# Role of the DHH1 Gene in the Regulation of Monocarboxylic Acids Transporters Expression in Saccharomyces cerevisiae

Sandra Mota, Neide Vieira, Sónia Barbosa, Thierry Delaveau, Claire Torchet, Agnès Le Saux, Mathilde Garcia, Ana Pereira, Sophie Lemoine, Fanny Coulpier, et al.

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## To cite this version:

Sandra Mota, Neide Vieira, Sónia Barbosa, Thierry Delaveau, Claire Torchet, et al. Role of the DHH1 Gene in the Regulation of Monocarboxylic Acids Transporters Expression in Saccharomyces cerevisiae. PLoS ONE, 2014, 9 (11), pp.e111589. 10.1371/journal.pone.0111589. hal-01527344

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# Role of the *DHH1* Gene in the Regulation of Monocarboxylic Acids Transporters Expression in *Saccharomyces cerevisiae*

Sandra Mota¹,²*, Neide Vieira¹*, Sónia Barbosa¹, Thierry Delaveau³,⁴, Claire Torchet⁵,⁶, Agnès Le Saux⁷, Mathilde Garcia³,⁴, Ana Pereira¹, Sophie Lemoine⁸,⁹,¹⁰, Fanny Coulpier⁸,⁹,¹⁰, Xavier Darzacq⁸,⁹,¹⁰, Lionel Benard⁵,⁶, Margarida Casal¹, Frédéric Devaux³,⁴*, Sandra Paiva¹*

¹Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, Braga, Portugal, ²Centre of Health and Environmental Research (CISA), School of Allied Health Sciences, Polytechnic Institute of Porto, Vila Nova de Gaia, Portugal, ³Sorbonne Universités, Université Pierre et Marie Curie, UMR7238, Laboratoire de Biologie computationnelle et quantitative, Paris, France, ⁴CNRS, UMR7238, Laboratoire de Biologie computationnelle et quantitative, Paris, France, ⁵CNRS, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, Paris, France, ⁶Sorbonne Universités, Université Pierre et Marie Curie UPMC, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, Paris, France, ⁷CNRS, FRE3630, Laboratoire de l'Expression Génétique Microbienne, Institut de Biologie Physico-Chimique, Paris, France, ⁸École normale supérieure, Institut de Biologie de l'ENS, IBENS, Paris, France, ⁹Inserm, U1024, Paris, France, ¹⁰CNRS, UMR 8197, Paris, France

## Abstract

Previous experiments revealed that *DHH1*, a RNA helicase involved in the regulation of mRNA stability and translation, complemented the phenotype of a *Saccharomyces cerevisiae* mutant affected in the expression of genes coding for monocarboxylic-acids transporters, *JEN1* and *ADY2* (Paiva S, Althoff S, Casal M, Leao C. FEMS Microbiol Lett, 1999, 170:301–306). In wild type cells, *JEN1* expression had been shown to be undetectable in the presence of glucose or formic acid, and induced in the presence of lactate. In this work, we show that *JEN1* mRNA accumulates in a *dhh1* mutant, when formic acid was used as sole carbon source. *Dhh1* interacts with the decapping activator *Dcp1* and with the deadenylase complex. This led to the hypothesis that *JEN1* expression is post-transcriptionally regulated by *Dhh1* in formic acid. Analyses of *JEN1* mRNAs decay in wild-type and *dhh1* mutant strains confirmed this hypothesis. In these conditions, the stabilized *JEN1* mRNA was associated to polysomes but no *Jen1* protein could be detected, either by measurable lactate carrier activity, *Jen1*-GFP fluorescence detection or western blots. These results revealed the complexity of the expression regulation of *JEN1* in *S. cerevisiae* and evidenced the importance of *DHH1* in this process. Additionally, microarray analyses of *dhh1* mutant indicated that *Dhh1* plays a large role in metabolic adaptation, suggesting that carbon source changes triggers a complex interplay between transcriptional and post-transcriptional effects.

**Citation:** Mota S, Vieira N, Barbosa S, Delaveau T, Torchet C, et al. (2014) Role of the *DHH1* Gene in the Regulation of Monocarboxylic Acids Transporters Expression in *Saccharomyces cerevisiae*. PLoS ONE 9(11): e111589. doi:10.1371/journal.pone.0111589

**Editor:** Marie-Joelle Virolle, University Paris South, France

**Received** July 21, 2014; **Accepted** September 26, 2014; **Published** November 3, 2014

**Copyright:** © 2014 Mota et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability:** The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. The *S. cerevisiae* microarrays used are fully described in Array express (www.ebi.ac.uk/microarray-as/aer/entry; accession number A-MEXP-337).

**Funding:** This study was supported by the Portuguese grant POCI/BIA-BCM/57812/2004 (Eixo 2, Medida 2.3, QCAIII – FEDER). N.V. received a FCT PhD fellowship (SFRH/BD/23503/2005). S.M. received a FCT PhD fellowship (SFRH/BD/74790/2010). F.D.'s work is supported by a grant from the Agence pour la Recherche contre le Cancer (ARC). Support to C.B.M.A. by FEDER through POFC-COMPETE and by Portuguese funds from FCT through the project PEST-OE/BIA/UI4050/2014 is also acknowledged. The authors thank Josette Banroques and Kyle Tanner for their advice regarding polysome gradients. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing Interests:** The authors have declared that no competing interests exist.

## Introduction

The cellular metabolism of most yeasts, including *Saccharomyces cerevisiae*, is set to run essentially on glucose. When this yeast encounters harsh conditions in niches deprived from glucose, the ability to transport and metabolize non-fermentable carbon sources is crucial for its survival. In this manner, the uptake of short-chain carboxylic acids across the plasma membrane plays a defining role in the metabolism of yeast cells and in its pH-stasis [1]. Physiological studies, carried out in this baker's yeast, identified two distinct monocarboxylate proton symporters,

strongly repressed by glucose, with different specificities and regulation. A permease involved in the uptake of lactate-pyruvate-acetate and propionate was identified in lactic or pyruvic acid-S. *cerevisiae* grown cells [2,3], being encoded by *JEN1* [4], whereas, an acetate-propionate-formate permease was found in ethanol or acetic acid grown cells, with no obvious gene candidate at that time [5,6]. Later, *ADY2* was identified as the acetate permease encoding gene in *S. cerevisiae* [7].

In an early attempt to identify the genes involved in acetate-propionate-formate transport, classical genetic studies were carried out. The strain *S. cerevisiae* W303-1A was subjected to

<footnote>* Email: spaiva@bio.uminho.pt (SP); frederic.devaux@upmc.fr (FD)</footnote>

<footnote>¶ These authors contributed equally to this work.</footnote>
UV mutagenesis, in order to obtain mutants affected in the ability to utilize acetic acid, but unaffected on the capacity to grow in ethanol, as the sole carbon and energy source [8]. According to this strategy, it was hypothesised that mutants specifically affected in monocarboxylate permease(s) activity could be found. A mutant clone, exhibiting growth on ethanol, but with pronounced growth defect in a medium with acetic acid, as the sole carbon and energy source, was isolated (Ace8 strain) [8]. Further genotypic characterization of the Ace8 mutant led to the identification of the *DHH1* gene as a most likely candidate for explaining the Ace8 phenotype. Indeed, the transformation of Ace8 cells with a genomic fragment containing *DHH1* restored their capacity to grow on acetate and the deletion of *DHH1* presented slower growth rates than the isogenic wild-type on acetic acid (Paiva, S. 2002 PhD thesis, Fig. S1 in File S1).

*DHH1* encodes a RNA helicase of the DEAD-box subfamily [9,10]. Several homologs have been described in a broad range of organisms, namely in: *S. pombe*, *STE13* [11], in *Xenopus laevis*, *XP54* [12], in *Drosophila melanogaster*, *ME31B* [13], in mouse, *DDX6* [14,15], in humans, *DDX6/P54/RCK* (encoding for an oncoprotein) [14] and in *Caenorhabditis elegans CGH-1* [16]. Dhh1 has been involved in the formation of specific dynamic cytoplasmic loci, the Processing Bodies (P-bodies). P-bodies have been observed in yeasts, insect cells, nematodes and mammalian cells as cytoplasmic foci accumulating translationally silent mRNAs and containing proteins involved in mRNA decay and translation inhibition, including the deccaping enzymes (Dcp1/Dcp2), as well as general activators of deccaping, like Dhh1, Pat1, Lsm1-7 complex, Edc3, the 5'-3' exonuclease Xrn1, the Non sense mediated mRNA Decay (NMD) regulator Nam7/Upf1, components of the deadenylation machinery, other translational inhibitors, but also translational elongators and ribosomal subunits [17-21] (reviewed in [22]). P-bodies were implicated in mRNA decay, mRNA storage and translation repression [23-25], miRNA-mediated repression [26,27], nonsense-mediated decay [28,29] and viral packaging [30]. In consequence, P-bodies have been proposed to be important players of the cytoplasmic "mRNA cycle", where normal or aberrant mRNAs having reduced translational rates and enhanced decapping and deadenylation activities are targeted and where they can either be degraded or stored for further translation [31]. However, it should be noted that their role as loci where cytoplasmic mRNA decay actually occurs is still controversial [31]. Namely, many studies have shown that the formation of microscopically detectable P-bodies does not seem to be required for most mRNA degradation pathways and that the mRNAs stored in P-bodies are more stable than the mRNAs found free in the cytoplasm (reviewed in [32]).

Dhh1 plays a fundamental role in regulating the balance between active translation, accumulation in P-bodies and cytoplasmic 5'-3' decay of mRNAs [33]. Dhh1 physically interacts with the decapping enzyme activator Dcp1, the deccaping enhancers Pat1, Lsm1, Edc3 and the Ccr4-Pop2-Not deadenylase complex [34-39]. Mutants deleted for *DHH1*, showed a deficient mRNA decay, longer half-life times of several mRNAs and accumulated capped deadenylated transcripts, indicating that Dhh1 acts as an activator of decapping [35,40]. More precisely, Dhh1 has been proposed to act on mRNA translation rates [24,41,42], based on the reported competition between mRNA deccaping and translation initiation [23,36]. Former experiments suggested that Dhh1 uses its ATPase activity to release eIF4F complex from the mRNP, or somehow destabilize this eIF4F-mRNA cap complex, and in this manner repress translation initiation with concomitant deccaping stimulation [36]. However, this model has been recently challenged by data indicating that

Dhh1 could promote decapping by slowing translation elongation downstream to the initiation step [43] and that its ATPase activity is required for regulating P-bodies dynamics but not translation inhibition [33]. Hence, Dhh1 plays a role in the regulation of several cellular processes [44] including mating [45], filamentous growth [46] and iron deficiency [47]. Moreover, Dhh1 controls the turn-over of the mRNA encoding the decapping enhancer Edc1 [44] and *DHH1* is epistatic on the *DCS1* gene, encoding a decapping enzyme scavenger [48]. Furthermore, *DHH1* has also been involved in mRNA and tRNA nuclear export [41,49-51]. Dhh1 is required for the efficient retrotransposition of Ty1 elements in yeast [52]. Finally, overexpression of *DHH1* also suppresses defects of the mitochondrial Rnase P subunit Rpm2 [53].

In order to elucidate the involvement of this Dead-box RNA helicase in monocarboxylate transport and in the regulation of non-fermentable carbon sources utilization in *S. cerevisiae*, we studied the role of Dhh1 in the expression of the Jen1 permease. We showed that, in the presence of formic acid as sole carbon source, *JEN1* expression is negatively controlled at the post-transcriptional level by a Dhh1-dependent mechanism. The deletion of *DHH1* led to the accumulation of *JEN1* mRNAs which were associated to polysomes but for which no Jen1 protein could be detected, questioning the fact that they are eventually translated. Furthermore, analyses of the wild-type and *dhh1* mutant cell transcriptomes evidenced the broad involvement of this RNA helicase in the control of various cellular pathways.

## Materials and Methods

### Yeast strains, plasmids and growth conditions

*S. cerevisiae* strains used in this work are listed in table 1 and the plasmids in table 2. The cultures were maintained on plates of yeast extract (1%, w/v), peptone (1%, w/v), glucose (2%, w/v) and agar (2%, w/v). Yeast cells were grown in YNB glucose 2.0% (w/v), supplemented with adequate requirements for prototrophic growth. Carbon sources were glucose (2%, w/v), lactic acid (0.5%, v/v, pH 5.0), acetic acid (0.5%, v/v, pH 6.0), formic acid (0.5%, w/v, pH 5.0) and propionic acid (0.5%, v/v, pH 5.0). Solid media were prepared adding agar (2%, w/v) to the respective liquid media. Growth was carried out at 30°C, both in solid or liquid media. The cells were also directly grown in rich media, YP lactic acid 0.5% pH 5.0, or YP acetic acid 0.5% pH 6.0. YNB glucose-containing media was used for growth of yeast cells under repression conditions. For derepression conditions glucose-grown cells were harvested during the exponential phase of growth, centrifuged, washed twice in ice-cold deionised water and cultivated into fresh YNB medium supplemented with a carbon source of choice.

### Strains construction

The yeast strains, the plasmids and the primers used in this work are listed respectively in Tables 1, 2 and 3. The mutant strain, *dhh1*, carrying a *dhh1::kanMX4* locus, was transformed with the hygromycin resistance gene *hphMX4* resulting in a marker switch producing the *dhh1::hphMX4 locus* [54]. The same procedure was made for the strain W303-1A: *JEN1::GFP-kanMX4* resulting in the strain W303-1A: *JEN1::GFP-hphMX4*. The *S. cerevisiae* strain BLC 491-U2, was used to amplify the genetic chimaera, *JEN1::GFP-kanMX4*, using the primers W303-1A forward and W303.1A reverse [55]. The *dhh1::hphMX4* strain was subsequently transformed with the 2.8 Kb *JEN1::GFP-kanMX4* PCR product resulting in strain NV2. Transformed cells were grown in
<table><caption>**Table 1.** *S. cerevisiae* strains used in this work.</caption><thead><tr><th>Strains</th><th>Genotype</th><th>Reference</th></tr></thead><tbody><tr><td>W303-1A</td><td>MATa ade2-1; leu2-3, 112; his3-11, 15; trp1Δ2; ura3-1; can 1-100</td><td>[90]</td></tr><tr><td>ACE 147</td><td>W303-1A; dhh1Δ::kanMX4</td><td>Paiva S., 2002 PhD thesis</td></tr><tr><td>jen1</td><td>W303-1A; jen1Δ::His3</td><td>(Casal et al. 1999)</td></tr><tr><td>BLC 491-U2</td><td>MATa ura3-52 JEN1::GFP-kanMX4</td><td>[55]</td></tr><tr><td>NV1</td><td>ACE 147; dhh1Δ::hphMX4</td><td>This work</td></tr><tr><td>NV2</td><td>NV1; JEN1::GFP-kanMX4</td><td>This work</td></tr><tr><td>ACE 145</td><td>W303-1A; JEN1::GFP-kanMX4</td><td>Paiva S., 2002 PhD thesis</td></tr><tr><td>BY 1 (YJL124c)</td><td>BY4742; MAT alpha; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YJL124c::kanMX4</td><td>Euroscarf</td></tr><tr><td>BY 2 (YCR077c)</td><td>BY4741; MAT a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YCR077c::kanMX4</td><td>Euroscarf</td></tr><tr><td>BY 3 (YMR080c)</td><td>BY4741; MAT a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YMR080c::kanMX4</td><td>Euroscarf</td></tr><tr><td>BY 4 (YOR076c)</td><td>BY4741; MAT a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YOR076c::kanMX4</td><td>Euroscarf</td></tr><tr><td>MAR 5</td><td>W303-1A; JEN1::GFP-hphMX4</td><td>This work</td></tr><tr><td>MAR 6</td><td>MAR 5; lsm1Δ</td><td>This work</td></tr><tr><td>MAR 7</td><td>MAR 5; pat1Δ</td><td>This work</td></tr><tr><td>MAR 8</td><td>MAR 5; nam7Δ</td><td>This work</td></tr><tr><td>MAR 9</td><td>MAR 5; ski7Δ</td><td>This work</td></tr><tr><td>MAR 14</td><td>W303-1A; lsm1Δ</td><td>This work</td></tr><tr><td>MAR 15</td><td>W303-1A; pat1Δ</td><td>This work</td></tr><tr><td>MAR 16</td><td>W303-1A; nam7Δ</td><td>This work</td></tr><tr><td>MAR 17</td><td>W303-1A; ski7Δ</td><td>This work</td></tr></tbody></table>

doi:10.1371/journal.pone.0111589.t001

YPD media, for 4 hours, and spread on YPD plates, containing 200 mg L⁻¹ of Geneticin (G418 from Invitrogen) and 300 mg L⁻¹ of Hygromycin (Hygromycin B from Invitrogen). The obtained transformants were confirmed by analytical PCR, with primers A1 and GFP rev [56].

To obtain the *lsm1*, *pat1*, *nam7* and *ski7* mutants in the W303-1A background, strains BY 1 to 4 were used to amplify the corresponding deletion cassettes, using the respective A and D primers. The strains W303-1A and W303-1A: *JEN1::GFP-hphMX4* were then transformed with this PCR product. The transformed cells were grown in YPD media for 4 hours and spread on YPD plates, containing 200 mg L⁻¹ of Geneticin and/or 300 mg L⁻¹ of Hygromycin.

Cloning and PCR amplification analyses were performed as previously described [57].

## Transport assays

YNB glucose-containing media was used for growth of yeast cells under repression conditions. For derepression conditions glucose-grown cells were harvested during the exponential phase of growth, centrifuged, washed twice in ice-cold deionized water and cultivated into fresh YNB medium supplemented with a

carbon source of choice. Cells were harvested by centrifugation, washed twice and resuspended in ice-cold deionized water to a final concentration of 20–40 mg dry weight/ml. Conical centrifuge tubes containing 30 µl of 0.1 M KH₂PO₄ buffer at pH 5.0 and 10 µl of the yeast suspension were incubated for 2 min at 26°C. The reaction was started by the addition of 10 µl of an 2 mM aqueous solution (saturation concentration) of 4000 d.p.m./nmol of radiolabeled [¹⁴C] lactic or [¹⁴C] acetic acid (sodium salt; GE Healthcare) at pH 5.0. The reaction was stopped by dilution with 5 ml of ice-cold water. The reaction mixtures were filtered immediately through GF/C membranes (GE Healthcare) and the filters were washed with 10 ml of ice-cold water and transferred to scintillation fluid (Opti-Phase HiSafe II; Pharmacia LKB). Radioactivity was measured in a liquid scintillation spectrophotometer (Tri-Carb 2200 CA; Packard Instrument Co.) equipped with a d.p.m. correction facility. For nonspecific adsorption of [¹⁴C] lactic acid was added at time zero after the cold water. All experiments were repeated at least three times, and the data reported represents average values. Data obtained is represented as the mean ± SD of triplicate measurements.

<table><caption>**Table 2.** Plasmids used in this study.</caption><thead><tr><th>Plasmids</th><th>Source or references</th></tr></thead><tbody><tr><td>pT12</td><td>[4]</td></tr><tr><td>pPDA1</td><td>Andrade, R. (This work)</td></tr><tr><td>pAG32</td><td>[54]</td></tr></tbody></table>

doi:10.1371/journal.pone.0111589.t002
<table><caption>**Table 3.** Primers used in this work.</caption><thead><tr><th>Primers</th><th>Sequence</th></tr></thead><tbody><tr><td>W303-1A forward</td><td>GATTTGTCCTCTCCTGTTATGAAG</td></tr><tr><td>W303-1A reverse</td><td>ATCTTGCTAGTGTTAACGGCTGTTA</td></tr><tr><td>A1</td><td>GGCCTATCCAAGGATGCTGTC</td></tr><tr><td>GFP_rev</td><td>AACATCACCATCTAATTCAAC</td></tr><tr><td>A LSM1</td><td>ACCGTATGGGTCTTTGATACACTTA</td></tr><tr><td>D LSM1</td><td>GGTCTACTGAGCTTACAATAGCAGC</td></tr><tr><td>A PAT1</td><td>CATTTTAATGGAGTAATTGTCCTGG</td></tr><tr><td>D PAT1</td><td>TCAAATAGTCGTTCTCCTCAAGTTC</td></tr><tr><td>A NAM7</td><td>TTTAGTATCATCAGTTTCCCTTTGC</td></tr><tr><td>D NAM7</td><td>TGATTAAACGAGCTTCAATTTTTC</td></tr><tr><td>A SKI7</td><td>GTGATTTTCTACAATCAAACAACCC</td></tr><tr><td>D SKI/</td><td>GAAATTCTCAATGGCTACTTTACGA</td></tr><tr><td>K2</td><td>CGATAGATTGTCGCACCTG</td></tr><tr><td>K3</td><td>CCATCCTATGGAACTGCCTC</td></tr></tbody></table>

doi:10.1371/journal.pone.0111589.t003

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

**Figure 1.** *JEN1* expression profile. A- Transcription analyses of *JEN1* in *S. cerevisiae* W303-1A wild-type and *dhh1* cells. Total RNA was isolated from YNB Glucose 2% (w/v) grown cells, collected at mid exponential phase, and after induction for 4 or 6 hours in different non-fermentable carbon sources: G – glucose; L – lactic acid (4 hours); E – ethanol (4 hours); Py – pyruvic acid (4 hours); A – Acetic acid (4 hours); F – formic acid (4 hours); F* – formic acid (6 hours); Pr – propionic acid (4 hours); Pr* – propionic acid (6 hours); Gly – glycerol (4 hours). An internal *JEN1* fragment was used as probe. *PDA1* was used as a reference, for relatively constant transcription. B- RT-qPCR analyses of *JEN1* mRNA expression levels in wild type and *dhh1* cells grown on glucose, lactic acid, acetic acid or formic acid. The *JEN1* expression levels indicated here are relative to *SCR1*, a RNA pol III transcript which is not supposed to be sensitive to the deletion of *DHH1*. A different control was used for Q-PCR (*SCR1*) and northern blot (*PDA1*) analyses to ensure that the measured effects were not a bias coming from the control. The levels of *JEN1* mRNAs measured in wild-type cells in lactic acid were used as a reference and arbitrarily set up at a value of 1. The experiments were performed three times on biologically independent samples.
doi:10.1371/journal.pone.0111589.g001
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Real time quantitative RT-PCR analyses of JEN1 mRNA steady states in different carbon sources and genetic contexts. Wild-type, *dhh1*, *pat1*, *lsm1*, *nam7* and *ski7* strains were grown in formic, acetic or lactic acid as sole carbon sources. The levels of JEN1 mRNA were measured by real time PCR and normalized by the levels of *SCR1* RNA. The levels of JEN1 mRNAs measured in wild-type cells in lactic acid were used as a reference and arbitrarily set up at a value of 1. The experiments were performed three times on biologically independent samples.
doi:10.1371/journal.pone.0111589.g002</figcaption></figure>

Microscopy

S. cerevisiae living cells were examined with a Leica Microsystems DM-5000B epifluorescence microscope with appropriate filter settings. Images were acquired with a Leica DCF350FX digital camera and processed with LAS AF Leica Microsystems software.

RNA analysis

Total RNA was isolated using the standard hot acidic phenol
protocol. In a 1.5% (w/v) agarose/MOPS/formaldehyde gel,
samples of 20 µg RNA were electrophorised and blotted onto a
Hybond- N+ membrane [58]. An internal fragment of 844 bp,
obtained by the digestion of the pT12 plasmid (Table 2) with the
restriction enzymes NcoI and PstI, was ³²P-labelled and used as a
JEN1 probe. As internal control RNA of PDA1 was also used. For
mRNA relative half-life times (t ¹/₂ mRNA) determination,
inhibition of transcription was accomplished by the addition of
1,10-phenanthroline (0.1 mg.ml⁻¹) for 0, 4, 10 and 20 minutes
[59]. Real time quantitative RT PCR experiments were conducted
as previously described (Garcia et al., MBC 2007) using primer
pairs specific of JEN1 (sequences), ACT1 (sequence) or SCR1
(sequences). Relative half-live times were determined by measuring
the ratio between amounts of JEN1 mRNA and those of SCR1 at
each time point and applying, a linear regression equation to the
Log (JEN1/SCR1) = f(t) function. The calculation of the slope

directly gave access to the relative half-life of *JEN1* in the mutant
and the wild-type strain. The reported half-live times represent a
mean value obtained from three experiments performed on
independent biological samples.

Polysome gradients

dhh1 mutant cells were grown in YNB glucose media until they
reached an optical density of 0.8. Then, they were washed twice in
sterile water and transferred into an equivalent volume of either
YNB lactic acid or YNB formic acid media. After 5 hours,
cycloheximide was added to the cells at a final concentration of
100 µg/ml. After 5 minutes of incubation on ice, the cultures were
centrifuged 5 minutes at 3000 g, washed once with sterile water
and a second time with lysis buffer (Tris-HCl pH 7.4 20 mM,
NaCl 50 mM, MgCl₂ 5 mM, DTT 1 mM, cycloheximide
100 µg/ml). After the last centrifugation, the cell pellet was
resuspended in 800 µl of lysis buffer including 1X protease
inhibitor cocktail (Roche). An equivalent volume of glass beads
were added and the cells were disrupted by vortexing 6 times
30 seconds, at 4°C). The supernatant was collected and centri-
fuged 5 minutes at 5000 g (4°C). The supernatant was cleared by
two rounds of centrifugation 10 minutes at 12000 g (4°C). The
final supernatant was stored at -80°C after adding 10% glycerol.
About 600 µl of this solution was loaded on a 10%-50% sucrose
gradient, centrifuged at 39000 g for 3 hours and 0.5 ml fractions
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Real time quantitative RT-PCR analyses of *JEN1* mRNA stability in YP lactic acid-grown wild-type, *nam7* and *dhh1* mutant cells. Mutant and wild-type cells were grown in YP lactic acid and collected immediately before (time zero) or 4, 10 or 20 minutes after the addition of 1,10-phenantroline (0.1 mg/ml). The expression levels are expressed relatively to the *SCR1* expression level used as a control. These relative expression levels were set to one at time zero for both strains, in order to normalize differences in mRNA levels between the two strains at the beginning of the experiment. Squares represent the values obtained for the *dhh1* mutant, triangles represent the values obtained for the *nam7* mutant and diamonds represent the values obtained for the wild-type. The experiments were performed three times on biologically independent samples.</figcaption></figure>

doi:10.1371/journal.pone.0111589.g003

were collected in 2 ml tubes using a Retriever 500 (ISCO) fraction collector and a Type 11 Optical unit (ISCO) with 254 nm filters. The RNAs contained in the fractions were precipitated by added 50 µl of ammonium acetate 3 M pH 5.3 and 1.2 ml of absolute ethanol. The mixtures were stored overnight at -20°C, and then they were centrifuged for 20 minutes at 15000 g (4°C). The pellets corresponding to the polysomes fractions were resuspended with the same 100 µl of water. The pellets corresponding to the other

fractions were pooled in another 100 µl of water. The RNA was then purified and cleaned up using the RNA easy midi kit (Qiagen), following the supplier's recommendations. The RNA was quantified by spectrometry and 0.5 µg (for lactic acid) or 1.5 µg (for formic acid) were used for reverse transcription and real-time quantitative PCR analyses using the *JEN1* and *ACT1* primer probes as described above.

<figure><img src="image_7.png" /><figcaption>**Figure 4.** Transport activity of lactic acid in *S. cerevisiae* W303-1A strains: wild-type, *dhh1*, *jen1* (A), *lsm1*, *ski7*, *pat1* and *nam7* (B). The results are percentages of initial activities of 2 mM [¹⁴C] lactic acid uptake, pH 5.0. Cells were grown in YNB glucose and derepressed in YNB lactic acid or YNB formic acid. Wild-type and *dhh1* YNB lactic acid derepressed cells were used as a control.</figcaption></figure>

doi:10.1371/journal.pone.0111589.g004
A

<figure><img src="image_8.png" /></figure>

B

<figure><img src="image_9.png" /></figure>

**Figure 5. Jen1 protein expression.** A - Subcellular localization of Jen1::GFP in *S. cerevisiae* living cells. Wild-type, W303-1A, *dhh1*, *lsm1*, *pat1*, *nam7* and *ski7* mutant cells harboring Jen1::GFP were used to follow Jen1 expression after growth in YNB glucose, and derepression for 4 hours in YNB lactic 0.5%, pH 5.0, or YNB formic acid 0.5%, pH 5.0. B - Total protein extracts from wild type and *dhh1* mutant cells harboring Jen1::GFP were used to follow Jen1 expression after growth in YNB glucose, and derepression for 4 hours in YNB lactic 0.5%, pH 5.0, or YNB formic acid 0.5%, pH 5.0 and immunoblotted with the indicated antibodies. We add a sample of extracts from lactic acid 50 fold diluted.
doi:10.1371/journal.pone.0111589.g005

Microarray analysis

Detailed protocols are described at http://www.transcriptome.
ens.fr/sgdb/protocols/. The *S. cerevisiae* microarrays used are
fully described in Array express (www.ebi.ac.uk/microarray-as/
aer/entry; accession number A-MEXP-337). The microarray
experiments were conducted as previously described [60]. Raw
data were normalized using global lowess followed by print-tip
median methods, with background removal, as implemented in
Goulphar [61]. Experiments were carried out 2 times, with dye

swapping. The microarray data are available in Table S2 and fully
available at the GEO database (accession number: GSE60983).
The statistical significance of the expression variations measured
was addressed by using the TMEV version of SAM with a FDR of
5%, a S0 calculated by the Tusher method and using the exact
number of permutation [62–64]. Only genes that passed the SAM
filter and had an average log2 of ratio above 0.9 in glucose or in
formic acid were considered as significantly changing their
expression in the mutant compared with the wild type. These
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 6. Polysome gradient analyses of *dhh1* mutant cells grown in lactic or formic acids.** A: Absorbance profiles along a polysomes gradient obtained with cells grown in lactic acid. The fractions corresponding to the polysomes are indicated. B: the polysomes fractions obtained in acetic and lactic acid were pooled and the percentage of *JEN1* mRNA contained in this fractions compared to the rest of the gradient was quantified by RT-QPCR. In contrast to previous Q-PCR experiments, *SCR1* could not be used as a reference because it is not translated. Hence, *ACT1* was used as a control for an mRNA which was actively transcribed in both lactic and formic acids. The experiments were performed three times on biologically independent samples. The difference of *JEN1* abundance in polysomal fractions between lactic and formic acid was not significant, according to a Student test.</figcaption></figure>

doi:10.1371/journal.pone.0111589.g006

<figure><img src="image_12.png" /><figcaption>**Figure 7. Transcriptome analyses of Dhh1 impact on gene expression.** Venn diagram representing the overlap of down (left) and up (right) regulation effects in a *dhh1* mutant, grown either in glucose or in formic acid. The main functional categories enriched in each group are indicated. They were determined using the FUNSPEC web tool (funspec.med.utoronto.ca/). The complete set of genes in each category, together with their functional annotation, can be found in Table S1.</figcaption></figure>

doi:10.1371/journal.pone.0111589.g007
<figure><img src="image_13.png" /><figcaption>**Figure 8.** *ADY2* expression profile. A- Transcription analyses of *ADY2* in *S. cerevisiae* W303-1A and *dhh1* cells. Total RNA was isolated from YNB Glucose 2%-grown cells, collected at mid exponential phase, and after induction for 4 or 6 hours in different non-fermentable carbon sources: G - glucose; L - lactic acid (4 hours); E - ethanol (4 hours); Py - pyruvic acid (4 hours); A - Acetic acid (4 hours); F - formic acid (4 hours); F* - formic acid (6 hours); Pr - propionic acid (4 hours); Pr* - propionic acid (6 hours); Gly - glycerol (4 hours). An internal *ADY2* fragment was used as probe. *PDA1* was used as a reference, for relatively constant transcription. B- Densiometry analysis of *ADY2* Northern blots was performed on scanned films using ImageJ gel analysis tool (public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/)). Absolute intensities were calculated for both *ADY2* and the *PDA1* control. Relative intensities were calculated for each experimental band by normalizing the absolute intensity to the corresponding control intensity.
doi:10.1371/journal.pone.0111589.g008</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 9.** Transport activity of Ady2 in *S. cerevisiae* W303-1A strains: wild-type, *dhh1*, *lsm1*, *ski7*, *pat1* and *nam7*. The results are percentages of initial activities of 2 mM [¹⁴C] acetic acid uptake, pH 5.0. Cells were grown in YNB glucose and derepressed in YNB acetic acid or YNB formic acid. Wild-type and *dhh1* YNB acetic acid derepressed cells were used as a control.
doi:10.1371/journal.pone.0111589.g009</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure 10. Model for JEN1 expression regulation.** This model is inferred from the data presented in this work. Briefly, in glucose, *JEN1* is transcriptionally silent. In lactate or acetate, *JEN1* is transcriptionally activated by *Cat8* and *JEN1* mRNA are actively translated, which results in the accumulation of *Jen1* in the plasma membrane and in an active transport of carboxylic acids. In formic acid, *JEN1* is transcriptionally active, but the *JEN1* mRNAs are targeted to degradation in P-bodies and therefore barely detectable by northern blots. In the absence of *Dhh1* or *Pat1*, *JEN1* mRNA are no more degraded but still the *Jen1* protein was not detectable, which result in an accumulation of mRNA with no or very few *Jen1* protein in the membrane and no or very few active transport of carboxylic acids. Our data cannot tell if this low protein level is due to the low RNA level or to actual translation inhibition.</figcaption></figure>

doi:10.1371/journal.pone.0111589.g010

genes were listed in Table S1. Global functional analyses were
performed using FUNSPEC [65].

## Results

### JEN1 expression profile

W303-1A and *dhh1* cells were grown in different carbon and energy sources, in an effort to clarify the mechanisms involved in the expression regulation of *JEN1* by *DHH1*. The expression pattern of *JEN1* was studied by Northern blot analyses (Fig. 1A). In the wild-type strain, *JEN1* was highly expressed in lactate, acetate and glycerol, poorly expressed in ethanol and almost totally absent in glucose, formic acid and propionic acid. In the *dhh1* strain, the *JEN1* expression profile was similar to the wild-type with the notable exceptions of pyruvic, acetic, formic and propionic acids. In the presence of formate and propionate, the *JEN1* mRNA largely accumulated in the mutant, indicating either

a derepression of *JEN1* transcription or a stabilization of *JEN1* mRNA in this strain as compared with the wild-type. In the presence of pyruvate and acetate, an opposite behavior was observed: the *JEN1* mRNA was less abundant in the *dhh1* strain than in the wild-type. *JEN1* expression was also quantified by real-time quantitative PCR in wild-type and *dhh1* cells grown in glucose, lactic acid, acetic acid or formic acid, which confirmed the conclusions taken from the northern blots (Fig. 1B). These results show that *DHH1* regulates the expression of *JEN1* at the mRNA level, pointing to an involvement of this RNA helicase in the regulation of monocarboxylic acids utilization, in *S. cerevisiae*.

### Involvement of other players in the mRNA degradation pathways

To further investigate the mechanisms by which *Dhh1* controls *JEN1* expression, we measured the *JEN1* mRNA levels in cells
mutated for genes involved in various aspects of cytoplasmic
mRNA degradation: decapping activation (*pat1* and *lsm1*), 3'-5'-
exosome-mediated degradation and the non-stop decay (*ski7*) and
non sense mediated decay (*nam7*). The inactivation of *PAT1* or
*NAM7* led to an accumulation of *JEN1* mRNA in formic acid of 6
and 4 fold, respectively, as compared with the wild-type. This
effect was similar to what was observed in the *dhh1* strain (Fig. 2,
left panel). The *lsm1* strain exhibited an increase in *JEN1* mRNA,
which was significant, but lower for the one observed for *pat1* and
*nam7* strains (about 2 fold). In contrast, the deletion of *SK17* had
no effect on *JEN1* expression in these growth conditions. With
acetic acid as the sole carbon source, the deletion of *DHH1* or
*PAT1* led to a significant decrease of *JEN1* expression of 5 and 2
fold, respectively (Fig. 2, right panel). The other mutants
examined showed no significant differences as compared with
the wild-type. Finally, in lactic acid, the mutations tested had few
effects on *JEN1* mRNA levels (Fig. 2, lower panel). These results
suggested contrasted roles of *Dhh1*, *Pat1* and *Nam7* on *JEN1*
expression, which largely vary depending on the carbon source.
We further investigated the regulation of *JEN1* in formic acid.

Decay of JEN1 mRNA in the S. cerevisiae *dhh1* and *nam7*
strains

The relative stability of *JEN1* mRNA was analysed in the *S. cerevisiae* wild-type strain and in a *dhh1* or *nam7* genetic background. A pulse of 1,10-phenanthroline (0.1 mg/ml) was added to YP lactic acid-grown cells (*JEN1* inducing conditions) to stop transcription. RNA samples were prepared 0, 4, 10 and 20 minutes after the pulse. *JEN1* mRNA levels were measured using real time quantitative PCR. The results were normalized to the signals obtained from the *SCR1* mRNA (a stable RNA pol III transcript) and time zero was used as a reference to normalize for RNA steady state differences between wild-type and *dhh1* strains before the phenanthrolin pulse (Fig. 3). The relative half-live times (t ½ mRNA) of *JEN1* mRNA were calculated in each strain. This relative half-life increased by two fold in the *dhh1* mutant as compared with the wild-type and *nam7* mutants (15+/-1.05 minutes in *dhh1* mutant, 8.5+/-0.9 and 8.7+/-1.2 minutes in the wild type and *nam7* mutant, respectively) (Fig. 3). These results suggest that *dhh1* actively participates to the regulation of the stability of *JEN1* mRNA in formic acid. In contrast, the increase of *JEN1* mRNA seen in the *nam7* mutant is not related to a higher stability, suggesting that *Dhh1* and *Nam7* act at different levels in the regulation of *JEN1*.

Activity of monocarboxylic acids transporters in S. cerevisiae *dhh1* strain

In order to determine whether *JEN1* mRNAs detected in *dhh1*, *pat1* and *nam7* mutant strains grown in formic acid was being translated to a functional protein, the uptake of 2 mM of labelled lactic acid pH 5.0, was assessed in wild-type and mutant cells grown on glucose and shifted to YNB formic acid 0.5%, pH 5.0, for 4 hours (Fig. 4B). As a control, wild-type and *dhh1* mutant cells were grown in glucose and shifted to YNB lactic acid 0.5%, for 4 hours and no significant differences were observed in the uptake of labelled lactic acid (Fig. 4A), in contrast to what was found for *jen1* mutant, where no active transport was observed. In formic acid, no lactate transporter activity was observed in the wild-type cells, which is in accordance with the fact that no mRNA expression was found in these conditions. In *dhh1*, *pat1* and *nam7* mutants, although the *JEN1* mRNA was accumulating, no active lactate transport could be detected (Fig. 4B), suggesting that the

accumulation of *JEN1* mRNAs in the absence of *DHH1* does not
lead to detectable Jen1 activity.

Jen1 protein is undetectable in the *dhh1*, *pat1* and *nam7*
mutant strains grown in formic acid

To try to detect Jen1 protein in formic acid, cells of the wild-
type and the *dhh1*, *nam7*, *pat1*, *lsm1* and *ski7* (Table 1) mutant
strains, harboring a gene encoding a Jen1::GFP chimera in their
genome, were grown in YNB formic acid or in YNB lactic acid
(positive control) for 4 hours. Cells were harvested and equal
volumes of cell suspension were resuspended in low-melt agarose
(1.0%, w/v), and observed by epifluorescence microscopy. In lactic
acid, the fluorescence was unambiguously localized to the plasma
membrane in all tested cells as previously described for the wild-
type strain [55] (Fig. 5A). The same experiments were conducted
with cells grown in formic acid as sole carbon and energy source
for 4 and 6 hours. In these conditions, there was no fluorescence of
Jen1::GFP in any of the strains tested (Fig. 5A). Again, this result
was expected for the wild-type, in which almost no JEN1 mRNA
is present, but not for *dhh1*, *pat1* or *nam7* mutants, in which
significant amounts of JEN1 mRNA were detected by Q-PCR
and northern blots. However, this may just be a problem of
detection sensitivity, because, even in the *dhh1*, *pat1* and *nam7*
mutants, the JEN1 mRNA levels in formic acid are still 50 fold
lower than the one measured in lactic acid. Hence, we used a more
sensitive technique than GFP fluorescence to try to detect Jen1.
We performed western blots, using anti-GFP antibodies, in wild
type and *dhh1* mutant grown in glucose, lactic acid or formic acid
(Fig. 5B). As expected, the Jen1-GFP protein was detected in no
strain in glucose and in all strains in lactic acid. No signal was
detected in formic acid, even in the *dhh1* strain, which supported
the fact that the jen1 protein was not produced in these conditions
(Fig. 5B). Still, these experiments did not exclude the possibility
that Jen1 is actually produced but below the detection sensitivity of
the method. Indeed, with a simple 50 fold dilution of the lactic
acid sample (which roughly mimics the amount of protein that
may be expected in the formic acid grown mutant cells, based on
the mRNA levels measured in Fig. 1B), the protein became barely
detectable (Fig. 5B).

JEN1 mRNA is associated with polysomes in the *dhh1*
mutant grown in formic acid

To clarify the translational status of the *JEN1* mRNA, we
performed polysome gradients in formic acid grown *dhh1* mutant
cells (Fig. 6A). Similar experiments were conducted in lactic acid
grown *dhh1* cells, as a control for a condition in which *JEN1*
mRNA are actively translated. *ACT1* was used as a control for an
mRNA which is actively translated in both formic and lactic acid
grown cells. The mRNA levels were estimated using real-time
quantitative PCR. Because the *JEN1* mRNA levels were very low
in formic acid, we had to pool all the fractions corresponding to
polysomes on one hand and all the other fractions on the other
hand, and make a rough estimate of the percentage of *ACT1* or
*JEN1* mRNAs present in each of the two categories of fractions
(Fig. 6B). As expected, *JEN1* mRNAs were enriched in the
polysome fractions in cells grown in lactic acid. Interestingly, it was
also clearly enriched in the polysomes fractions in cells grown in
formic acid. These experiments indicated that, although we could
not detect any Jen1 activity, the *JEN1* mRNAs, which accumulate
in formic acid in the absence of Dhh1, are associated with
polysomes.
## Genome-wide analyses of the *dhh1* role in metabolic adaptation

To highlight the role of Dhh1 in the gene regulation associated with carboxylic acids and non fermentative growth conditions, we performed DNA microarray analyses of the transcriptome of yeast wild-type and *dhh1* mutant cells, grown in glucose or shifted from glucose to formic acid 0.5%, pH 5.0, for 4 hours. About 920 genes were identified as being significantly up or down regulated in the *dhh1* mutant compared with the wild-type, in at least one of the two tested conditions (Fig. 7). The mRNAs which amounts increased in the mutant were mostly involved in proteasomal and vacuolar proteolysis, respiration, oxidative and general stress responses and carbohydrate metabolism. The mRNA which steady-state decreased in the mutant were involved in ammonia and amino acid metabolism (including most of the corresponding transcriptional regulators), DNA topology and the maintenance and silencing of telomeres, aminoacyl-tRNA synthesis, translational elongation and mating (Fig. 7). About 75% of these effects were independent of the carbon source, i.e. they were found both in glucose and formic acid. Among the genes that accumulated independently of the carbon source, we found the previously identified targets of Dhh1: EDC1, COX17 [25,44] and SDH4 [47]. Also, the decrease in expression of genes involved in mating and in tRNA metabolism is reminiscent of the roles of Dhh1 in Ste12 induction [46] and tRNA maturation [51], respectively. Interestingly, several genes involved in mRNA decay were up-(EDC1, EDC2, DCS1, DCS2, PUF3, PUF2) or down-(POP1 and the subunits of the CCR4-NOT complex CAF16, CAF4 and NOT3) regulated in the mutant, suggesting the existence of feedback controls between the activity of Dhh1 and the components of the mRNA degradation pathways. Moreover, some translation regulators exhibited increased (SRO9, PET122, PPQ1, SUI1, CBP6) or decreased (TPA1, RPS31, GCN1, RBG2, MDM38, GCN3, ECM32) expression in the *dhh1* mutant. Intriguingly enough, many genes encoding RNA helicases (SLH1, BRR2, DED1, DBP1) and telomeric DNA helicases (Table S1) showed significant expression changes in the mutant.

Finally, microarray results confirmed the accumulation of the *JEN1* mRNA in the mutant strain, only in the presence of formic acid, as previously that was described in this work. Then, we focused our attention on the genes which, like *JEN1*, are repressed in the presence of glucose and induced by acetate [7] (Table S1). We found that 32 of these genes behaved like *JEN1* in the *dhh1* mutant. This is for instance the case of the other carboxylic acid transporter, *ADY2*, of *CAT8*, the transcriptional regulator of *JEN1*, and of the positive regulator of respiratory gene expression, *HAP4*. Northern blot analyses confirmed that the *ADY2* mRNA indeed accumulated in the *dhh1* mutant only in the presence of formic acid (Fig. 8). Additionally, transport activity experiments in formic acid derepressed cells showed that the accumulation of *ADY2* mRNA did not produce detectable amounts of Ady2 protein, nor in *dhh1* mutant nor in the other mutants tested in this work, similar to what was found for *Jen1* (Fig. 9). These results suggest that the post-transcriptional regulation that we characterized for *JEN1* is shared by several other genes involved in carbon source metabolism. Surprisingly, several genes that were known to be similarly subjected to glucose repression already accumulated in the *dhh1* mutant in the presence of glucose (Table S1). This is for instance the case of *ADR1*, a transcription factor which collaborates with *CAT8* under non fermentative growth conditions. Noteworthy, *ADR1* mRNA had been already shown to be post-transcriptionally down-regulated by the non-sense mediated mRNA decay machinery and the decapping enzyme Dcp1 in presence of glucose [66]. This raises the interesting hypothesis that

the balance between transcriptional and post-transcriptional regulations ensuring glucose catabolic repression may largely differ from one gene to another.

## Discussion

*Jen1* is localized at the plasma membrane of *S. cerevisiae* cells and it is involved in the transport of lactic, pyruvic, acetic and propionic acids. This permease is induced in the presence of non-fermentable carbon and energy sources, like lactic and pyruvic acids and its expression is undetectable in the presence of glucose, formic or propionic acids [4,67]. The disruption of the RNA helicase encoding gene *DHH1* attenuated growth on acetic acid. Dhh1 was known to participate in the mRNA cycle [25] controlling, together with the *Pat1-Lsm* complex, the balance between translation and mRNA degradation by inhibiting translation initiation, targeting mRNAs to the P-bodies and contributing to the recruitment of the decapping machinery [68]. In this work, we showed that Dhh1 in particular, and the decapping complex in general, have roles in the post-transcriptional regulation of *JEN1* expression, which depend on carbon source. In the absence of Dhh1, *Pat1* or *Lsm1*, *JEN1* mRNAs accumulated in formic acid and associated with polysomes, although we could not detect the translated functional protein. Hence, the translational status of *JEN1* mRNAs in these conditions remains an open question. The same phenomena occurred in a mutant for *Nam7/Upf1*, which is an important actor of the NMD pathway. Additionally, we confirmed that the half-lives of the *JEN1* mRNA actually increased in the absence of Dhh1, but not in the *nam7* mutant. In contrast, in acetic acid, the inactivation of *Pat1* or *Dhh1* had a negative effect on *JEN1* mRNA expression. Our microarray experiments suggest that other key genes of metabolic adaptation, like the transcription factor encoding gene *CAT8* or the acetate transporter encoding gene *ADY2* (Fig. 8), may encounter similar regulations.

Hence, the model that we can draw from our results and from previous studies is the following (Fig. 10). In glucose, *JEN1* is transcriptionally silent, as described previously. In lactic, its transcription is induced by *Cat8* and *Adr1*, which results in high levels of *Jen1* protein. In formic acid, the glucose transcriptional repression is also released, but *JEN1* mRNAs are rapidly degraded. This degradation requires *Dhh1*, *Pat1* and *Lsm1*, which are known to collaborate in the activation of decapping and 5'-3' mRNA decay, but not *Ski7*, which is involved in the 3'-5' degradation of cytoplasmic mRNA by the exosome. Notably, the stability of the *JEN1* mRNA increase in the *dhh1* mutant was only two fold, when its accumulation was about 6 fold, suggesting additional levels of controls of Dhh1 on *JEN1* mRNA steady-state. This accumulation of *JEN1* mRNA in formic acid is also dependent on the presence of *Nam7*, but *Nam7* does not act at the level of *JEN1* mRNA stability. *NAM7/UPF1* is involved in the NMD pathway which degrades aberrant mRNAs exhibiting a premature stop codon and "normal" mRNAs which present particular features (long 3'UTRs, alternative translation initiation sites, upstream ORFs) [69], reviewed in [31]. However, our results suggest that *JEN1* mRNA is not a target of *Nam7*. One possibility is that *Nam7* acts indirectly on *JEN1* expression by regulating the levels of a transcriptional regulator of *JEN1* in formic acid.

In acetic acid, the regulation of *JEN1* seems to be totally different. In the wild type, the *JEN1* mRNA is highly expressed. Mutations of *DHH1* or *PAT1* decreased this expression level (Fig. 1 and 2). GFP-fusion experiments showed that the *JEN1* mRNAs are translated in the *dhh1* mutant but that this lower level of mRNA expression resulted in a lower permease activity, as
measured by lactate transport assays (Fig. S2 in File S1). These
observations may explain the slow-growth phenotype of the *dhh1*
mutant in acetic acid. This effect on the mRNA levels of *JEN1* in
acetic acid was independent from Nam7 (Fig. 2). The fact that the
inactivation of a degradation pathway can lead to a decrease in
gene expression may seem counter intuitive. It was shown recently
that the inactivation of the cytoplasmic 5'-3' exonuclease Xrn1 or
of the decapping enzyme Dcp2 leads to accumulation of long non
coding RNAs (lncRNAs), some of which being located in the
promoter or in antisense position of coding genes [70,71]. In some
cases, this accumulation can lead to the transcriptional silencing of
the overlapping genes. This system seems to preferentially target
inducible genes, as for instance the GAL system [70,72]. The
*JEN1* genomic region has been shown to be able to produce two
stable unannotated transcripts (SUTs) [73] in sense and antisense
positions (www.yeastgenome.org). Moreover, it overlaps with one
large Xrn1 sensitive lncRNA (XUT) antisense to the *JEN1*
mRNA sequence [71] (www.yeastgenome.org). Therefore, it was
tempting to speculate that the negative effects of Dhh1 and Pat1
deletion on *JEN1* expression in acetic acid could be mediated by
an accumulation of one or several of these intergenic or antisense
lncRNAs. Northern blot analyses of the three non coding RNAs
overlapping the *JEN1* locus could not show any difference of
expression between the wild type and the *dhh1* mutant grown in
acetic acid (data not shown). This suggested that Dhh1 does not
act on *JEN1* expression in acetic acid by degrading overlapping
transcripts. This is consistent with previous observations that Dhh1
and Pat1 had no role in the transcriptional silencing by the
accumulation of lncRNAs [70].

More generally, we pointed out about 900 potential targets for Dhh1, which are involved in many, different cellular pathways. These results emphasized the large role of Dhh1 in gene expression regulation. Still, this number (about 15% of the genes) is relatively small, considering that Dhh1 participates to a general mRNA degradation pathway. Interestingly, in trypanosomes, microarray analyses of *dhh1* mutants suggested that it controls the expression of only 1% of the genes, several of them being involved specifically in developmental processes [74,75]. More recently, CLIP-seq experiments have shown that Dhh1 was able to bind about 300 mRNAs in standard growth conditions [76]. Our microarray results and the model of *JEN1* regulation discussed above support the idea that, besides its general role in the global cytoplasmic mRNA decay, Dhh1, like Xrn1 or Dcp2 [70–72] may have more specific roles in the post-transcriptional and/or transcriptional regulation of some genes, in response to environmental stimuli. Moreover, our list of genes whose expression is affected in the *dhh1* deletion strain provides explanations for the various phenotypes reported for *DHH1* mutations, including defects in G1/S checkpoint recovery, filamentous growth, stress responses, membrane asymmetry, sporulation, ion homeostasis, apoptosis, vacuolar trafficking, ethanol, 2-deoxyglucose and zinc resistance [41,46,77–86]. However, the interpretation of these mRNA steady-state measurements in terms of direct and indirect effects is not straightforward, since Dhh1 impacts on the expression of a large number of transcriptional and post-transcriptional regulators and of their target genes (Table S1). For instance, the expression of the transcription factor encoding gene *WAR1* (involved in weak acid resistance) and of its main target gene *PDR12* decreased in the *dhh1* mutant. Noteworthy,

References

1. Casal M, Paiva S, Queiros O, Soares-Silva I (2008) Transport of carboxylic acids in yeasts. FEMS Microbiol Rev 32: 974–994.

the level of expression of *DHH1* increased in a *WAR1* gain of function mutant [87]. Similarly, Dhh1 apparently controls the level of expression of several proteins regulating mRNA stability, including for instance *PUF2* and *PUF3*. Some PUF proteins have been shown to promote mRNA decay depending on Dhh1 [88,89]. This suggests a complex interplay between transcriptional and post-transcriptional effects, with regulatory feedbacks between them. Clearly, further genome-wide mRNA stability and pro- teome studies of the *dhh1* mutant will be required to decipher the global regulatory roles of Dhh1.

In conclusion, this study revealed that he regulation of *JEN1*, *ADY2* and possibly many other mRNA in carboxylic acids is much more complex than a simple relieve of glucose repression, and that the mechanisms which control this expression considerably vary from one carbon source to another.

**Supporting Information**

**File S1 Figures S1 and S2.** Figure S1. Representative growth curves of wild-type and *dhh1* cells grown in YNB glucose 2% (A) and in YP acetic acid 0.5% (B) media. Figure S2. Transport activity and subcellular localization of *Jen1::GFP* in *S. cerevisiae* W303-1A strains. A – Percentages of initial activities of 2 mM lactic acid uptake, at pH 5.0, in cells grown in YNB glucose and derepressed in YNB acetic acid 0.5%, pH 6.0. B – Wild-type and *dhh1* cells harboring Jen1-GFP were used to monitor Jen1 expression after growth in YNB glucose and derepression in YNB acetic acid 0.5% pH 6.0 for 6 hours or YNB lactic acid 0.5% pH 5.0 for 4 hours.
(DOCX)

**Table S1 Lists of genes with significant expression variations.** The criteria used to select these genes can be found in the material and methods. There are three sheets corresponding to the following categories: gene changing expression 1- only in formic acid, 2- only in glucose or 3- in both glucose and formic acid. Column 1: ORF ID, column 2: fold change in acetate compared with glucose (data from Paiva *et al.*, Yeast 2004), Column 3: average log of fold change (mutant/wild-type) in glucose, Column 4: average log of fold change (mutant/wild-type) in formic acid, Column 5: gene name, Column 6: functional annotation taken from the SGD.
(XLS)

**Table S2 Complete microarray results.** Column 1: ORF ID. Column 2 to 4: Log2 of normalized fluorescence ratios (*dhh1* mutant/wild-type) for the 4 experiments (two growth conditions in duplicate).
(XLS)

Acknowledgments

We thank Josette Banroques and Kyle Tanner for their advices regarding
polysome gradients.

Author Contributions

Conceived and designed the experiments: NV MC SP FD. Performed the experiments: SM NV SB TD CT AS MG AP SL FC LB. Analyzed the data: NV SM MC SP FD. Contributed reagents/materials/analysis tools: XD. Contributed to the writing of the manuscript: NV SM SP FD.

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