1 encodes a putative transcriptional regulator, and as such it likely has an impact in the regulation of C. albicans gene expression during host infection. Here, recent advances in RNA-seq technologies were used to obtain a detailed analysis of the impact of MBF1 on C. albicans gene expression both in vitro and during infection. MBF1 was involved in the regulation of several genes with a role in glycolysis and response to stress, particularly to nutritional stress. We also investigated whether an interaction existed between MBF1 and GCN4, a master regulator of response to starvation, and found that both genes were needed for resistance to amino acid starvation, suggesting some level of interaction between the two. Reinforcing this idea, we showed that the proteins encoded by both genes could interact. Consistent with the role of MBF1 in virulence, we also established that GCN4 was necessary for virulence in the mouse model of systemic infection as well as in the Galleria mellonella infection model.

OBJECTIVE: Mitochondrial pyruvate carrier (MPC) is a pyruvate transporter that plays a crucial role in regulating the carbon metabolic flow and is considered an essential mechanism for microorganisms to adapt to environmental changes. However, it remains unclear how MPC responds to environmental stress in organisms. General control non-derepressible 4 (GCN4), a key regulator of nitrogen metabolism, plays a pivotal role in the growth and development of fungi. In this study, we report that GCN4 can directly bind to the promoter region and activate the expression of GlMPC, thereby regulating the tricarboxylic acid cycle and secondary metabolism under nitrogen limitation conditions in Ganoderma lucidum. These findings provide significant insights into the regulation of carbon and nitrogen metabolism in fungi, highlighting the critical role of GCN4 in coordinating metabolic adaptation to environmental stresses.

In addition to the main, protein-coding, open reading frame (mORF), many eukaryotic mRNAs contain upstream ORFs (uORFs) initiated at AUG or near-cognate codons residing 5\' of the mORF start site. Whereas translation of uORFs generally represses translation of the mORFs, a subset of uORFs serves as a nexus for regulating translation of the mORF. In this review, we summarize the mechanisms by which uORFs can repress or stimulate mRNA translation, highlight uORF-mediated translational repression involving ribosome queuing, and critically evaluate recently described alternatives to the delayed reinitiation model for uORF-mediated regulation of the GCN4/ATF4 mRNAs.

Visualizing RNA dynamics is important for understanding RNA function. Catalytically dead (d) CRISPR-Cas13 systems have been established to image and track RNAs in living cells, but efficient dCas13 for RNA imaging is still limited. Here, we analyzed metagenomic and bacterial genomic databases to comprehensively screen Cas13 homologies for their RNA labeling capabilities in living mammalian cells. Among eight previously unreported dCas13 proteins that can be used for RNA labeling, dHgm4Cas13b and dMisCas13b displayed comparable, if not higher, efficiencies to the best-known ones when targeting endogenous MUC4 and NEAT1_2 by single guide (g) RNAs. Further examination of the labeling robustness of different dCas13 systems using the GCN4 repeats revealed that a minimum of 12 GCN4 repeats was required for dHgm4Cas13b and dMisCas13b imaging at the single RNA molecule level, while >24 GCN4 repeats were required for reported dLwaCas13a, dRfxCas13d and dPguCas13b. Importantly, by silencing pre-crRNA processing activity of dMisCas13b (ddMisCas13b) and further incorporating RNA aptamers including PP7, MS2, Pepper or BoxB to individual gRNAs, a CRISPRpalette system was developed to successfully achieve multi-color RNA visualization in living cells.

This study aimed to explore the roles of SAP2 and GCN4 in itraconazole (ITR) resistance of C. albicans under different conditions, and their correlations. A total of 20 clinical strains of C. albicans, including 10 ITR resistant strains and 10 sensitive strains, were used. Then, SAP2 sequencing and GCN4 sequencing were performed, and the biofilm formation ability of different C. albicans strains was determined. Finally, real-time quantitative PCR was used to measure the expression of SAP2 and GCN4 in C. albicans under planktonic and biofilm conditions, as well as their correlation was also analyzed. No missense mutations and three synonymous mutation sites, including T276A, G543A, and A675C, were found in SAP2 sequencing. GCN4 sequencing showed one missense mutation site (A106T (T36S)) and six synonymous mutation sites (A147C, C426T, T513C, T576A, G624A and C732T). The biofilm formation ability of drug-resistant C. albicans strains was significantly higher than that of sensitive strains (P < 0.05). Additionally, SAP2 and GCN4 were up-regulated in the ITR-resistant strains, and were both significantly higher in C. albicans under biofilm condition. The mRNA expression levels of SAP2 and GCN4 had significantly positive correlation. The higher expression levels of SAP2 and GCN4 were observed in the ITR-resistant strains of C. albicans under planktonic and biofilm conditions, as well as there was a positive correlation between SAP2 and GCN4 mRNA expression.