Translational control of gene expression by eIF2 modulates proteostasis and extends lifespan.

Although the stress response in eukaryotes depends on early events triggered in cells by environmental insults, long-term processes such as aging are also affected. The loss of cellular proteostasis greatly impacts aging, which is regulated by the balancing of protein synthesis and degradation systems. As translation is the input event in proteostasis, we decided to study the role of translational activity on cell lifespan. Our hypothesis was that a reduction on translational activity or specific changes in translation may increase cellular longevity. Using mutant strains of Schizosaccharomyces pombe and various stress conditions, we showed that translational reduction caused by phosphorylation of eukaryotic translation initiation factor 2 (eIF2) during the exponential growth phase enhances chronological lifespan (CLS). Furthermore, through next-generation sequence analysis, we found eIF2α phosphorylation-dependent translational activation of some specific genes, especially those involved in autophagy. This fact, together with the observed regulation of autophagy, points to a conserved mechanism involving general and specific control of translation and autophagy as mediators of the role of eIF2α phosphorylation in aging.

Analyzing Cdc2/Cdk1 activation during stress response in Schizosaccharomyces pombe.

Stress leads to multiple changes in the physiology of the cell. One of the most important is the adaptation of the cell cycle to the changing conditions of the environment. Cellular responses after stress can be followed by cellular synchronization previous to the insult. In this chapter, we use centrifugal elutriation to synchronize Schizosaccharomyces pombe cells and outline methods to investigate the hallmarks of cell cycle progression upon stress. These include analyses of cyclin-dependent kinase phosphorylation and cell size change.

Modification of tRNA(Lys) UUU by elongator is essential for efficient translation of stress mRNAs.

The Elongator complex, including the histone acetyl transferase Sin3/Elp3, was isolated as an RNA polymerase II-interacting complex, and cells deficient in Elongator subunits display transcriptional defects. However, it has also been shown that Elongator mediates the modification of some tRNAs, modulating translation efficiency. We show here that the fission yeast Sin3/Elp3 is important for oxidative stress survival. The stress transcriptional program, governed by the Sty1-Atf1-Pcr1 pathway, is affected in mutant cells, but not severely. On the contrary, cells lacking Sin3/Elp3 cannot modify the uridine wobble nucleoside of certain tRNAs, and other tRNA modifying activities such as Ctu1-Ctu2 are also essential for normal tolerance to H2O2. In particular, a plasmid over-expressing the tRNA(Lys) UUU complements the stress-related phenotypes of Sin3/Elp3 mutant cells. We have determined that the main H2O2-dependent genes, including those coding for the transcription factors Atf1 and Pcr1, are highly expressed mRNAs containing a biased number of lysine-coding codons AAA versus AAG. Thus, their mRNAs are poorly translated after stress in cells lacking Sin3/Elp3 or Ctu2, whereas a mutated atf1 transcript with AAA-to-AAG lysine codons is efficiently translated in all strain backgrounds. Our study demonstrates that the lack of a functional Elongator complex results in stress phenotypes due to its contribution to tRNA modification and subsequent translation inefficiency of certain stress-induced, highly expressed mRNAs. These results suggest that the transcriptional defects of these strain backgrounds may be a secondary consequence of the deficient expression of a transcription factor, Atf1-Pcr1, and other components of the transcriptional machinery.

Functional characterization of Upf1 targets in Schizosaccharomyces pombe.

Nonsense-mediated mRNA decay (NMD) is a highly conserved mechanism of mRNA degradation. NMD eliminates mRNAs containing premature termination codons (PTCs), preventing the production of truncated proteins with possible deleterious effects. However, there is mounting evidence that NMD factors, like Upf1, Upf2 and Upf3, participate in general regulation of gene expression, affecting the expression of genes lacking PTCs. We have used the fission yeast Schizosaccharomyces pombe to identify mRNAs directly regulated by NMD. Using a combination of genetic and biochemical approaches, we have defined a population of fission yeast mRNAs specifically regulated by Upf1. We show that other components of the Upf complex, Upf2 and Upf3, are required for binding of Upf1 to its RNA targets and for the proper response of fission yeast to oxidative stress. Finally, we investigated the physiological importance of this phenomenon, and demonstrate that the Upf1-dependent downregulation of some of its direct targets is necessary for normal resistance to oxidative stress.

Is oxidized thioredoxin a major trigger for cysteine oxidation? Clues from a redox proteomics approach.

Cysteine oxidation mediates oxidative stress toxicity and signaling. It has been long proposed that the thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (Trr), is not only involved in recycling classical Trx substrates, such as ribonucleotide reductase, but it also regulates general cytoplasmic thiol homeostasis. To investigate such a role, we have performed a proteome-wide analysis of cells expressing or not the two components of the Trx system. We have compared the reversibly oxidized thiol proteomes of wild-type Schizosaccharomyces pombe cells with mutants lacking Trx or Trr. Specific Trx substrates are reversibly-oxidized in both strain backgrounds; however, in the absence of Trr, Trx can weakly recycle its substrates at the expense of an alternative electron donor. A massive thiol oxidation occurs only in cells lacking Trr, with 30% of all cysteine-containing peptides being reversibly oxidized; this oxidized cysteine proteome depends on the presence of Trxs. Our observations lead to the hypothesis that, in the absence of its reductase, the natural electron donor Trx becomes a powerful oxidant and triggers general thiol oxidation.

Response to arsenate treatment in Schizosaccharomyces pombe and the role of its arsenate reductase activity.

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.

The RNA binding protein Csx1 promotes sexual differentiation in Schizosaccharomyces pombe.

Sexual differentiation is a highly regulated process in the fission yeast Schizosaccharomyces pombe and is triggered by nutrient depletion, mainly nitrogen source. One of the key regulatory proteins in fission yeast sexual differentiation is the transcription factor Ste11. Ste11 regulates the transcription of many genes required for the initial steps of conjugation and meiosis, and its deficiency leads to sterility. Ste11 activity is mainly regulated at two levels: phosphorylation and abundance of its mRNA. Csx1 is an RNA binding protein that we have previously described to bind and regulate the turnover rate of the mRNA encoding the transcription factor Atf1 in the presence of oxidative stress. We have observed that Csx1-deficient cells have defects in sexual differentiation and are partially sterile. We investigated how Csx1 is regulating this process in S. pombe. Csx1 associates with ste11+ mRNA and cells lacking Csx1 are sterile with a reduced amount of ste11+ mRNA. Overexpression of ste11+ mRNA completely rescues the mating deficiencies of csx1Δ cells. Here, we present a novel mechanism of ste11+ mRNA positive regulation through the activity of Csx1, an RNA binding protein that also have key functions in the response to oxidative stress in fission yeast. This finding opens interesting question about the possible coordination of sexual differentiation and oxidative stress response in eukaryotes and the role of RNA binding proteins in the adaptation to environmental signals.

Slt2 MAPK pathway is essential for cell integrity in the presence of arsenate.

Arsenate is a common toxic metalloid found in drinking water worldwide that causes several human diseases. The biochemical action underlying cellular response to arsenate, however, is not yet completely understood. Here we used Saccharomyces cerevisiae as an eukaryotic model system to identify proteins essential for adaptation to arsenate treatment. Previous studies have demonstrated a function for Hog1 MAPK in modulating the cellular response to arsenite. Our results, however, showed that cells deficient in Hog1 did not show increased sensitivity to arsenate, suggesting that perhaps other MAPKs may be involved in the response to this particular arsenic species. Here, we found that Slt2 MAPK and several of its upstream regulators are essential in modulating the response to arsenate, and that Slt2 is phosphorylated after arsenate treatment. Furthermore, whole-genome transcriptional analysis showed that Slt2 is required for the induction of several genes in response to arsenate exposure. Many of these genes are involved in the cellular response to heat, suggesting an overlap between these two stress response pathways, and pointing toward a common response to both arsenate and heat exposure in Saccharomyces cerevisiae. Furthermore, our results support the idea that cellular exposure to arsenate results in induction of cellular signalling pathways different from those induced under arsenite treatment.

Activation of Srk1 by the mitogen-activated protein kinase Sty1/Spc1 precedes its dissociation from the kinase and signals its degradation.

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. The Schizosaccharomyces pombe SAPK Sty1/Spc1 orchestrates general changes in gene expression in response to diverse forms of cytotoxic stress. Here we show that Sty1/Spc1 is bound to its target, the Srk1 kinase, when the signaling pathway is inactive. In response to stress, Sty1/Spc1 phosphorylates Srk1 at threonine 463 of the regulatory domain, inducing both activation of Srk1 kinase, which negatively regulates cell cycle progression by inhibiting Cdc25, and dissociation of Srk1 from the SAPK, which leads to Srk1 degradation by the proteasome.

Control of mRNA stability by SAPKs.

Control of mRNA turnover is an essential step in the regulation of gene expression in eukaryotes. The concerted action of many enzymes regulates the way each mRNA is degraded. Moreover, the degradation of each mRNA is influenced by the environment surrounding the cell. The conection between the environment and changes in the half-lifes of mRNAs is regulated by the activity of stress activated MAP kinases (SAPKs) and their substrates. Here we will describe some of those mechanisms, and how SAPKs regulate mRNA stability in eukaryotic cells.

Upf1, an RNA helicase required for nonsense-mediated mRNA decay, modulates the transcriptional response to oxidative stress in fission yeast.

In the fission yeast Schizosaccharomyces pombe, oxidative stress triggers the activation of the Spc1/Sty1 mitogen-activated protein kinase, which in turn phosphorylates the Atf1/Pcr1 heterodimeric transcription factor to effect global changes in the patterns of gene expression. This transcriptional response is also controlled by Csx1, an RNA-binding protein that directly associates with and stabilizes atf1(+) mRNA. Here we report the surprising observation that this response also requires Upf1, a component of the nonsense-mediated mRNA decay (NMD) system. Accordingly, upf1Delta and csx1Delta strains are similarly sensitive to oxidative stress, and the effects of the mutations are not additive, suggesting that Upf1 and Csx1 work in the same pathway to stabilize atf1(+) mRNA during oxidative stress. Consistent with these observations, whole-genome expression profiling studies have shown that Upf1 controls the expression of more than 100 genes that are transcriptionally induced in response to oxidative stress, the large majority of which are also controlled by Atf1 and Csx1. The unexpected connection between an NMD factor and the oxidative stress response in fission yeast may provide important new clues about the physiological function of NMD in other species.

Mitogen-activated protein kinase Hog1 is essential for the response to arsenite in Saccharomyces cerevisiae.

Here we describe, for the first time, that budding yeast mitogen-activated protein kinase Hog1 and its upstream activators Pbs2 and Ssk1 are essential for the response to arsenite. Hog1 is rapidly phosphorylated in response to arsenite and triggers a transcriptional response that involves the upregulation of genes essential for arsenite detoxification.

Cip1 and Cip2 are novel RNA-recognition-motif proteins that counteract Csx1 function during oxidative stress.

Eukaryotic cells reprogram their global patterns of gene expression in response to stress. Recent studies in Schizosaccharomyces pombe showed that the RNA-binding protein Csx1 plays a central role in controlling gene expression during oxidative stress. It does so by stabilizing atf1(+) mRNA, which encodes a subunit of a bZIP transcription factor required for gene expression during oxidative stress. Here, we describe two related proteins, Cip1 and Cip2, that were identified by multidimensional protein identification technology (MudPIT) as proteins that coprecipitate with Csx1. Cip1 and Cip2 are cytoplasmic proteins that have RNA recognition motifs (RRMs). Neither protein is essential for viability, but a cip1Delta cip2Delta strain grows poorly and has altered cellular morphology. Genetic epistasis studies and whole genome expression profiling show that Cip1 and Cip2 exert posttranscriptional control of gene expression in a manner that is counteracted by Csx1. Notably, the sensitivity of csx1Delta cells to oxidative stress and their inability to induce expression of Atf1-dependent genes are partially rescued by cip1Delta and cip2Delta mutations. This study emphasizes the importance of a modulated mRNA stability in the eukaryotic stress response pathways and adds new information to the role of RNA-binding proteins in the oxidative stress response.

Distinct signaling pathways respond to arsenite and reactive oxygen species in Schizosaccharomyces pombe.

Exposure to certain metal and metalloid species, such as arsenic, cadmium, chromium, and nickel, has been associated with an increased risk of cancer in humans. The biological effects of these metals are thought to result from induction of reactive oxygen species (ROS) and inhibition of DNA repair enzymes, although alterations in signal transduction pathways may also be involved in tumor development. To better understand metal toxicity and its connection to ROS, we have compared the effects of arsenite and hydrogen peroxide in wild-type and mutant strains of the fission yeast Schizosaccharomyces pombe. An atf1Delta pap1Delta strain, which is defective in two transcription factors that control stress responses, is extremely sensitive to hydrogen peroxide but not to arsenite. A strain that lacks the transcription factor Zip1 has the opposite relationship. Spc1 (Sty1) mitogen-activated protein kinase (MAPK), a homologue of mammalian p38 MAPK, and the upstream MAPK kinase (MAPKK) Wis1 are essential for survival of both arsenite and hydrogen peroxide. Inactivation of two MAPKK kinases, Win1 and Wis4, almost completely eliminates Spc1 activation by arsenite, yet these cells survive arsenite treatment. The two-component phosphorelay protein Mcs4, which acts upstream of Win1 and Wis4 and is required for Spc1 activation in response to oxidative stress, is not required for Spc1 activation in response to arsenite. We conclude that the toxic effects of arsenic are not strongly connected to oxidative stress and that although Spc1 is activated by arsenic exposure, the basal activity of Spc1 is largely sufficient for the survival of arsenic.

RNA-binding protein Csx1 mediates global control of gene expression in response to oxidative stress.

Fission yeast Spc1 (Sty1), a stress-activated mitogen-activated protein kinase (MAPK) homologous to human p38, orchestrates global changes in gene expression in response to diverse forms of cytotoxic stress. This control is partly mediated through Atf1, a transcription factor homologous to human ATF2. How Spc1 controls Atf1, and how the cells tailor gene expression patterns to different forms of stress, are unknown. Here we describe Csx1, a novel protein crucial for survival of oxidative but not osmotic stress. Csx1 associates with and stabilizes atf1+ mRNA in response to oxidative stress. Csx1 controls expression of the majority of the genes induced by oxidative stress, including most of the genes regulated by Spc1 and Atf1. These studies reveal a novel mechanism controlling MAPK-regulated transcription factors and suggest how gene expression patterns can be customized to specific forms of stress. Csx1-like proteins in humans may perform similar tasks.