eIF4F complex dynamics are important for the activation of the integrated stress response.

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.

Translational regulation by uORFs and start codon selection stringency.

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.

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes.

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

Gcn4 Binding in Coding Regions Can Activate Internal and Canonical 5' Promoters in Yeast.

Gcn4 is a yeast transcriptional activator induced by amino acid starvation. ChIP-seq analysis revealed 546 genomic sites occupied by Gcn4 in starved cells, representing ∼30% of Gcn4-binding motifs. Surprisingly, only ∼40% of the bound sites are in promoters, of which only ∼60% activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining ∼300 Gcn4-bound sites are within coding sequences (CDSs), with ∼75 representing the only bound sites near Gcn4-induced genes. Many such unconventional sites map between divergent antisense and sub-genic sense transcripts induced within CDSs adjacent to induced TBP peaks, consistent with Gcn4 activation of cryptic bidirectional internal promoters. Mutational analysis confirms that Gcn4 sites within CDSs can activate sub-genic and full-length transcripts from the same or adjacent genes, showing that functional Gcn4 binding is not confined to promoters. Our results show that internal promoters can be regulated by an activator that functions at conventional 5'-positioned promoters.

Inability to rescue stalled ribosomes results in overactivation of the integrated stress response.

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.

The transcription factor GCN4 contributes to maintaining intracellular amino acid contents under nitrogen-limiting conditions in the mushroom Ganoderma lucidum.

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. CONCLUSION: 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.

Multiomics of GCN4-Dependent Replicative Lifespan Extension Models Reveals Gcn4 as a Regulator of Protein Turnover in Yeast.

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.

Wide mutational analysis to ascertain the functional roles of eL33 in ribosome biogenesis and translation initiation.

An extensive mutational analysis of RPL33A, encoding the yeast ribosomal protein L33A (eL33) allowed us to identify several novel rpl33a mutants with different translational phenotypes. Most of the rpl33a mutants are defective in the processing of 35S and 27S pre-rRNA precursors and the production of mature rRNAs, exhibiting reductions in the amounts of ribosomal subunits and altered polysome profiles. Some of the rpl33a mutants exhibit a Gcd- phenotype of constitutive derepression of GCN4 translation and strong slow growth phenotypes at several temperatures. Interestingly, some of the later mutants also show a detectable increase in the UUG/AUG translation initiation ratio that can be suppressed by eIF1 overexpression, suggesting a requirement for eL33 and a correct 60S/40S subunit ratio for the proper recognition of the AUG start codon. In addition to producing differential reductions in the rates of pre-rRNA maturation and perhaps in r-protein assembly, most of the point rpl33a mutations alter specific molecular interactions of eL33 with the rRNAs and other r-proteins in the 60S structure. Thus, rpl33a mutations cause distinctive effects on the abundance and/or functionality of 60S subunits, leading to more or less pronounced defects in the rates and fidelity of mRNA translation.

GCN4 Enhances the Transcriptional Regulation of AreA by Interacting with SKO1 To Mediate Nitrogen Utilization in Ganoderma lucidum.

Fungi utilize a wide range of nitrogen to adapt their metabolism. The transcription factor GCN4 has a pivotal role in nitrogen metabolism. However, the mechanism by which GCN4 regulates nitrogen utilization in Ganoderma lucidum is not well understood. In this study, we found that GCN4 physically interacts with SKO1, a bZIP (basic leucine zipper) transcription factor. GCN4 cooperated with SKO1 to positively regulate nitrogen utilization, especially for the expression of areA. Electrophoretic mobility shift assays (EMSA) indicate that GCN4 directly binds to the areA promoter region. Further affinity analysis through biolayer interferometry (BLI) experiments and surface plasmon resonance (SPR) confirmed that GCN4 specifically binds to the promoter region of areA with a strong binding affinity to activate the transcription of areA. In contrast, SKO1 showed no specified binding effect on the areA promoter. However, SKO1 activates the expression of the areA by forming a complex with GCN4, which exhibits a 14.2-fold-higher affinity than GCN4 alone. Furthermore, the presence of SKO1 promotes the stability of GCN4 protein. Accordingly, our study found that the transcription factor SKO1 enhances the transcriptional activity of GCN4 on its target gene areA by interacting with GCN4. Our study illustrates a specific regulatory mechanism for the involvement of GCN4 and SKO1 in nitrogen utilization, which provides innovative insight into the regulation of nitrogen utilization in fungi. IMPORTANCE Nitrogen is an essential nutrient for cell growth and proliferation. Limitations of nitrogen availability in organisms elicit a series of rapid transcriptional reprogramming mechanisms, which involve the participation of many transcription factors. However, the specific mechanism of coordination between different transcription factors regulating nitrogen metabolism has not been explored. Our study revealed that GCN4 interacts with SKO1 and that they are both involved in regulating nitrogen utilization by affecting the transcription level of areA. We also found that GCN4 activates transcription by directly binding to the promoter recognition region of areA. SKO1 facilitates the transcription of areA by GCN4 by forming a more stable complex with GCN4. Our study deepens our understanding of the regulatory network of nitrogen metabolism and demonstrates a further level of regulation for transcription factors.

Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program.

Methionine, through S-adenosylmethionine, activates a multifaceted growth program in which ribosome biogenesis, carbon metabolism, and amino acid and nucleotide biosynthesis are induced. This growth program requires the activity of the Gcn4 transcription factor (called ATF4 in mammals), which facilitates the supply of metabolic precursors that are essential for anabolism. However, how Gcn4 itself is regulated in the presence of methionine is unknown. Here, we discover that Gcn4 protein levels are increased by methionine, despite conditions of high cell growth and translation (in which the roles of Gcn4 are not well-studied). We demonstrate that this mechanism of Gcn4 induction is independent of transcription, as well as the conventional Gcn2/eIF2α-mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 phosphorylation is decreased, which reduces its ubiquitination and therefore degradation. Gcn4 is dephosphorylated by the protein phosphatase 2A (PP2A); our data show that when methionine is abundant, the conserved methyltransferase Ppm1 methylates and alters the activity of the catalytic subunit of PP2A, shifting the balance of Gcn4 toward a dephosphorylated, stable state. The absence of Ppm1 or the loss of the PP2A methylation destabilizes Gcn4 even when methionine is abundant, leading to collapse of the Gcn4-dependent anabolic program. These findings reveal a novel, methionine-dependent signaling and regulatory axis. Here methionine directs the conserved methyltransferase Ppm1 via its target phosphatase PP2A to selectively stabilize Gcn4. Through this, cells conditionally modify a major phosphatase to stabilize a metabolic master regulator and drive anabolism.

GCN4 Regulates Secondary Metabolism through Activation of Antioxidant Gene Expression under Nitrogen Limitation Conditions in Ganoderma lucidum.

Nitrogen limitation has been widely reported to affect the growth and development of fungi, and the transcription factor GCN4 (general control nonderepressible 4) is involved in nitrogen restriction. Here, we found that nitrogen limitation highly induced the expression of GCN4 and promoted the synthesis of ganoderic acid (GA), an important secondary metabolite in Ganoderma lucidum. The activated GCN4 is involved in regulating GA biosynthesis. In addition, the accumulation of reactive oxygen species (ROS) also affects the synthesis of GA under nitrogen restrictions. The silencing of the gcn4 gene led to further accumulation of ROS and increased the content of GA. Further studies found that GCN4 activated the transcription of antioxidant enzyme biosynthesis genes gr, gst2, and cat3 (encoding glutathione reductase, glutathione S-transferase, and catalase, respectively) through direct binding to the promoter of these genes to reduce the ROS accumulation. In conclusion, our study found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. This provides an essential insight into the understanding of GCN4 transcriptional regulation of the ROS signaling pathway and enriches the knowledge of nitrogen regulation mechanisms in fungal secondary metabolism of G. lucidum.IMPORTANCE Nitrogen has been widely reported to regulate secondary metabolism in fungi. Our study assessed the specific nitrogen regulatory mechanisms in Ganoderma lucidum. We found that GCN4 directly interacts with the ROS signaling pathway to negatively regulate GA biosynthesis under nitrogen-limiting conditions. Our research highlights a novel insight that GCN4, the nitrogen utilization regulator, participates in secondary metabolism through ROS signal regulation. In addition, this also provides a theoretical foundation for exploring the regulation of other physiological processes by GCN4 through ROS in fungi.

The Yeast eIF2 Kinase Gcn2 Facilitates H2O2-Mediated Feedback Inhibition of Both Protein Synthesis and Endoplasmic Reticulum Oxidative Folding during Recombinant Protein Production.

Recombinant protein production is a known source of oxidative stress. However, knowledge of which reactive oxygen species are involved or the specific growth phase in which stress occurs remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, microcultivation, and continuous measurements in batch culture, we observed H2O2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H2O2, we find that Gcn2-dependent phosphorylation of eIF2α increases alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains but not in the wild type. Gcn2 deficiency furthermore reduced intracellular H2O2 levels and the Hac1 splicing ratio, while expression of antioxidants and the endoplasmic reticulum (ER) disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding are coupled and subject to feedback inhibition by H2O2. IMPORTANCE Recombinant protein production is a multibillion dollar industry. Optimizing the productivity of host cells is, therefore, of great interest. In several hosts, oxidants are produced as an unwanted side product of recombinant protein production. The buildup of oxidants can result in intracellular stress responses that could compromise the productivity of the host cell. Here, we document a novel protein synthesis inhibitory mechanism that is activated by the buildup of a specific oxidant (H2O2) in the cytosol of yeast cells upon the production of recombinant proteins. At the center of this inhibitory mechanism lies the protein kinase Gcn2. By removing Gcn2, we observed a doubling of recombinant protein productivity in addition to reduced H2O2 levels in the cytosol. In this study, we want to raise awareness of this inhibitory mechanism in eukaryotic cells to further improve protein production and contribute to the development of novel protein-based therapeutic strategies.

The evolution of the 9aaTAD domain in Sp2 proteins: inactivation with valines and intron reservoirs.

The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.

Autophagy modulates Aß accumulation and formation of aggregates in yeast.

Intracellular accumulation of amyloid-ß protein (Aß) is an early event in Alzheimer's disease (AD). The autophagy-lysosomal pathway is an important pathway for maintaining cellular proteostasis and for the removal of damaged organelles and protein aggregates in all eukaryotes. Despite mounting evidence showing that modulating autophagy promotes clearance of Aß aggregates, the regulatory mechanisms and signalling pathways underlying this process remain poorly understood. In order to gain better insight we used our previously characterised yeast model expressing GFP-Aß42 to identify genes that regulate the removal of Aß42 aggregates by autophagy. We report that GFP-Aß42 is sequestered and is selectively transported to vacuole for degradation and that autophagy is the prominent pathway for clearance of aggregates. Next, to identify genes that selectively promote the removal of Aß42 aggregates, we screened levels of GFP-Aß42 and non-aggregating GFP-Aß42 (19:34) proteins in a panel of 192 autophagy mutants lacking genes involved in regulation and initiation of the pathway, cargo selection and degradation processes. The nutrient and stress signalling genes RRD1, SNF4, GCN4 and SSE1 were identified. Deletion of these genes impaired GFP-Aß42 clearance and their overexpression reduced GFP-Aß42 levels in yeast. Overall, our findings identify a novel role for these nutrient and stress signalling genes in the targeted elimination of Aß42 aggregates, which offer a promising avenue for developing autophagy based therapies to suppress amyloid deposition in AD.

General amino acid control in fission yeast is regulated by a nonconserved transcription factor, with functions analogous to Gcn4/Atf4.

Eukaryotes respond to amino acid starvation by enhancing the translation of mRNAs encoding b-ZIP family transcription factors (GCN4 in Saccharomyces cerevisiae and ATF4 in mammals), which launch transcriptional programs to counter this stress. This pathway involves phosphorylation of the eIF2 translation factor by Gcn2-protein kinases and is regulated by upstream ORFs (uORFs) in the GCN4/ATF4 5' leaders. Here, we present evidence that the transcription factors that mediate this response are not evolutionarily conserved. Although cells of the fission yeast Schizosaccharomyces pombe respond transcriptionally to amino acid starvation, they lack clear Gcn4 and Atf4 orthologs. We used ribosome profiling to identify mediators of this response in S. pombe, looking for transcription factors that behave like GCN4 We discovered a transcription factor (Fil1) translationally induced by amino acid starvation in a 5' leader and Gcn2-dependent manner. Like Gcn4, Fil1 is required for the transcriptional response to amino acid starvation, and Gcn4 and Fil1 regulate similar genes. Despite their similarities in regulation, function, and targets, Fil1 and Gcn4 belong to different transcription factor families (GATA and b-ZIP, respectively). Thus, the same functions are performed by nonorthologous proteins under similar regulation. These results highlight the plasticity of transcriptional networks, which maintain conserved principles with nonconserved regulators.

Is Gcn4-induced autophagy the ultimate downstream mechanism by which hormesis extends yeast replicative lifespan?

The number of times a cell divides before irreversibly arresting is termed replicative lifespan. Despite discovery of many chemical, dietary and genetic interventions that extend replicative lifespan, usually first discovered in budding yeast and subsequently shown to apply to metazoans, there is still little understanding of the underlying molecular mechanisms involved. One unifying theme is that most, if not all, interventions that extend replicative lifespan induce "hormesis", where a little inflicted damage makes cells more able to resist similar challenges in the future. One of the many cellular changes that occur during hormesis is a global reduction in protein synthesis, which has been linked to enhanced longevity in many organisms. Our recent study in budding yeast found that it was not the reduction in protein synthesis per se, but rather the subsequent induction of the conserved Gcn4 transcriptional regulator and its ability to induce autophagy that was responsible for extending replicative lifespan. We propose that Gcn4-dependent induction of autophagy occurring downstream of reduced global protein synthesis may be a unifying molecular mechanism for many interventions that extend replicative lifespan.

Sequence-Specific DNA Binding by Noncovalent Peptide-Azocyclodextrin Dimer Complex as a Suitable Model for Conformational Fuzziness.

Transcription factors are proteins lying at the endpoint of signaling pathways that control the complex process of DNA transcription. Typically, they are structurally disordered in the inactive state, but in response to an external stimulus, like a suitable ligand, they change their conformation, thereby activating DNA transcription in a spatiotemporal fashion. The observed disorder or fuzziness is functionally beneficial because it can add adaptability, versatility, and reversibility to the interaction. In this context, mimetics of the basic region of the GCN4 transcription factor (Tf) and their interaction with dsDNA sequences would be suitable models to explore the concept of conformational fuzziness experimentally. Herein, we present the first example of a system that mimics the DNA sequence-specific recognition by the GCN4 Tf through the formation of a non- covalent tetra-component complex: peptide-azoß-CyD(dimer)-peptide-DNA. The non-covalent complex is constructed on the one hand by a 30 amino acid peptide corresponding to the basic region of GCN4 and functionalized with an adamantane moiety, and on the other hand an allosteric receptor, the azoCyDdimer, that has an azobenzene linker connecting two ß-cyclodextrin units. The azoCyDdimer responds to light stimulus, existing as two photo-states: the first thermodynamically stable with an E:Z isomer ratio of 95:5 and the second obtained after irradiation with ultraviolet light, resulting in a photostationary state with a 60:40 E:Z ratio. Through electrophoretic shift assays and circular dichroism spectroscopy, we demonstrate that the E isomer is responsible for dimerization and recognition. The formation of the non-covalent tetra component complex occurs in the presence of the GCN4 cognate dsDNA sequence ('5-..ATGA cg TCAT..-3') but not with ('5-..ATGA c TCAT..-3') that differs in only one spacing nucleotide. Thus, we demonstrated that the tetra-component complex is formed in a specific manner that depends on the geometry of the ligand, the peptide length, and the ds DNA sequence. We hypothesized that the mechanism of interaction is sequential, and it can be described by the polymorphism model of static fuzziness. We argue that chemically modified peptides of the GCN4 Tf are suitable minimalist experimental models to investigate conformational fuzziness in protein-DNA interactions.

Rules of UGA-N decoding by near-cognate tRNAs and analysis of readthrough on short uORFs in yeast.

The molecular mechanism of stop codon recognition by the release factor eRF1 in complex with eRF3 has been described in great detail; however, our understanding of what determines the difference in termination efficiencies among various stop codon tetranucleotides and how near-cognate (nc) tRNAs recode stop codons during programmed readthrough in Saccharomyces cerevisiae is still poor. Here, we show that UGA-C as the only tetranucleotide of all four possible combinations dramatically exacerbated the readthrough phenotype of the stop codon recognition-deficient mutants in eRF1. Since the same is true also for UAA-C and UAG-C, we propose that the exceptionally high readthrough levels that all three stop codons display when followed by cytosine are partially caused by the compromised sampling ability of eRF1, which specifically senses cytosine at the +4 position. The difference in termination efficiencies among the remaining three UGA-N tetranucleotides is then given by their varying preferences for nc-tRNAs. In particular, UGA-A allows increased incorporation of Trp-tRNA whereas UGA-G and UGA-C favor Cys-tRNA. Our findings thus expand the repertoire of general decoding rules by showing that the +4 base determines the preferred selection of nc-tRNAs and, in the case of cytosine, it also genetically interacts with eRF1. Finally, using an example of the GCN4 translational control governed by four short uORFs, we also show how the evolution of this mechanism dealt with undesirable readthrough on those uORFs that serve as the key translation reinitiation promoting features of the GCN4 regulation, as both of these otherwise counteracting activities, readthrough versus reinitiation, are mediated by eIF3.

In-depth analysis of cis-determinants that either promote or inhibit reinitiation on GCN4 mRNA after translation of its four short uORFs.

Translational control in eukaryotes is exerted by many means, one of which involves a ribosome translating multiple cistrons per mRNA as in bacteria. It is called reinitiation (REI) and occurs on mRNAs where the main ORF is preceded by a short upstream uORF(s). Some uORFs support efficient REI on downstream cistrons, whereas some others do not. The mRNA of yeast transcriptional activator GCN4 contains four uORFs of both types that together compose an intriguing regulatory mechanism of its expression responding to nutrients' availability and various stresses. Here we subjected all GCN4 uORFs to a comprehensive analysis to identify all REI-promoting and inhibiting cis-determinants that contribute either autonomously or in synergy to the overall efficiency of REI on GCN4. We found that the 3' sequences of uORFs 1-3 contain a conserved AU1-2A/UUAU2 motif that promotes REI in position-specific, autonomous fashion such as the REI-promoting elements occurring in 5' sequences of uORF1 and uORF2. We also identified autonomous and transferable REI-inhibiting elements in the 3' sequences of uORF2 and uORF3, immediately following their AU-rich motif. Furthermore, we analyzed contributions of coding triplets and terminating stop codon tetranucleotides of GCN4 uORFs showing a negative correlation between the efficiency of reinitiation and efficiency of translation termination. Together we provide a complex overview of all cis-determinants of REI with their effects set in the context of the overall GCN4 translational control.

Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs.

In the present study, we utilized TALEN- and CRISPR/Cas9-induced mutations to analyze the promoter of the barley phytase gene HvPAPhy_a. The purpose of the study was dual, validation of the PAPhy_a enzyme as the main contributor of the mature grain phytase activity (MGPA), as well as validating the importance of a specific promoter region of the PAPhy_a gene which contains three overlapping cis-acting regulatory elements (GCN4, Skn1 and the RY-element) known to be involved in gene expression during grain filling. The results confirm that the barley PAPhy_a enzyme is the main contributor to the MGPA as grains of knock-out lines show very low MGPA. Additionally, the analysis of the HvPAPhy_a promoter region containing the GCN4/Skn1/RY motif highlights its importance for HvPAPhy_a expression as the MGPA in grains of plant lines with mutations within this motif is significantly reduced. Interestingly, lines with deletions located downstream of the motif show even lower MGPA levels, indicating that the GCN4/SKn1/RY motif is not the only element responsible for the level of PAPhy_a expression during grain maturation. Mutant grains with very low MPGA showed delayed germination as compared to grains of wild type barley. As grains with high levels of preformed phytases would provide more readily available phosphorous needed for a fast germination, this indicates that faster germination may be implicated in the positive selection of the ancient PAPhy gene duplication that lead to the creation of the PAPhy_a gene.