Translation Regulation of the Glutamyl-prolyl-tRNA Synthetase Gene EPRS through Bypass of Upstream Open Reading Frames with Noncanonical Initiation Codons.

In the integrated stress response, phosphorylation of eIF2α (eIF2α-P) reduces protein synthesis while concomitantly promoting preferential translation of specific transcripts associated with stress adaptation. Translation of the glutamyl-prolyl-tRNA synthetase gene EPRS is enhanced in response to eIF2α-P. To identify the underlying mechanism of translation control, we employed biochemical approaches to determine the regulatory features by which upstream ORFs (uORFs) direct downstream translation control and expression of the EPRS coding region. Our findings reveal that translation of two inhibitory uORFs encoded by noncanonical CUG and UUG initiation codons in the EPRS mRNA 5'-leader serve to dampen levels of translation initiation at the EPRS coding region. By a mechanism suggested to involve increased translation initiation stringency during stress-induced eIF2α-P, we observed facilitated ribosome bypass of these uORFs, allowing for increased translation of the EPRS coding region. Importantly, EPRS protein expression is enhanced through this preferential translation mechanism in response to multiple known activators of eIF2α-P and likely serves to facilitate stress adaptation in response to a variety of cellular stresses. The rules presented here for the regulated ribosome bypass of noncanonical initiation codons in the EPRS 5'-leader add complexity into the nature of uORF-mediated translation control mechanisms during eIF2α-P and additionally illustrate the roles that previously unexamined uORFs with noncanonical initiation codons can play in modulating gene expression.

Crystal structures of GCN2 protein kinase C-terminal domains suggest regulatory differences in yeast and mammals.

In response to amino acid starvation, GCN2 phosphorylation of eIF2 leads to repression of general translation and initiation of gene reprogramming that facilitates adaptation to nutrient stress. GCN2 is a multidomain protein with key regulatory domains that directly monitor uncharged tRNAs which accumulate during nutrient limitation, leading to activation of this eIF2 kinase and translational control. A critical feature of regulation of this stress response kinase is its C-terminal domain (CTD). Here, we present high resolution crystal structures of murine and yeast CTDs, which guide a functional analysis of the mammalian GCN2. Despite low sequence identity, both yeast and mammalian CTDs share a core subunit structure and an unusual interdigitated dimeric form, albeit with significant differences. Disruption of the dimeric form of murine CTD led to loss of translational control by GCN2, suggesting that dimerization is critical for function as is true for yeast GCN2. However, although both CTDs bind single- and double-stranded RNA, murine GCN2 does not appear to stably associate with the ribosome, whereas yeast GCN2 does. This finding suggests that there are key regulatory differences between yeast and mammalian CTDs, which is consistent with structural differences.

Influence of increasing temperature and salinity on herbicide toxicity in estuarine phytoplankton.

Ecological risk assessments are, in part, based on results of toxicity tests conducted under standard exposure conditions. Global climate change will have a wide range of effects on estuarine habitats, including potentially increasing water temperature and salinity, which may alter the risk assessment of estuarine pollutants. We examined the effects of increasing temperature and salinity on the toxicity of common herbicides (irgarol, diuron, atrazine, and ametryn) to the phytoplankton species Dunaliella tertiolecta. Static 96-h algal bioassays were conducted for each herbicide under four exposure scenarios: standard temperature and salinity (25°C, 20 ppt), standard temperature and elevated salinity (25°C, 40 ppt), elevated temperature and standard salinity (35°C, 20 ppt), and elevated temperature and elevated salinity (35°C, 40 ppt). The endpoints assessed were algal cell density at 96 h, growth rate, chlorophyll a content, lipid content, and starch content. Increasing exposure temperature reduced growth rate and 96-h cell density but increased the cellular chlorophyll and lipid concentrations of the control algae. Exposure condition did not alter starch content of control algae. Herbicides were found to decrease growth rate, 96 h cell density, and cellular chlorophyll and lipid concentrations, while starch concentrations increased with herbicide exposure. Herbicide effects under standard test conditions were then compared with those observed under elevated temperature and salinity. Herbicide effects on growth rate, cell density, and starch content were more pronounced under elevated salinity and temperature conditions. To encompass the natural variability in estuarine temperature and salinity, and to account for future changes in climate, toxicity tests should be conducted under a wider range of environmental conditions.

Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation.

In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the integrated stress response (ISR). The ISR increases the expression of genes important for alleviating stress or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this study, we show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5'-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF due to its poor start site context, and instead it allows scanning ribosomes to translate CHOP. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.

Both transcriptional regulation and translational control of ATF4 are central to the integrated stress response.

In response to different environmental stresses, phosphorylation of eIF2 (eIF2∼P) represses global translation coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of the integrated stress response, a program of gene expression involved in metabolism, nutrient uptake, anti-oxidation, and the activation of additional transcription factors, such as CHOP/GADD153, that can induce apoptosis. Although eIF2-P elicits translational control in response to many different stress arrangements, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2∼P. In this study we addressed the underlying mechanism for variable expression of ATF4 in response to eIF2∼P during different stress conditions and the biological significance of omission of enhanced ATF4 function. We show that in addition to translational control, ATF4 expression is subject to transcriptional regulation. Stress conditions such as endoplasmic reticulum stress induce both transcription and translation of ATF4, which together enhance expression of ATF4 and its target genes in response to eIF2∼P. By contrast, UV irradiation represses ATF4 transcription, which diminishes ATF4 mRNA available for translation during eIF2∼P. eIF2∼P enhances cell survival in response to UV irradiation. However, forced expression of ATF4 and its target gene CHOP leads to increased sensitivity to UV irradiation. This combination of transcriptional regulation and translational control allows the eIF2 kinase pathway to selectively repress or activate key regulatory genes subject to preferential translation, providing the integrated stress response versatility to direct the transcriptome that is essential for maintaining the balance between stress remediation and apoptosis.

Descriptive and mechanistic toxicity of conazole fungicides using the model test alga Dunaliella tertiolecta (Chlorophyceae).

Conazole fungicides are commonly used to prevent fungal growth on turf grass and agricultural crops. As many of these sites are adjacent to coastal waterways and estuaries, there exists the potential for nontarget effects of runoff on marine organisms. This study reports 96 h EC(50) values for four selected conazole fungicides (triadimefon = 5.98 mg/L; triadimenol = 5.51 mg/L; propiconazole = 2.33 mg/L; hexaconazole = 0.91 mg/L) to the model test alga Dunaliella tertiolecta. We further investigated possible mechanisms of toxicity by examining sublethal effects of exposure on cell morphology, osmoregulatory function, and lipid composition. These mechanistic studies revealed that conazole exposure does not inhibit synthesis of the cell's glycerol osmolyte, but does result in an overall increase in cellular volume and total lipid content. Both fungi and chlorophytes rely on ergosterol to maintain membrane structure and fluidity, and we provide evidence that the sterol-inhibiting conazoles may interfere with ergosterol biosynthesis in the cell membrane of Dunaliella. These findings suggest that green algae may be especially susceptible to nontarget effects of sterol-inhibiting fungicides in marine systems.

Temperature and salinity effects on the toxicity of common pesticides to the grass shrimp, Palaemonetes pugio.

This study investigated the effects of increased temperature and salinity, two potential impacts of global climate change, on the toxicity of two common pesticides to the estuarine grass shrimp, Palaemonetes pugio. Larval and adult grass shrimp were exposed to the fungicide chlorothalonil and the insecticide Scourge under standard toxicity test conditions, a 10 degrees C increase in temperature, a 10 ppt increase in salinity, and a combined increased temperature and salinity exposure. Toxicity of the fungicide chlorothalonil increased with temperature and salinity. Toxicity of the insecticide Scourge also increased with temperature; while increased salinity reduced Scourge toxicity, but only in adult shrimp. These findings suggest that changes in temperature and salinity may alter the toxicity of certain pesticides, and that the nature of the effect will depend on both the organism's life stage and the chemical contaminant. Standard toxicity bioassays may not be predictive of actual pesticide toxicity under variable environmental conditions, and testing under a wider range of exposure conditions could improve the accuracy of chemical risk assessments.

Using Tet-Off Cells and RNAi Knockdown to Assay mRNA Decay.

Cellular mRNA levels are determined by the competing forces of transcription and decay. A wide array of cellular mRNA decay pathways carry out RNA turnover either on a constitutive basis or in response to changing cellular conditions. Here, we outline a method to investigate mRNA decay that employs RNAi knockdown of known or putative decay factors in commercially available Tet-off cell systems. Reporter mRNAs of interest are expressed under the control of a tetracycline-regulated promoter, allowing pulse-chase mRNA decay assays to be conducted. Levels of reporter and constitutively expressed control RNAs throughout the decay assay time course are detected by traditional northern blot analysis and used to calculate mRNA half-lives. We describe the utility of this approach to study nonsense-mediated mRNA decay substrates and factors, but it can be readily adapted to investigate key mechanistic features that dictate the specificity and functions of any mRNA decay pathway.

ICE1 promotes the link between splicing and nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway detects aberrant transcripts containing premature termination codons (PTCs) and regulates expression of 5-10% of non-aberrant human mRNAs. To date, most proteins involved in NMD have been identified by genetic screens in model organisms; however, the increased complexity of gene expression regulation in human cells suggests that additional proteins may participate in the human NMD pathway. To identify proteins required for NMD, we performed a genome-wide RNAi screen against >21,000 genes. Canonical members of the NMD pathway were highly enriched as top hits in the siRNA screen, along with numerous candidate NMD factors, including the conserved ICE1/KIAA0947 protein. RNAseq studies reveal that depletion of ICE1 globally enhances accumulation and stability of NMD-target mRNAs. Further, our data suggest that ICE1 uses a putative MIF4G domain to interact with exon junction complex (EJC) proteins and promotes the association of the NMD protein UPF3B with the EJC.

Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKα.

Disruption of protein folding in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a transcriptional and translational control network designed to restore protein homeostasis. Central to the UPR is PKR-like ER kinase (PERK/EIF2AK3) phosphorylation of the α subunit of eIF2 (eIF2α∼P), which represses global translation coincident with preferential translation of mRNAs, such as activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP), that serve to implement UPR transcriptional regulation. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary over a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2α∼P, whereas a notable cohort of key regulators are subject to preferential translation. From the latter group, we identified the α isoform of inhibitor of Bruton's tyrosine kinase (IBTKα) as being subject to both translational and transcriptional induction during eIF2α∼P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKα mRNA involves stress-induced relief of two inhibitory upstream open reading frames in the 5'-leader of the transcript. Depletion of IBTKα by short hairpin RNA reduced viability of cultured cells coincident with increased caspase 3/7 cleavage, suggesting that IBTKα is a key regulator in determining cell fate during the UPR.

Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism.

Regulation of mRNA translation is a rapid and effective means to couple changes in the cellular environment with global rates of protein synthesis. In response to stresses, such as nutrient deprivation and accumulation of misfolded proteins in the endoplasmic reticulum, phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α~P) reduces general translation initiation while facilitating the preferential translation of select transcripts, such as that encoding activating transcription factor 4 (ATF4), a transcriptional activator of genes subject to the integrated stress response (ISR). In this review, we highlight the translational control processes regulated by nutritional stress, with an emphasis on the events triggered by eIF2α~P, and describe the family of eukaryotic initiation factor 2 kinases and the mechanisms by which each sense different stresses. We then address 3 questions. First, what are the mechanisms by which eIF2α~P confers preferential translation on select mRNA and what are the consequences of the gene expression induced by the ISR? Second, what are the molecular processes by which certain stresses can differentially activate eIF2α~P and ATF4 expression? The third question we address is what are the modes of cross-regulation between the ISR and other stress response pathways, such as the unfolded protein response and mammalian target of rapamycin, and how do these regulatory schemes provide for gene expression programs that are tailored for specific stresses? This review highlights recent advances in each of these areas of research, emphasizing how eIF2α~P and the ISR can affect metabolic health and disease.

Methods for analyzing eIF2 kinases and translational control in the unfolded protein response.

Endoplasmic reticulum (ER) stress induces a program of translational and transcriptional regulation, designated the unfolded protein response (UPR), that collectively remedies stress damage and restores ER homeostasis. The protein kinase PERK facilitates the translational control arm of the UPR by phosphorylation of eIF2, a translation initiation factor that combines with GTP to escort initiator Met-tRNA(i)(Met) to the ribosomal machinery during the initiation of protein synthesis. Phosphorylation of the alpha subunit of eIF2 on serine-51 inhibits global translation initiation, which reduces the influx of nascent polypeptides into the overloaded ER. eIF2 phosphorylation also facilitates the preferential translation of stress-related mRNAs, such as ATF4 which in turn activates the transcription of UPR genes. In this chapter, we present experimental strategies and methods for establishing and characterizing global and gene-specific translation control induced by eIF2 phosphorylation (eIF2α~P) during ER stress. These methods include assays for the detection of eIF2α~P and its target genes. We also discuss strategies to address whether a given ER stress condition triggers eIF2α~P through PERK, as opposed to other stress conditions activating alternative members of the eIF2 kinase family. Additionally, experimental descriptions are provided for detecting and quantifying a repression in global translation initiation, and identifying stress-induced preferential translation, such as that described for ATF4. Together, these experimental descriptions will provide a useful molecular "toolkit" to study each feature of the translational control processes invoked during ER stress.