Physical interactions between specifically regulated subpopulations of the MCM and RNR complexes prevent genetic instability.

The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinase Dun1. These physical interactions encompass small subpopulations of MCM and RNR, are independent of the major subcellular locations of these two complexes, augment in response to DNA damage and, in the case of the Rnr2 and Rnr4 subunits of RNR, depend on Dun1. Partial disruption of the MCM/RNR interactions impairs the release of Rad52 -but not RPA-from the DNA repair centers despite the lesions are repaired, a phenotype that is associated with hypermutagenesis but not with alterations in the levels of dNTPs. These results suggest that a specifically regulated pool of MCM and RNR complexes plays non-canonical roles in genetic stability preventing persistent Rad52 centers and hypermutagenesis.

SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells.

BACKGROUND: The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. METHODS: SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1-GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student's t-test, or one-way or two-way analysis of variance (ANOVA). RESULTS: We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. CONCLUSIONS: SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.

Genome-wide imaging screen uncovers molecular determinants of arsenite-induced protein aggregation and toxicity.

The toxic metalloid arsenic causes widespread misfolding and aggregation of cellular proteins. How these protein aggregates are formed in vivo, the mechanisms by which they affect cells and how cells prevent their accumulation is not fully understood. To find components involved in these processes, we performed a genome-wide imaging screen and identified Saccharomyces cerevisiae deletion mutants with either enhanced or reduced protein aggregation levels during arsenite exposure. We show that many of the identified factors are crucial to safeguard protein homeostasis (proteostasis) and to protect cells against arsenite toxicity. The hits were enriched for various functions including protein biosynthesis and transcription, and dedicated follow-up experiments highlight the importance of accurate transcriptional and translational control for mitigating protein aggregation and toxicity during arsenite stress. Some of the hits are associated with pathological conditions, suggesting that arsenite-induced protein aggregation may affect disease processes. The broad network of cellular systems that impinge on proteostasis during arsenic stress identified in this current study provides a valuable resource and a framework for further elucidation of the mechanistic details of metalloid toxicity and pathogenesis. This article has an associated First Person interview with the first authors of the paper.

Etp1 confers arsenite resistance by affecting ACR3 expression.

In a high-throughput yeast two-hybrid screen of predicted coiled-coil motif interactions in the Saccharomyces cerevisiae proteome, the protein Etp1 was found to interact with the yeast AP-1-like transcription factors Yap8, Yap1 and Yap6. Yap8 plays a crucial role during arsenic stress since it regulates expression of the resistance genes ACR2 and ACR3. The function of Etp1 is not well understood but the protein has been implicated in transcription and protein turnover during ethanol stress, and the etp1∆ mutant is sensitive to ethanol. In this current study, we investigated whether Etp1 is implicated in Yap8-dependent functions. We show that Etp1 is required for optimal growth in the presence of trivalent arsenite and for optimal expression of the arsenite export protein encoded by ACR3. Since Yap8 is the only known transcription factor that regulates ACR3 expression, we investigated whether Etp1 regulates Yap8. Yap8 ubiquitination, stability, nuclear localization and ACR3 promoter association were unaffected in etp1∆ cells, indicating that Etp1 affects ACR3 expression independently of Yap8. Thus, Etp1 impacts gene expression under arsenic and other stress conditions but the mechanistic details remain to be elucidated.

Yeast Cth2 protein represses the translation of ARE-containing mRNAs in response to iron deficiency.

In response to iron deficiency, the budding yeast Saccharomyces cerevisiae undergoes a metabolic remodeling in order to optimize iron utilization. The tandem zinc finger (TZF)-containing protein Cth2 plays a critical role in this adaptation by binding and promoting the degradation of multiple mRNAs that contain AU-rich elements (AREs). Here, we demonstrate that Cth2 also functions as a translational repressor of its target mRNAs. By complementary approaches, we demonstrate that Cth2 protein inhibits the translation of SDH4, which encodes a subunit of succinate dehydrogenase, and CTH2 mRNAs in response to iron depletion. Both the AREs within SDH4 and CTH2 transcripts, and the Cth2 TZF are essential for translational repression. We show that the role played by Cth2 as a negative translational regulator extends to other mRNA targets such as WTM1, CCP1 and HEM15. A structure-function analysis of Cth2 protein suggests that the Cth2 amino-terminal domain (NTD) is important for both mRNA turnover and translation inhibition, while its carboxy-terminal domain (CTD) only participates in the regulation of translation, but is dispensable for mRNA degradation. Finally, we demonstrate that the Cth2 CTD is physiologically relevant for adaptation to iron deficiency.

Dissecting mRNA decay and translation inhibition during iron deficiency.

Iron participates as a vital cofactor in multiple metabolic pathways. Despite its abundance, iron bioavailability is highly restricted in aerobic and alkaline environments. Therefore, living organisms have evolved multiple adaptive mechanisms to respond to iron scarcity. These strategies include a global remodeling of iron metabolism directed to optimize iron utilization. In the baker's yeast Saccharomyces cerevisiae, this metabolic reorganization is accomplished to a large extent by an mRNA-binding protein called Cth2. Yeast Cth2 belongs to a conserved family of tandem zinc finger containing proteins that specifically bind to transcripts with AU-rich elements and promote their turnover. A recent study has revealed that Cth2 also inhibits the translation of its target mRNAs (Ramos-Alonso et al., PLoS Genet 14:e1007476, https://doi.org/10.1371/journal.pgen.1007476 , 2018). Interestingly, the mammalian Cth2 ortholog known as tristetraprolin (aka TTP/TIS11/ZFP36), which is also implicated in controlling iron metabolism, promotes the decay and prevents the translation of its regulated transcripts. These observations open the possibility to study the relative contribution of altering mRNA stability and translation to the physiological adaptation to iron deficiency, the function played by the different domains within the mRNA-binding protein, and the potential factors implicated in coordinating both post-transcriptional events.

Regulation of yeast fatty acid desaturase in response to iron deficiency.

Unsaturated fatty acids (UFA) are essential components of phospholipids that greatly contribute to the biophysical properties of cellular membranes. Biosynthesis of UFAs relies on a conserved family of iron-dependent fatty acid desaturases, whose representative in the model yeast Saccharomyces cerevisiae is Ole1. OLE1 expression is tightly regulated to adapt UFA biosynthesis and lipid bilayer properties to changes in temperature, and in UFA or oxygen availability. Despite iron deficiency being the most extended nutritional disorder worldwide, very little is known about the mechanisms and the biological relevance of fatty acid desaturases regulation in response to iron starvation. In this report, we show that endoplasmic reticulum-anchored transcription factor Mga2 activates OLE1 transcription in response to nutritional and genetic iron deficiencies. Cells lacking MGA2 display low UFA levels and do not grow under iron-limited conditions, unless UFAs are supplemented or OLE1 is overexpressed. The proteasome, E3 ubiquitin ligase Rsp5 and the Cdc48Npl4/Ufd1 complex are required for OLE1 activation during iron depletion. Interestingly, Mga2 also activates the transcription of its own mRNA in response to iron deficiency, hypoxia, low temperature and low UFAs. MGA2 up-regulation contributes to increase OLE1 expression in these situations. These results reveal the mechanism of OLE1 regulation when iron is scarce and identify the MGA2 auto-regulation as a potential activation strategy in multiple stresses.

Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Small Subunit Localization in Response to Iron Deficiency.

Ribonucleotide reductase (RNR) is an essential iron-dependent enzyme that catalyzes deoxyribonucleotide synthesis in eukaryotes. Living organisms have developed multiple strategies to tightly modulate RNR function to avoid inadequate or unbalanced deoxyribonucleotide pools that cause DNA damage and genome instability. Yeast cells activate RNR in response to genotoxic stress and iron deficiency by facilitating redistribution of its small heterodimeric subunit Rnr2-Rnr4 from the nucleus to the cytoplasm, where it forms an active holoenzyme with large Rnr1 subunit. Dif1 protein inhibits RNR by promoting nuclear import of Rnr2-Rnr4. Upon DNA damage, Dif1 phosphorylation by the Dun1 checkpoint kinase and its subsequent degradation enhances RNR function. In this report, we demonstrate that Dun1 kinase triggers Rnr2-Rnr4 redistribution to the cytoplasm in response to iron deficiency. We show that Rnr2-Rnr4 relocalization by low iron requires Dun1 kinase activity and phosphorylation site Thr-380 in the Dun1 activation loop, but not the Dun1 forkhead-associated domain. By using different Dif1 mutant proteins, we uncover that Dun1 phosphorylates Dif1 Ser-104 and Thr-105 residues upon iron scarcity. We observe that the Dif1 phosphorylation pattern differs depending on the stimuli, which suggests different Dun1 activating pathways. Importantly, the Dif1-S104A/T105A mutant exhibits defects in nucleus-to-cytoplasm redistribution of Rnr2-Rnr4 by iron limitation. Taken together, these results reveal that, in response to iron starvation, Dun1 kinase phosphorylates Dif1 to stimulate Rnr2-Rnr4 relocalization to the cytoplasm and promote RNR function.

Soybean Ferritin Expression in Saccharomyces cerevisiae Modulates Iron Accumulation and Resistance to Elevated Iron Concentrations.

UNLABELLED: Fungi, including the yeast Saccharomyces cerevisiae, lack ferritin and use vacuoles as iron storage organelles. This work explored how plant ferritin expression influenced baker's yeast iron metabolism. Soybean seed ferritin H1 (SFerH1) and SFerH2 genes were cloned and expressed in yeast cells. Both soybean ferritins assembled as multimeric complexes, which bound yeast intracellular iron in vivo and, consequently, induced the activation of the genes expressed during iron scarcity. Soybean ferritin protected yeast cells that lacked the Ccc1 vacuolar iron detoxification transporter from toxic iron levels by reducing cellular oxidation, thus allowing growth at high iron concentrations. Interestingly, when simultaneously expressed in ccc1Δ cells, SFerH1 and SFerH2 assembled as heteropolymers, which further increased iron resistance and reduced the oxidative stress produced by excess iron compared to ferritin homopolymer complexes. Finally, soybean ferritin expression led to increased iron accumulation in both wild-type and ccc1Δ yeast cells at certain environmental iron concentrations. IMPORTANCE: Iron deficiency is a worldwide nutritional disorder to which women and children are especially vulnerable. A common strategy to combat iron deficiency consists of dietary supplementation with inorganic iron salts, whose bioavailability is very low. Iron-enriched yeasts and cereals are alternative strategies to diminish iron deficiency. Animals and plants possess large ferritin complexes that accumulate, detoxify, or buffer excess cellular iron. However, the yeast Saccharomyces cerevisiae lacks ferritin and uses vacuoles as iron storage organelles. Here, we explored how soybean ferritin expression influenced yeast iron metabolism, confirming that yeasts that express soybean seed ferritin could be explored as a novel strategy to increase dietary iron absorption.

Responses of Saccharomyces cerevisiae Strains from Different Origins to Elevated Iron Concentrations.

Iron is an essential micronutrient for all eukaryotic organisms. However, the low solubility of ferric iron has tremendously increased the prevalence of iron deficiency anemia, especially in women and children, with dramatic consequences. Baker's yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, a fermentative microorganism, and a feed supplement. In this report, we explore the genetic diversity of 123 wild and domestic strains of S. cerevisiae isolated from different geographical origins and sources to characterize how yeast cells respond to elevated iron concentrations in the environment. By using two different forms of iron, we selected and characterized both iron-sensitive and iron-resistant yeast strains. We observed that when the iron concentration in the medium increases, iron-sensitive strains accumulate iron more rapidly than iron-resistant isolates. We observed that, consistent with excess iron leading to oxidative stress, the redox state of iron-sensitive strains was more oxidized than that of iron-resistant strains. Growth assays in the presence of different oxidative reagents ruled out that this phenotype was due to alterations in the general oxidative stress protection machinery. It was noteworthy that iron-resistant strains were more sensitive to iron deficiency conditions than iron-sensitive strains, which suggests that adaptation to either high or low iron is detrimental for the opposite condition. An initial gene expression analysis suggested that alterations in iron homeostasis genes could contribute to the different responses of distant iron-sensitive and iron-resistant yeast strains to elevated environmental iron levels.

Ribosome profiling reveals the role of yeast RNA-binding proteins Cth1 and Cth2 in translational regulation.

Iron serves as a cofactor for enzymes involved in several steps of protein translation, but the control of translation during iron limitation is not understood at the molecular level. Here, we report a genome-wide analysis of protein translation in response to iron deficiency in yeast using ribosome profiling. We show that iron depletion affects global protein synthesis and leads to translational repression of multiple genes involved in iron-related processes. Furthermore, we demonstrate that the RNA-binding proteins Cth1 and Cth2 play a central role in this translational regulation by repressing the activity of the iron-dependent Rli1 ribosome recycling factor and inhibiting mitochondrial translation and heme biosynthesis. Additionally, we found that iron deficiency represses MRS3 mRNA translation through increased expression of antisense long non-coding RNA. Together, our results reveal complex gene expression and protein synthesis remodeling in response to low iron, demonstrating how this important metal affects protein translation at multiple levels.

Post-transcriptional regulation of iron homeostasis in Saccharomyces cerevisiae.

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in a wide variety of biological processes. Recent studies in Saccharomyces cerevisiae have shown that in response to iron deficiency, an RNA-binding protein denoted Cth2 coordinates a global metabolic rearrangement that aims to optimize iron utilization. The Cth2 protein contains two Cx8Cx5Cx3H tandem zinc fingers (TZFs) that specifically bind to adenosine/uridine-rich elements within the 3' untranslated region of many mRNAs to promote their degradation. The Cth2 protein shuttles between the nucleus and the cytoplasm. Once inside the nucleus, Cth2 binds target mRNAs and stimulates alternative 3' end processing. A Cth2/mRNA-containing complex is required for export to the cytoplasm, where the mRNA is degraded by the 5' to 3' degradation pathway. This post-transcriptional regulatory mechanism limits iron utilization in nonessential pathways and activates essential iron-dependent enzymes such as ribonucleotide reductase, which is required for DNA synthesis and repair. Recent findings indicate that the TZF-containing tristetraprolin protein also functions in modulating human iron homeostasis. Elevated iron concentrations can also be detrimental for cells. The Rnt1 RNase III exonuclease protects cells from excess iron by promoting the degradation of a subset of the Fe acquisition system when iron levels rise.

Iron drives anabolic metabolism through active histone demethylation and mTORC1.

All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.

Changes in mRNA stability play an important role in the adaptation of yeast cells to iron deprivation.

Eukaryotic cells rely on iron as an indispensable cofactor for multiple biological functions including mitochondrial respiration and protein synthesis. The budding yeast Saccharomyces cerevisiae utilizes both transcriptional and posttranscriptional mechanisms to couple mRNA levels to the requirements of iron deprivation. Thus, in response to iron deficiency, transcription factors Aft1 and Aft2 activate the expression of genes implicated in iron acquisition and mobilization, whereas two mRNA-binding proteins, Cth1 and Cth2, posttranscriptionally control iron metabolism. By using a genome-wide approach, we describe here a global stabilization of mRNAs, including transcripts encoding ribosomal proteins (RPs), when iron bioavailability diminishes. mRNA decay assays indicate that the mRNA-binding protein Pub1 contributes to RP transcript stabilization during adaptation to iron limitation. In fact, Pub1 becomes critical for growth and translational repression in low-iron conditions. Remarkably, we observe that pub1Δ cells also exhibit an increase in the transcription of RP genes that evidences the crosstalk between transcription and degradation mechanisms to maintain the appropriate mRNA balance under iron deficiency conditions.

Deficiency of the RNA-binding protein Cth2 extends yeast replicative lifespan by alleviating its repressive effects on mitochondrial function.

Iron dyshomeostasis contributes to aging, but little information is available about the molecular mechanisms. Here, we provide evidence that in Saccharomyces cerevisiae, aging is associated with altered expression of genes involved in iron homeostasis. We further demonstrate that defects in the conserved mRNA-binding protein Cth2, which controls stability and translation of mRNAs encoding iron-containing proteins, increase lifespan by alleviating its repressive effects on mitochondrial function. Mutation of the conserved cysteine residue in Cth2 that inhibits its RNA-binding activity is sufficient to confer longevity, whereas Cth2 gain of function shortens replicative lifespan. Consistent with its function in RNA degradation, Cth2 deficiency relieves Cth2-mediated post-transcriptional repression of nuclear-encoded components of the electron transport chain. Our findings uncover a major role of the RNA-binding protein Cth2 in the regulation of lifespan and suggest that modulation of iron starvation signaling can serve as a target for potential aging interventions.

Iron in Translation: From the Beginning to the End.

Iron is an essential element for all eukaryotes, since it acts as a cofactor for many enzymes involved in basic cellular functions, including translation. While the mammalian iron-regulatory protein/iron-responsive element (IRP/IRE) system arose as one of the first examples of translational regulation in higher eukaryotes, little is known about the contribution of iron itself to the different stages of eukaryotic translation. In the yeast Saccharomyces cerevisiae, iron deficiency provokes a global impairment of translation at the initiation step, which is mediated by the Gcn2-eIF2α pathway, while the post-transcriptional regulator Cth2 specifically represses the translation of a subgroup of iron-related transcripts. In addition, several steps of the translation process depend on iron-containing enzymes, including particular modifications of translation elongation factors and transfer RNAs (tRNAs), and translation termination by the ATP-binding cassette family member Rli1 (ABCE1 in humans) and the prolyl hydroxylase Tpa1. The influence of these modifications and their correlation with codon bias in the dynamic control of protein biosynthesis, mainly in response to stress, is emerging as an interesting focus of research. Taking S. cerevisiae as a model, we hereby discuss the relevance of iron in the control of global and specific translation steps.

Iron Regulatory Mechanisms in Saccharomyces cerevisiae.

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in many cellular processes. However, excess iron can damage cells since it promotes the generation of reactive oxygen species. The budding yeast Saccharomyces cerevisiae has been used as a model organism to study the adaptation of eukaryotic cells to changes in iron availability. Upon iron deficiency, yeast utilizes two transcription factors, Aft1 and Aft2, to activate the expression of a set of genes known as the iron regulon, which are implicated in iron uptake, recycling and mobilization. Moreover, Aft1 and Aft2 activate the expression of Cth2, an mRNA-binding protein that limits the expression of genes encoding for iron-containing proteins or that participate in iron-using processes. Cth2 contributes to prioritize iron utilization in particular pathways over other highly iron-consuming and non-essential processes including mitochondrial respiration. Recent studies have revealed that iron deficiency also alters many other metabolic routes including amino acid and lipid synthesis, the mitochondrial retrograde response, transcription, translation and deoxyribonucleotide synthesis; and activates the DNA damage and general stress responses. At high iron levels, the yeast Yap5, Msn2, and Msn4 transcription factors activate the expression of a vacuolar iron importer called Ccc1, which is the most important high-iron protecting factor devoted to detoxify excess cytosolic iron that is stored into the vacuole for its mobilization upon scarcity. The complete sequencing and annotation of many yeast genomes is starting to unveil the diversity and evolution of the iron homeostasis network in this species.

The lipid composition of yeast cells modulates the response to iron deficiency.

Iron is a vital micronutrient for all eukaryotes because it participates as a redox cofactor in multiple metabolic pathways, including lipid biosynthesis. In response to iron deficiency, the Saccharomyces cerevisiae iron-responsive transcription factor Aft1 accumulates in the nucleus and activates a set of genes that promote iron acquisition at the cell surface. In this study, we report that yeast cells lacking the transcription factor Mga2, which promotes the expression of the iron-dependent Δ9-fatty acid desaturase Ole1, display a defect in the activation of the iron regulon during the adaptation to iron limitation. Supplementation with exogenous unsaturated fatty acids (UFAs) or OLE1 expression rescues the iron regulon activation defect of mga2Δ cells. These observations and fatty acid measurements suggest that the mga2Δ defect in iron regulon expression is due to low UFA levels. Subcellular localization studies reveal that low UFAs cause a mislocalization of Aft1 protein to the vacuole upon iron deprivation that prevents its nuclear accumulation. These results indicate that Mga2 and Ole1 are essential to maintain the UFA levels required for Aft1-dependent activation of the iron regulon in response to iron deficiency, and directly connect the biosynthesis of fatty acids to the response to iron depletion.

Global translational repression induced by iron deficiency in yeast depends on the Gcn2/eIF2α pathway.

Iron is an essential element for all eukaryotic organisms because it participates as a redox active cofactor in a wide range of biological processes, including protein synthesis. Translation is probably the most energy consuming process in cells. Therefore, one of the initial responses of eukaryotic cells to stress or nutrient limitation is the arrest of mRNA translation. In first instance, the budding yeast Saccharomyces cerevisiae responds to iron deficiency by activating iron acquisition and remodeling cellular metabolism in order to prioritize essential over non-essential iron-dependent processes. We have determined that, despite a global decrease in transcription, mRNA translation is actively maintained during a short-term exposure to iron scarcity. However, a more severe iron deficiency condition induces a global repression of translation. Our results indicate that the Gcn2-eIF2α pathway limits general translation at its initiation step during iron deficiency. This bulk translational inhibition depends on the uncharged tRNA sensing Gcn1-Gcn20 complex. The involvement of the Gcn2-eIF2α pathway in the response to iron deficiency highlights its central role in the eukaryotic response to stress or nutritional deprivation, which is conserved from yeast to mammals.

The yeast Aft1 transcription factor activates ribonucleotide reductase catalytic subunit RNR1 in response to iron deficiency.

Eukaryotic ribonucleotide reductases are iron-dependent enzymes that catalyze the rate-limiting step in the de novo synthesis of deoxyribonucleotides. Multiple mechanisms regulate the activity of ribonucleotide reductases in response to genotoxic stresses and iron deficiency. Upon iron starvation, the Saccharomyces cerevisiae Aft1 transcription factor specifically binds to iron-responsive cis elements within the promoter of a group of genes, known as the iron regulon, activating their transcription. Members of the iron regulon participate in iron acquisition, mobilization and recycling, and trigger a genome-wide metabolic remodeling of iron-dependent pathways. Here, we describe a mechanism that optimizes the activity of yeast ribonucleotide reductase when iron is scarce. We demonstrate that Aft1 and the DNA-binding protein Ixr1 enhance the expression of the gene encoding for its catalytic subunit, RNR1, in response to iron limitation, leading to an increase in both mRNA and protein levels. By mutagenesis of the Aft1-binding sites within RNR1 promoter, we conclude that RNR1 activation by iron depletion is important for Rnr1 protein and deoxyribonucleotide synthesis. Remarkably, Aft1 also activates the expression of IXR1 upon iron scarcity through an iron-responsive element located within its promoter. These results provide a novel mechanism for the direct activation of ribonucleotide reductase function by the iron-regulated Aft1 transcription factor.