Cell, Vol. 120, 99—110, January 14, 2005, Copyright ©2005 by Elsevier lnc.
`
`DOI 10.1 01 6/].cell.2004.11.032
`
`Coordinated Remcdeling sf Gelluiar
`Metabolism during Iron fieficieacy
`through Targeted mRMA Degradation
`
`Sergi Puig,“ Eric Askeland,‘ and Dennis J. Thiele’”
`‘Department of Pharmacology and Cancer Biology
`Sarah W. Stedman Nutrition and Metabolism Center
`Duke University Medical Center
`Research Drive-LSRC-0351
`Durham, North Carolina 27710
`
`Summary
`
`Iron (Fe) is an essential micronutrient for virtually all
`organisms and serves as a cofactor for a wide variety
`of vital cellular processes. Although Fe deficiency is
`the primary nutritional disorder in the world, cellular
`responses to Fe deprivation are poorly understood.
`We have discovered a posttranscriptional regulatory
`process controlled by Fe deficiency, which coordi-
`nately drives widespread metabolic reprogramming.
`We demonstrate that, in response to Fe deficiency,
`the Saccharomyces cerevisiae Cth2 protein specifi-
`cally downregulates mRNAs encoding proteins that
`participate in many Fe-dependent processes. mRNA
`turnover requires the binding of Cth2, an RNA binding
`protein conserved in plants and mammals, to specific
`AU~rich elements in the 3’ untranslated region of
`mRNAs targeted for degradation. These studies eluci-
`date coordinated global metabolic reprogramming in
`response to Fe deiiciency and identify a mechanism
`for achieving this by targeting specific mRNA mole-
`cules for degradation, thereby facilitating the utiliza-
`tion of limited cellular Fe levels.
`
`introduction
`
`Iron (Fe) is an essential nutrient for virtually all organ-
`isms. Fe serves as a cofactorfor a wide variety of cellular
`processes, including oxygen transport, cellular respira-
`tion, the tricarboxylic acid (TCA) cycle, lipid metabolism,
`synthesis of metabolic intermediates, gene regulation,
`and DNA replication and repair. Despite its abundance
`in the earth’s crust, Fe bioavailability is highly restricted
`due to its extreme insolubility at physiological pH. In-
`deed, Fe deficiency is the primary nutritional disorder
`in the world, estimated to affect over two billion people
`and resulting in iron deficiency anemia (Baynes and
`Bothwell, 1990). Alterations in iron homeostasis underlie
`many human diseases, including Friedreich’s ataxia, he-
`reditary hemochromatosis, aceruloplasminemia, Par-
`kinson’s disease, aging, microbial pathogenesis, and
`cancer (Hentze et al., 2004; Nittis and Gitlin, 2002; Roy
`and Andrews, 2001).
`Elegant genetic, biochemical, and physiological stud-
`ies have elucidated many of the components that func-
`tion in Fe uptake, efflux, and distribution and their mechs
`
`‘Correspondence: dennis.thiele@duke.edu
`1 Present address: Departament de Bioquimica i Biologia Molecular,
`Universitat de Valencia, Avenida Dr. Moliner 50, E461 00 Buriassot,
`Valencia. Spain.
`
`anisms of action in both prokaryotic and eukaryotic cells
`(Escolar et al., 1909; Hentze et al., 2004; Van Ho et al.,
`2002). Studies with the baker’s yeast Saccharomyces
`cerevisiae have demonstrated that, in response to Fe
`deprivation, cells utilize the Fe-responsive transcription
`factors Am and Aim to induce expression of the so-
`called iron regulon (Rutherford et al., 2003; Shakoury-
`Elizeh et al., 2004), which includes proteins involved in
`Fe reduction at the plasma membrane, uptake, mobiliza-
`tion from intracellular stores, and utilization from heme,
`among others (Van Ho et al., 2002). Less attention has
`been dedicated to the characterization of metabolic
`pathways that are specifically downregulated by Fe
`depletion. Recent studies have shown that mRNA levels
`of genes involved in biotin synthesis, glutamate metabo-
`lism and heme assembly are downregulated under low
`Fe conditions (Lesuisse et al., 2003; Shakoury-Elizeh et
`al., 2004). However, the mechanisms controlling the Fe
`deprivation-dependent downregulation of these genes,
`and other global metabolic pathways altered as a conse
`quence of Fe deficiency, have not been elucidated.
`In mammals, one response to iron scarcity is posttran-
`scriptionally controlled by the iron—regulatory proteins
`IRP1 and lRP2. In response to Fe deprivation, lRP1 binds
`to specific mRNA stem-loop structures known as iron-
`responsive elements (IRES). lRPi binding to IRES in the
`5’ untranslated region inhibits translation of erythroid
`aminolevulinic acid synthase, mitochondrial aconitase,
`the ferroportin Fe efflux pump, and subunits of the Fe
`storage protein ferritin. IRP1 binding to lREs in the 3’
`untranslated region (3’UTR) of the transferrin receptor 1
`isoform stabilizes the mRNA, thereby increasing protein
`levels and enhancing Fe uptake via Fe loaded transferrin
`(Hentze et al., 2004; Theil, 2000). A posttranscriptional
`downregulation of Fe-dependent pathways, which de-
`pends on small antisense RNAs, has recently been de-
`scribed in bacteria (Masse and Gottesman, 2002; Wil-
`derman et al., 2004).
`While several dozen metabolic enzymes require Fe
`for catalysis in eukaryotic cells, little is known about
`global reprogramming and regulatory mechanisms gov-
`erning this process in response to Fe deficiency. We
`have discovered a mechanism that mediates global
`posttranscriptional control of multiple components of
`Fe-dependent pathways to respond in a concerted fash-
`ion to Fe deficiency. The Fe—regulated protein Cth2 coor—
`clinates this process by binding to and targeting specific
`mRNA molecules for degradation under Fe deficiency,
`thereby facilitating the utilization of limited available Fe
`for normal growth.
`
`Results
`
`Genome-Wide Response of Saccharomyces
`cerevisiae to iron Deprivation
`Although Fe plays a crucial role in a wide array of ceilular
`processes, little is known about how Fe deprivation af-
`fects metabolic pathways on a global scale in eukaryotic
`cells. To investigate the response of S. cerevisiae to Fe
`
`BUTAMAX 1006
`
`

`

`Cell
`1 00
`
`Downregu‘iated
`
`Upregulated
`
`iron reguion'
`
`Sterol and unsaturated fatty acid synthesis
`
`, Trécarboxylic acid cycle
`:- Electron transport chain
`Heme and Fees proteinslbiosynthesis
`
`Biotin synthesis
`
`DNA replication and repair
`
`.3,
`
`i
`
`;;
`
`I
`
`
`
`
`20
`
`1O
`
`0
`
`10
`
`20
`
`Number of genes
`Figure 1 . Response of Pea-Dependent Processes to Fe Depletion in Yeast
`B4741 wild—type cells were grown in SC containing 300 pM Fe or 100 pM BPS, and RNA was analyzed with DNA microarrays as detailed in
`Experimental Procedures. Only components of multiple Fe-dependent pathways with a fold change greater than two have been represented.
`A list of the genes grouped in each functional family Es shown in Supplemental Tables 82 and SS.
`
`deprivation, we compared the mRNA expression profile
`of wild-type cells grown under Fe—replete conditions to
`cells grown under Fe scarcity achieved by addition of
`the Fe(ll) chelator bathophenantholine disulfonic acid
`(BPS). We observed that, in addition to changes in other
`processes (data not shown), key components of multiple
`Fe-dependent metabolic pathways are significantly al-
`tered by Fe availability (Figure 1 and see Supplemental
`Tables 82 and $3 at http://www.celLoom/cgi/content/
`full/120l1/99/DC‘H’). in addition to the induction of the
`Aft1/2-dependent Fe regulon previously described
`(Blajseau et al., 2001; Rutherford et al., 2003; Shakoury-
`Elizeh et al., 2004; Yamaguchi-lwai et al., 1996), genes
`involved in sterol biosynthesis (ERG genes) and the fatty
`acid desaturase OLE1 are induced under Fe deprivation.
`In addition, key components of multiple Fe=dependent
`pathways and proteins including (1) the TCA cycle; (2)
`the mitochondrial electron transport chain; (3) Fe—S clus-
`ter, di-Fe-tyrosyl, and heme—containing proteins; and, (4)
`as recently described (Lesuisse et al., 2003; Shakoury-
`Elizeh et al., 2004), HEM15 encoding ferrochelatase, the
`last step in heme biosynthesis, and two enzymes in-
`volved in biotin synthesis are coordinately downregu-
`lated by Fe depletion (Figure1 and Supplemental Table
`83). Taken together, these results demonstrate that
`mFiNA levels of multiple components of Fe—dependent
`metabolic pathways in S. cerevisiae are coordinately
`regulated in response to Fe deprivation.
`
`The Aft1-A’ft2 Target CTH2 is important for Growth
`under Fe Limitation
`
`Previous DNA microarray experiments strongly suggest
`that the CTH2 gene, which encodes a protein related to
`the mammalian tandem zinc finger (TZF) protein triste—
`traprolin or TTP (Figure 2A), is transcriptionally induced
`under Fe limitation (Foury and Talibi, 2001; Rutherford
`et al., 2003; Shakoury-Elizeh et al., 2004). As shown in
`Figures 28 and 2C, the steady-state levels of CTH2
`mRNA and a functional FLAG epitope-tagged 0ch pro-
`tein under Fez—adequate conditions are very low but dra-
`matically increase in response to Fe deprivation. Fur~
`
`thermore, CTH2 expression under low Fe conditions is
`significantly decreased in an aft1 strain and is undetect-
`able under either condition in the aft1aft2 double mutant.
`Mutagenesis of two putative Aft1-Aft2 binding sites (Ya—
`maguchi-lwai et al., 1996) from the CTH2 upstream
`sequence indicates that both sites cooperate in the acti-
`vation of CTH2 by Fe starvation, although these experi-
`ments do not exclude the participation of other cis-
`regulatory sequences in CTH2 regulation by Fe (see
`Suppiemental “Figure 82 on the Cell web site).
`Given that CTH2 mRNA levels are tightly regulated by
`Fe availability and the Aft1 -Aft2 Fe—responsive transcrip-
`tion factors, we assayed growth of cth2 deletion mutant
`cells under Fe deprivation conditions. cth2 cells exhib-
`ited a growth defect compared to wild-type cells in the
`presence of the intracellular Fe-speciféc chelator ferroz—
`ine (Figure 3A). The cch growth defect on ferrozine was
`reversed by addition of Fe (Figure 3A), demonstrating
`that the growth defect of cth2 cells occurs in response
`to Fe deprivation rather than to ferrozine administration.
`The yeast genome harbors a gene encoding a protein
`similar to Cth2, Cth1 (Thompson et al., 1996), whose
`transcription is independent of Fe levels (Figure 23).
`Although cth1 cells did not display a growth defect under
`Fe deprivation conditions, cells lacking both CTH1 and
`CTH2 exhibited a more severe growth defect than those
`lacking only 07222 (Figure 3A). Similarly, the cth1cth2
`growth defect in the presence of ferrozine was partially
`suppressed by CTH1 and completely recovered by
`coexpression of both CTH1 and CTH2 (Figure 3B). These
`results demonstrate that CTH2 is important for growth
`under Fe deprivation induced by the membrane perme-
`able Fe chelator ferrozine and suggest that Cth1 function
`in yeast cells may partially overlap with Cth2.
`
`CTH2 Coordinates the Downregulation of Multiple
`Fe-Dependent Pathways under Fe Deprivation
`A prominent feature of Cth2 is the presence of a
`stcxscst tandem zinc finger (TZF) domain near the
`carboxyl terminus of the protein (Figure 2A and Supple—
`mental Figure 81). This TZF motif is present in a family of
`
`

`

`1
`1
`06m Controls a Posttranscriptional Fe Regulon
`
`
`
`B
`
`0
`
`aft1
`WT aft1 aftz aftz
`Fe '+—..-'
`'+_-‘ T. at".
`g as
`.15:
`
`g»
`
`«sag
`
`CTH2
`
`FET3
`
`
`
`W“*‘§%§eww A03
`
`Flagz
`
`Cth2
`vector
`
`Fe+-+-
`
`Figure 2. Expression of CTH2 upon Fe Depletion ls Dependent on
`Both Aftt and Aft2 Transcription Factors
`(A) Model for the primary structure of S. cerevisiae Cth2 and Cth‘l
`and human tristetraprolin (hTi'P) protein. TZF, tandem zinc finger.
`(B) CM3260 wild-type, aft1, aft2, and aftlaft2 cells were grown in
`SC containing 100 pM Fe (Fe +) or 100 pM BPS (Fe —) and RNA
`extracted and analyzed by RNA blotting. The Aft1 target F573 was
`used as a control for Fe=regulated expression.
`(0) cth1cth2 cells transformed with pRS416-FLAG2-GTH2 or
`pFiS416 (vector) were grown in SC-Ura containing 300 pM Fe (Fe +)
`or 100 uM BPS (Fe -) and protein extracted and analyzed by immu-
`noblotting. Phosphoglycerate kinase (ng1) was used as a load—
`ing control.
`
`RNA binding proteins typified by the mammalian protein
`tristetraprolin (TTP). TTP mediates the targeted destabi-
`lization of tumor necrosis factor a UNFa), cyclooxygen-
`ass-2, interleukin-3, and granulocyte/macrophage col-
`ony-stimulating factor (GM-CSF) mRNAs (Blackshear,
`2002; Carballo et al.,
`1 998; Sawaoka et al., 2003;
`Stoecklin et al., 2001). An alignment of yeast Cth1 and
`Cth2 with human TTP shows that, while Cth1 and Cth2
`proteins share 46% identity, hTTP homology to Ctht
`and Cth2 is restricted to the TZF domains (Supplemental
`Figure S1). Despite little homology in the rest of the
`protein, we hypothesized that (3ch could be involved
`in posttranscriptional regulation of specific mRNAs un-
`der Fe deprivation. To test this hypothesis, we ascer-
`tained the effect of Cth2 on multiple mRNAs we ob-
`served in our microarray to be downregulated by Fe
`deficiency. As shown in Figure 3C, genes encoding pro«
`teins involved in the TCA cycle (SDH4), heme synthesis
`(HEM15), Fe-S cluster assembly (ISA 1), vacuolar Fe ac-
`cumulation (0001), and Fe—S proteins (LIP5) are dramat-
`ically downregulated under Fe starvation in a wild-type
`strain. The mRNA levels of RNR2, encoding a subunit
`of the essential di—Fe-tyrosil-dependent enzyme ribonu-
`
`cleotide reductase, are only modestly decreased by Fe
`deprivation (Figure 36). Interestingly, this coordinated
`mRNA downregulation does not occur in the absence
`of CTH2 (Figure 3C, wt versus cth2 mutant). While mRNA
`levels in cth1 cells did not change significantly with
`respect to wild-type cells, the cth1cth2 mutant dis-
`played reduced mRNA downregulation, suggesting that
`Cth1 directly or indirectly influences in this process.
`These results demonstrate that Cth2 functions in the
`downregulation of specific mRNAs under conditions of
`Fe deprivation.
`We used DNA microarrays to ascertain which mRNAs
`exhibit CTH2-dependent changes on a genome-wide
`scale by comparing the gene expression profiles under
`Fe deficiency of cth1cth2 cells expressing a plasmid-
`borne CTH2 gene or transformed with vector alone.
`Messenger RNAs corresponding to 84 genes were sig-
`nificantly upregulated in the absence of CTH2 (Table 1).
`lnterestingfy, 54% (45 of 84) of the upregulated genes
`are involved in obvious Feedependent processes, 14%
`(1 2 of 84) have other functions, and 32% (27 of 87) are
`genes of unknown function. Among the 45 Fe-related
`genes whose expression is increased in cth2 mutants
`under low Fe conditions compared to wild-type cells,
`we find (1) three members of the Fe regulon (FIT1, FIT2,
`and HMX1); (2) genes encoding key enzymes involved
`in heme biosynthesis (HEM15); (3) two genes encoding
`proteins involved in Fe—S cluster assembly (ISA1 and
`NFU1);
`(4) eight genes encoding enzymes that parti-
`cipate in the TCA cycle including aconitase (A001),
`succinate dehydrogenase subunits SDH2 and SDH4,
`a-ketoglutarate dehydrogenase (K601), and dihydroly-
`poyl transsuccinylase (KGD2); (5) 15 genes encoding
`proteins that participate in the electron transfer chain
`that include four subunits of the cytocrome c oxidase
`(COX4, COX6, COX8, COX9) and six subunits of the
`ubiquinol cytochrome c reductase complex (0031-5
`and RIP1); (6) eight members of the sterol and unsatu-
`rated fatty acid synthesis and metabolism pathways
`(ERG genes and OLE1); (7) ribonucleotide—diphosphate
`reductase subunits (RNR4); and (8) genes encoding ad-
`ditional Fe-S cluster-containing proteins (LIP5, encod-
`ing lipoic acid synthase; LEU1, required for leucine bio-
`synthesis; and RL! 1, related to RNase L inhibitor). Taken
`together, these results demonstrate that Cth2 functions
`in the coordinated downregulation of multiple Fe-depen-
`dent metabolic pathways, and potentially other as yet
`uncharacterized pathways, in yeast under conditions of
`Fe deficiency.
`
`A Conserved RNA Binding Motif Is Required
`for Cth2-Mediated mRNA Downregulation
`Studies with TTP in mammalian systems have demon-
`strated that the integrity of the zinc finger domains is
`required for binding and destabilization of specific
`mFtNAs (Blackshear, 2002; Lai et al., 1999, 2003). We
`tested the role of the CCCH zinc fingers in Cth2-depen-
`dent mRNA downregulation by mutagenizing conserved
`cysteine residues, located in both zinc finger motifs, to
`arginine. First, cells expressing CTH2-C190R or CTH2-
`0213R mutant alleles displayed a growth defect in the
`presence of the Fe-chelator ferrozine (Figure 3E). Sec-
`ond, Cth2-dependent downregulation of SDH4, HEM 15,
`
`

`

`Cell
`102
`
`A
`
`_
`Complete F'errozine
`
`Ferrozine
`+ Fe
`
`
`
` ctm
`
`'
`
`CTH2 .
`
`-
`
`»
`
`(:ch
`
`CTH1 + CTH2 ..
`
`cth1
`WT (:ch c1h'1 (:ch
`l"—II—!f_“"lr‘—"i
`Fe+-+-+-+-
`
`a!“ fit
`
`
`
`
`
`
`
`ctm cthz
`
`CTH2 vector C1981? 6213}?
`
`Figure 3. CTH2 Is Required for Fe—Limited Growth and Fe Deficiency-Dependent mRNA Downregulation
`(A) BY4741 wild-type, cthl, cth2, and cth1cth2 cells were assayed for growth on SC (Complete) and SC containing 750 uM ferrozine without
`or with 300 uM Fe (t Fe).
`(B) cth1cth2 cells cotransformed with pRS416 plus pRS415 (vector), pRS416-CTH1 plus pRS415 (CTH1), pRS416 plus pRS415—CTH2 (CTH2;
`and pRS416-CTH1 plus pRS415-CTH2 (CTH1 + CTH2) were assayed on SC-Ura-Leu (—Ura — Lou) and SC containing 750 pM ferrozine.
`(C) CTH2 is essential for Fe deficiency-dependent mRNA downregulation. Wild-type cth2, cth1, and cth1cth2 cells were grown in SC media
`containing 300 uM Fe (Fe +) or 100 uM BPS (Fe —) and RNA extracted and analyzed by RNA blotting.
`(D) Schematic representation of the CCCH TZF domain in 0ch protein. Cysteine residues 190 and 213 (white characters) were mutagenized
`to arginine.
`(E) The 0ch TZF domains are essential for growth in the presence of ferrozine. BY4741 wild-type and cth2 cells transformed with vector
`alone or expressing CTH2, CTH2-C1908, and C7742—C213Fl alleles were assayed fog growth on ferrozine plates.
`(F) The 6ch CCCH TZF motifs are essential for mRNA downregulation. cth1cth2 cells containing vector or expressing CTH2, CTH2-C190Fl,
`and 07142-0213}? alleles were analyzed by RNA blotting as described for (C).
`
`LIPS, COX6, and other mRNAs (data not shown) was
`abrogated in both (3ch mutants (Figure 3F). Similar re-
`sults were obtained when cysteine residues 190 and 213
`were mutagenized to alanine (data not shown). Control
`experiments showed that the cysteine mutant proteins
`are properly expressed (data not shown). Taken to-
`gether, these results demonstrate that the integrity of
`both CCCH zinc finger motifs is essential for 0ch func-
`tion in coordinated mRNA downregulation in response
`to Fe deprivation.
`
`Downregulation of Specific mRNAs by Fe
`Deprévation Requires AU—Rich Elements
`Human Tl'P binds to AU-rich elements (ARES) within the
`3’UTR of target mRNAs and induces RNA degradation
`(Blackshear et al., 2003; Lai et at, 1999). interestingly,
`in silico analysis and visual inspectfon reveals that ap-
`proximately 80% of the mFlNAs upregulated in cch cells
`
`under low Fe conditions contain one or more putative
`ARES, defined as 5'-UAUUUAUU-3’ and 5'-UUAUU
`UAU-S’ octamer sequences, located within 500 nucleo—
`tides after the translation termination codon (Table 1).
`To test whether Cth2-dependent mRNA downregulation
`during Fe deficiency occurs via ARES located within the
`3'UTR, we used the mRNA encoding the membrane-
`anchored heme'containing subunit of the succinate de-
`hydrogenase complex in mitochondria, SDH4, which is
`downregulated under Fe deprivation in a manner com-
`pletely dependent on 0ch (Figures 30 and 3F). The
`SDH4 3’UTFI contains three 5’-UUAUUUAUU-3’ se-
`quences beginning at 125, 135, and 158 nucleotides
`afterthe translation termination codon (Table 1 and Fig-
`ure 4A). The adenine nucleotides 127, 134, 141, and 160
`were mutated to cytosine in a plasmid-borne copy of
`the SDH4 gene (Figures 4A and 4B, SDH4-AREmt2) and
`mFlNA levels assessed under high and low Fe conditions
`
`

`

`Cth2 Controls 3 Posttranscn'ptional Fe Regulon
`1 03
`
`
`Putative AREs
`ORF
`Geno
`Function
`Fold : SD
`iron reguion
`YDR5346
`YOR382W
`YLR2OSC
`Heme biosynthesis
`YDR044W
`YOR176W
`
`Cell wall mannoprotein involved in sideroohore-Fe uptake
`Cell wall mannoprotein involved in siderophore-Fe uptake
`Home binding peroxidase involved in reuliiization of heme Fe
`
`2.0 :t 0.2
`1.6 1 0.1
`2.0 i 0.4
`
`263
`255
`
`Coproporphyrinogen Ill oxidase, oxygen-requiring enzyme
`Ferrochelatase, catalyzes insertion of Fe(ll) into
`protoporphyrin IX
`
`1.5 :t'. 0.3“
`2.2 1 0.2
`
`68, 89
`43, 99
`
`FiT1
`FiT2
`HMX1
`
`HEM13
`HEM15
`
`2.0 1 0.2
`1.9 + 0.1
`
`2.0 :c 0.2
`1 .7 .1- 0.4
`2.6 1: 0.3
`1.6 t 0.2
`1.8 t 0.2
`2.8 :- 0.8
`3.2 1 0.7
`
`1.6 x 0.2
`
`1.9 :l: 0.4
`2.2 I 0.2
`1.9 r 0.2
`1.8 1- 0.2
`
`2.0 i 0.2
`1.6 :r 0.2
`1.7 3: 0.3
`1.9 i 0.2
`1.8 1 0.3
`2.0 tr 0.1
`
`2.1 3: 0.2
`1.7 :1.- 0.3
`2.8 :r. 0.5
`1.6 r 0.2
`1.6 1 0.2
`
`2.0 3: 0.3
`1.6 't 0.1
`1.6 t 0.1
`1.7 I 0.4
`1.6 a“. 0.3
`
`191, 203
`46, 62
`
`32, 150, 177
`193, 230
`242
`162, 309, 328
`125, 135, 158
`
`53
`88
`104
`44
`
`140
`155
`31
`150, 239
`97, 114
`293, 355
`
`13. 37, 81
`42
`18, 41, 50, 59
`
`4, 60
`174, 203, 273
`19
`
`52
`
`1.6 i: 0.3
`1.7 + 0.2
`1.6 1 0.1
`
`151, 187
`89, 105
`18, 148
`
`1.4 t 0.2'
`1.6 t 0.4
`
`68
`39, 125
`
`1.4 :: 0.1 "
`2.0 x 0.3
`1.7 .t 0.3
`1.6 i 0.2
`1.7 2‘: 0.3
`1.6 f". 0.4
`1.7 i 0.2
`1.6 1" 0.2
`
`24, 144
`70, 92
`B5, 123
`280, 291
`275, 303
`
`16
`
`Fe~S cluster biogenesis
`YKLMOC
`NFU1
`YL£027W
`ISM
`TCA cycle
`YNR001C
`YPR001W
`YL3304C
`Yll..1 25W
`YDm 48C)
`YLL041C
`YDR178W
`
`(3111
`GETS
`A001
`KGDi
`K602
`SDH2
`SDH4
`
`NifU-like protein
`Member of Fo-S cluster biosynthesis machinery
`
`Subunit N of cytochrome c oxidase
`Subunit Vi of cytochrome c oxidase
`Subunit Vii! of cytochrome c oxidase
`Subunit Vila of cytochrome c oxidase
`
`Citrate synthase
`Mitochondrial isoiorm of citrate synthase
`Mitochondrial aconitase, Fe~s cluster protein
`Alpha-ketoglutarate dehydrogcnztse
`Dihydroiipoyi transsuccinyiasc
`Succinate dehydrogenase (ubiquinone) res cluster subunit
`Succinate dehydrogenase membrane anchor heme-binding
`subunk
`Mitochondrial and cytoplasmic iumarase, Fe-S cluster protein
`FUM1
`YPL262W
`Mitochondrial respiration/electron transport chain
`Cytochrome c oxidase
`YGL187’C
`COX4
`YHR051W
`COXB
`YLRSQSC
`COXB
`YDL067C
`COXS}
`Ubiquinoi cytochrome :2 reductase
`YBL045C
`(tom/com Core subunit l of ubiquinol cytochrome (2 reductase complex
`YPR191W
`QCRZ/CORZ
`Core subunit ll of ubiquinoi cytochrome (2 reductase complex
`YFFlOSSC
`QCRG/COR3
`Subunit Vi of ubiquinoi cytochrome c reductase complex
`YDRSEQC
`001271130114
`Subunit Vii of ubiquinol cytochrome c reduclase complex
`YJL166W
`QCRS/CORS
`Subunit Viil of ubiquinol cytochrome c reductase complex
`YEL024W
`RIF-’1
`Rieske Fe-S protein oi ubiquinol cytochrome c reduc’tase
`complex
`Putative mitochondrial dehydrogenase fiavoprotein
`YORSSSW
`Flavinudependent monooxygenase, ubiquinone biosynihesis
`COOS
`YGR2550
`Cytochrome c peroxidase
`CCPi
`YKR0660
`NADH dehydrogenase
`N051
`YMRMSC
`Mitochondrial ADP/ATP carrier
`PETQ/AACZ
`YBL0300
`Steroi and fatty acid synthesis and metabolism
`YHRO72W
`ERG?
`Lanosteroi syathaso
`YHROO7C
`ERGH
`Lanosteroi C-1 4 demelhyiase
`YMR208W
`ERG12
`Mevalonate kinase
`YGROSOW
`ERGZS
`(3—4 methyl steroi oxidase
`YERDMC
`ERG28
`ER membrane protein, may facilitate Erg26 and Erg27
`interactions
`Fatty acid desaturase
`Hydroxylalion of C-26 fatty acid in ceramide
`Damage response protein involved in stem! synthesis
`
`OLE1
`YGLOSSW
`FAHVSCS?
`YMR272C
`DAP1
`YPLWOW
`DNA replication and repair
`Ribonucleotide-diphosphate reductase, di«Fe—tyrosyl coiactor
`YJL026W
`RNRZ
`Ribonuclemidediphosphate reductase, Y4 subunit
`YGR1BOC
`RNR4
`Other Fe-, Cu». and oxygen-related function
`YLR220W
`(3061
`Transporter that mediates vacuolar Fe storage
`YOR19£SC
`LIPS
`Lipoic acid synthase. Fe~S cluster protein
`YGLOOQC
`LEU1
`lsopropylmalate isomerase, Fe—S oiuster protein
`YDROQ1 C
`RLH
`RNase 1. inhibitor, Fo~S cluster protein
`YKL109W
`HAP4
`Subunit of Hap transcriptional activator
`YHR055C
`CUP1~2
`Copper-binding metallothionoin
`YAROZOC
`PAU?
`Member of PAU family
`YORSSMW
`Member of PAU family
`Other functions
`1.6 i 0.3
`Nonmitochondriai citrate synthase
`0sz
`YCHOOSC
`1.8 .x 0.2
`Phosphoribosylanihranilate isomerase
`TFlP‘i
`YDROGYW
`1.6 t 0.3
`Leucine zipper transcriptional activator
`CAD1
`YDR423G
`54
`1.6 i 0.2
`Phosphomannose isomerase
`PMMO
`YEROOGG
`YHROOZW
`
`
`Mitochondrial carrier proteinLEU5 1.6 2; 0.3
`
`
`

`

`Cell
`
`Table 1. Continued
`
`
`
`
`
`Function
`
` Fold 2 so ORF Gene Putative AREs
`Other functions
`YJL172W
`YJR016C
`YLR121C
`YML028W
`YOFl230W
`YPL053C
`YPL1540
`Unknown function
`YBL048W
`YBR187W
`YCR017C
`YDR36GC
`YDR411C
`YER048W—A
`YER138W-A
`YER15SC
`YGL002W
`YGL1886
`YHRO45W
`YHR113W
`YJL171C
`YKR103W
`YKR104W
`YLROBSC
`YLR251W
`YMLOBQC
`YMR041C
`YMR11OC
`YNL320W
`YOL083W
`YOLOQZW
`YOR214C
`YORSOGC
`YPL2500
`YPR002W
`
`1.7 i 0.1
`1.6 I 0.2
`1.7 t 0.2
`2.0 t 0.2
`2.3 i 0.2
`1.6 i 0.2
`1.9 i 0.3
`
`1.3 : Of:
`1.8 t 0.1
`1.6 t 0.3
`1.6 i 0.2
`1.8 r 0.2
`1.6 :- 0.3
`1.7 :t 0.3
`1.8 i 0.3
`1.7 t 0.1
`1.7 t 0.1
`1.7 1- 0.2
`1.9 ft 0.1
`1.7 r 0.2
`3.3 x 0.4
`3.6 t 0.7
`1.6 1*. 0.2
`1.6 1 0.2
`1.8 :t 0.2
`1.6 r 0.3
`1.7 '3; 0.3
`1.9 :— 02
`1.8 t 0.3
`1.8 i 0.1
`1.6 :5 0.2
`1 .6 i 0.1
`1 .6 f. 0.1
`2.0 i 0.3
`
`98
`314
`
`145
`62
`
`109
`
`59
`71
`
`19
`33
`81
`
`43, 194
`
`327
`83
`92
`52
`15
`
`
`198
`
`CPS1
`lLV3
`YPSS
`TSAt
`WTMt
`KTRS
`PEP4
`
`509.413
`
`CWH43
`
`DFM1
`
`ERPS
`
`NFTt
`NFTi
`EMP70
`SYM1
`
`MCH5
`lCY2
`PDH1
`
`Vacuolar carboxypeptidase
`Dihydroxyacid dehydratase
`GPlvanchored aspartic protease
`Thioredoxin-persxidase
`WD repeat containing transca'ptional modulator I
`Mannosylphosphate transferase
`Vacuolar proteinase A
`
`Unknown function
`Unknown function
`Putative sensor/transporter protein
`Unknown function
`Den-like family member
`Unknown function
`Unknown function
`Unknown function
`Member of p24 family
`Unknown function
`Unknown function
`Putative vacuolar aminopeptidase
`Unknown function
`Merged with YKR104 in some backgrounds
`Putative MRP-type ABC transporter
`Endosomal membrane protein
`Stress-induced yeast MPV17 homologue
`Unknown function
`Unknown function
`Unknown function
`Unknown function
`Unknown function
`Unknown function
`Unknown function
`Monocar’ooxylate permease homologue
`interacts with the cytoskeleton
`
`Homologue to E. coli prpD
`
`
`
`cfh1cth2 cells expressing CTH2 or vector alone were independently grown by triplicate in the presesce of 100 uM BPS (Fe depletion) until
`exponential cell phase; RNA was extsacted, labeled, and hybridized to yeast DNA microarrays as described in Bperémental Procedures. The
`gene expression profile of cells containing vector alone versus expressing CTH2 (cth2 versus CTHZ was determined and the average fold
`induction represented. Only mRNAs with a fold induction in the cth2 mutant higher than 15-fold and a p value < 0.05 are shown. ORF,
`open reading frame systematic name; gene, common name; function, description of the biological function of the protein according to the
`Saccharomyces Genome Database, published data, and sequence homology; ARES. AU-rich elements (5’—UUAUUUAUU-3’ nonarner sequence)
`positioned within the 500 nucleotides after translation termination codon. 5’-UAUUUAUU-3' and 5'-UUAUUUAU—3‘ octamers were indicated
`in italics. SD, standard deviation. The complete set of data is available at http://data.cgt,duke.edufiron.php.
`‘Genes below the cutoff but confirmed by RNA blotting analysis.
`
`by RNA blotting. As shown in Figure 4C, wild-type SDH4
`mRNA levels are dramatécally downregulated under Fe
`depletion, while SDH4-AREmt2 mRNA levels are unaf-
`fected by Fe. Furthermore, downregulation of CCC1
`mRNA by Fe depletion was also completely dependent
`on the 3'UTR (Supplemental Figure 83). Taken together,
`these results demonstrate that CTH2—dependent mRNA
`downregulation under low Fe conditions is dependent
`on the presence of specific AREs located in the 3’UTR.
`To ascertain whether ARES are sufficient for mRNA
`downregulation in response to Fe deprivation, chimeric
`transcripts were expressed that contain the coding se—
`quence of GCN4, a gene not regulated by Cth2 (data
`not shown), and the 3’UTR from either SDH4 or ACO1
`(Figures 4A and 4D), two genes whose mRNA steady-
`state levels are regulated by Cth2. While wild-type GCN4
`mRNA levels were not significantly decreased by Fe
`
`starvation, GCN4-ACO1-3’UTR mRNA was dramatically
`downregulated under Fe deprivation (Figure 4E). A simi-
`lar result was obtained when the 3'UTR of the SDH4
`mRNA was fused to GCN4 (Figure 4E). lmportantly, mu-
`tagenesis of the ARES in the SDH4-3’UTR abrogated
`the Fe dependent downregulation of GCN4-SDH4-
`3’UTR mRNA (Figure 4E, GCN4-SDH4-AREmt2). In addi-
`tion, the downregulation of both GCN4-ACOi-3’UTR
`and GCN4-SDH4—3’UTR mRNAs was completely de-
`pendent of the presence of a functional Cth2 protein
`(Figure 4F). cth1cth29cn4 mutants expressing either
`GCN4-ACO1-3’UTR or GCN4-SDH4»3’UTH were cotrans-
`fomied with vector, wild-type CTH2, or the CTH2-C190Fl
`mutant. As shown in Figure 4F, the Fe starvation-depen-
`dent decrease in steady-state levels for both mRNA
`species was abrogated in cells lacking CTH2 (vector
`lanes) and in cells with a nonfunctional allele of CTH2
`
`

`

`1
`0622 Controls a Posttranscriptional Fe Regulon
`
`A
`
`i3
`
`C
`
`SDH4-3’ Region
`
`
`
`
`
`T
`ORF
`
`i som'ma.
`”2;; ease
`
`”gig/g?
`
`SDH4-AREth
`
`SDH4
`SDH4 k’REmtz
`l—1F—l.
`
`
`
` GCN4-SDH4
`
`'
`
`;. GCN4-SDH4mt2
`
`gc'n4
`
`GCN4
`GCN4 SDH4
`GCN4
`GCN4 ACO1 GCN4 SDH4-AREmtz
`r———rr—""Ir——Ir--—II—1
`F'e+-+-+-’+-+-
`
`
`
`cth1’cth29cn4
`y—‘WM
`GCN4-SDH4 3'UTR GCN4-ACO1 3’UTR
`r-—----—-1 r———-’——"—"1
`CTH2 vectorC190R' CTHZ vector C1907?
`
`
`
`Figure 4. ARES in the 3'UTR of CTHZ Targets Induce mRNA Destabilization of a Reporter Gene under Fe Scarcity
`(A) Sequence of the 3’ region of SDH4 and ACO1 genes. Putative AREs are shown underlined. SDH4-3'UTR adenine residues mutagenized
`to cytosine in GCN4-SDH4-AREmt2 are shown in bold characters.
`(B) Schematic representation of wild-type SDH4 and SDH4-3’UTR mutant 2 (SDH4-AREmt2).
`he ARES located in the 3’UTFl. sdh4 cells expressing SDH4 and SDH4-
`(G) SDH4 downregulation in low Fe is dependent on the integrity of t
`AREth were grown and analyzed by RNA blotting as described in Figure SC.
`(D) Schematic representation ofwild-type GCN4 and GCN4 with 3'UTR replaced byACO1-3’UTH(GCN44A001), wild—type SDH4—31F“? (GCN4-
`SDH4), and mutant SDH4-ARE-mt2 (GCN4-SDH4mQ).
`(E) gcn4 cells expressing GCN4, GCN4-ACO1-3’UTH, GCN4oSDH4-3’UTR, and GCN4-SDH4—AREmt2 were grown in SC-Ura media (Fe +) and
`SC-Ura containing 100 ”M BPS (Fe -) and analyzed by RNA blotting with GCN4 and ACT1 probes.
`(F) cth1cchgcn4 cells expressing either GCN4—SDH4-3'UTR or GCN4-ACO1-3’UTR were transformed with vector alone or containing CTH2
`or CTH2-0190!? mutant allele and grown and analyzed by RNA blotting as described (E).
`
`(C1903 lanes). Taken together, these results demon—
`strate that the AREs found in the 3’UTR of both ACO1
`and SDH4 are necessary and sufficient to induce the
`CTH2 and Fe limitation-dependent downregulation of
`GCN4 mRNA.
`
`Cth2 Accelerates the Rate of mRNA Decay
`Our data strongly implicate Cth2 and 3’UTR ARES in the
`coordinated downregulation of specific mRNAs by Fe
`deprivation. Steady-state mRNA measurements are the
`net consequence of both transcription and the rate of
`mRNA decay, and our analyses are consistent with Cth2
`acting at a posttranscriptéonal level. To evaluate the effects
`of 0ch on mRNA decay rates, two Cth2—dependent target
`mRNAs were conditionally expressed in yeast using the
`galactose-inducible and glucose-repressible GAL1 pro-
`moter. cth1cth29¢n4 cells were cotransformed with
`GCN4-AC01-3'UTR or GCN4-SDH4-3'UTR constructs
`dréven by the GAL1 promoter (Figure 5) and plasmid-
`bome CTH2 or empty vector. Cells were grown in galac-
`
`tose and the Fe chelator BPS to induce transcription
`of GCN4-ACO1/SDH4-3’UTR and CTHZ, respectively.
`Transcription of the GCN4-ACO1 or GCN4vSDH4-3’UTR
`genes was shut off by gluc05e addition and mRNA levels
`analyzed over time by RNA blotting (Figure 5). The half-
`life of GCN4—ACO1—3’UTR mRNA decreased from 7 min
`to 3 min when CTH2 was expressed (Figure 5A). A similar
`decrease in the half-life, from 9 to 4 min, was observed
`for GCN4-SDH4-3'UTFl mRNA in cells expressing CTH2
`(Figure SB). No change in mRNA half-life was observed
`when the cells expressed the CTHZ-Ci'QOR mutant allele
`or when they were grown in the presence of Fe, condi-
`tions that severely repress the expression of CTH2 (data
`not shown). Furthermore, while the half-life of a mRNA
`including the SDH4 codéng sequence and 3'UTR was
`7 min in wild-type cells growing under Fe-deficient con-
`ditions, it increased to 14 min in either cells lacking CTHZ,
`wild-type cells grown in Fe-replete conditions, or in CFH2
`wild-type cells expressing SDH4 mRNA with mutated
`ARES (Figure 50). Similar results were obtained for the
`
`

`

`Promote?
`cg]:
`3mm
`
`a
`str_
`
`
`W Haif-lif'e
`
`CTHz
`
`Vector
`
`\
`
`
`
`3
`
`?
`
`Z2“? ”iii:
`
`mrahzsdm
`
`9 5 1o 15- 20
`
`(mini
`
`
`
`
`vector
`
`-Fe
`
`'c-TH2
`
`-Fe
`
`SDI-l4
`
`sum
`
`mm '+ Fe
`
`sum
`
`cer
`
`~Fe st-
`AREmt2
`
`14
`
`7'
`
`14
`
`14
`
`Cell
`106
`
`A
`
`B
`
`Promoter
`ORF
`
`
`
`promoter
`ORF
`sum
`éfita
`CCP1
`"*””
`
`
`Min-(after shutoff
`.0 5_.10 1-5 20
`
`Bait-life
`(min)
`
`
`
`vector
`
`cthmmc-CP’
`
`Min. after shutoff
`o a 10 15 20 30
`
`Half-fife
`(min)
`
`vector
`
`CTHZ
`
`-Fe
`
`
`- Fe WW Wt;
`
`.
`
`..
`
`“WM-win
`
`17
`
`11
`
`Worm 637;” as assess w 18
`
`Figure 5. Cth2 Accelerates the Decay of