ara2Δ cellular responses to H2O2-mediated oxidative stress
To determine if eAsA is related to stress responses in yeast, the survival of WT and mutant ara2Δ cells was assessed using streaking and spotting assays. In the streaking assay, WT cells recovered more rapidly than ara2Δ cells when in the presence of 3.5 mM H2O2; no difference existed between cell types under normal conditions (Fig. 1a). Furthermore, the spotting assay showed that WT cells acquired increased stress tolerance when exposed to 15 mM H2O2 for 1 h at 28 ºC with shaking at 180 rpm, compared to ara2Δ cells (Fig. 1b). To further understand eAsA-associated stress responses, redox homeostasis was examined using H2O2-sensitive probes, DCFHDA and DHR123, under oxidative stress. Fluorescence of DCFHDA and DHR 123, indicators of cytosolic and mitochondrial (Mt) H2O2, respectively, was lower in WT, than in ara2Δ, when exposed to 15 mM H2O2 for 1 h (Fig. 1c); there was no difference in signal intensity between WT and ara2Δ cells under normal conditions (data not shown). These results suggest that ARA2-mediated eAsA in the S. cerevisiae eukaryotic model system responds to oxidative stress when cells are challenged with H2O2. Furthermore, eAsA was identified as a vital factor for redox homeostasis in the cytosol and mitochondria in the presence of H2O2.
Expression profiling of amino acid (AA)-associated tRNA genes, TEs, and tRNA metabolism under oxidative stress
ARA2 gene-deleted ara2Δ cells were more sensitive to H2O2-induced oxidative stress than WT cells. Based on these results, we used 3′-quant RNA-Seq analysis to demonstrate, at the transcriptional level, the eAsA-deficiency stress sensitivity mechanism present in mutant ara2Δ cells. Compared to WT cells, a total of 644 genes were significantly differentially expressed (greater than a two-fold change in ratio) in ara2Δ cells following H2O2 stress. Among these, approximately 370 and 274 genes were up- and downregulated, respectively. Differential gene expression profiling was presented in a scatter plot and heat map (Fig. S1). Among the genes that changed two-fold under H2O2 stress, a wide range of tRNA genes related to AAs were identified. tRNA genes encoding phenylalanine (Phe), cysteine (Cys), aspartate (Asp), glutamine (Gln), glycine (Gly), leucine (Leu), tyrosine (Tyr), alanine (Ala), lysine (Lys), serine (Ser), threonine (Thr), proline (Pro), arginine (Arg), and histidine (His) were upregulated in ara2Δ cells, while those encoding Pro, Leu, Ser, Gln, Lys, Gly, and Arg were downregulated in the same cells; the most abundant upregulated tRNA genes were associated with Asp, Cys, and Leu. Upregulated tRNA genes encoding Gly, Tyr, Ser, and Thr were also abundant in ara2Δ cells, while those related to Phe were downregulated. tRNA genes related to Asp, Cys, Tyr, Ala, Thr, Asp, and His were detected as only upregulated (Fig. S2). Interestingly, the tX(XXX)D gene encoding undetermined specificity, was identified only as upregulated (Table S2).
Next, 36 TE-encoding genes, including YNL284C-A, YML045W-A, and YDR098C-A, were only upregulated in ara2Δ cells (Fig. 2a). Oxidative stress also produced changes in the expression of genes associated with tRNA metabolism, ribosomal RNA (rRNA), and small nucleolar RNA (snoRNA) in ara2Δ cells. In tRNA metabolism, mutant ara2Δ cells activated various genes, including PRP18, encoding a splicing factor, DHH1, encoding cytoplasmic DEAD-box helicase and an mRNA decapping activator, LSR1, encoding U2 splicesomal RNA, HIT1, encoding a protein involved in C/D snoRNP assembly, PPM2, encoding tRNA-methyltransferase, SWT21, encoding mRNA splicing, RIM4, encoding an RNA-binding protein, SSN2, encoding the RNA polymerase II mediator complex, PUF2, encoding an mRNA-binding protein, ECM2, encoding a pre-mRNA splicing factor, and FIR1, encoding a protein involved in 3′-mRNA processing. Furthermore, these cells were also associated with WHI4, encoding a RNA-binding protein, SOL2, encoding tRNA transport, NAN1, encoding a U3 snoRNA protein, IPA1, encoding a protein implicated in pre-mRNA processing, TEX1, encoding mRNA export, CDC40, encoding a pre-mRNA splicing factor, THG1, encoding tRNA methyltransferase, NMD4, encoding nonsense-mediated mRNA decay, CBT1, encoding a protein involved in 5′-RNA end processing, SWT1, encoding an endonuclease, TFC8, encoding an RNA polymerase III transcription initiation factor, BI3, encoding Mt mRNA maturase, and LSM8, encoding an Lsm protein (Fig. 2b).
With respect to ribosomal RNA, mutant ara2Δ cells upregulated genes associated with small rRNA, including RDN51, RDN18-1, RDN18-2, RDN5-4, RDN5-5, RDN58-2, RDN37-1, RDN25-2, RDN25-1, and RDN58-1, in the presence of H2O2, compared to WT cells (Fig. 2c). In addition, ara2Δ cells elevated the expression of genes associated with snoRNA, including SNR36, SNR58, and SNR73 (Fig. 2d). These results indicate that ara2Δ cells upregulate the expression of multiple genes associated with TEs, small rRNA, and snoRNA, compared to WT cells, when exposed to H2O2 stress, while downregulating the expression of tRNA and mRNA export and transcription initiation factors.
Expression of TF, and zinc ion binding, and protein kinase genes under oxidative stress
Mutant ara2Δ cells exhibited changes in the expression of TF-associated genes under H2O2 stress. Compared with WT cells, ara2Δ cells, under oxidative stress, upregulated TF-associated genes, including YPR196W, encoding a putative maltose-responsive TF; PUL4, encoding a zinc-cluster TF; HAP5, encoding a subunit of the Hap2p/3p/4p/5p CCAAT-binding complex as a heme activator protein; RGM1, encoding a zinc-finger DNA binding TF; GAL3, encoding a transcriptional regulator; PIP2, encoding an oleate-activated TF; GIS1, encoding a histone demethylase and TF; RTS2, encoding a basic zinc-finger protein; SEF1, encoding a putative TF; HAL9, encoding a putative TF containing a zinc finger; RDR1, encoding a transcriptional repressor involved in regulating multidrug resistance; and TEA1, encoding a Ty1 enhancer activator involved in Ty enhancer-mediated transcription (Fig. 3a and Table 1). In contrast, compared with WT cells, ara2Δ cells downregulated various TFs, including RAP1, encoding a DNA-binding transcription regulator; MSA2, encoding a transcriptional activator; MKS1, encoding a pleiotropic negative transcriptional regulator; CRZ1, encoding TF-activating stress response genes; SIP4, encoding a C6 zinc cluster transcriptional activator; SMP1, encoding a MADS-box TF involved in osmotic stress response; MET28, encoding a bZIP transcriptional activator; MSS11 and SFP1, which regulate the transcription of ribosomal proteins and biogenesis genes; MSN2, encoding a stress-responsive transcriptional activator; SUT1, encoding a zinc(II)2Cys6 family TF; SKN7, encoding a nuclear response regulator and TF; PHO92, encoding a post-transcriptional regulator of phosphate and glucose metabolism, (Fig. 3a and Table 2). In particular, ara2Δ cells decreased the expression of the TFs involved in the activation of stress-responsive genes, such as CDC14, CRZ1, and MSN2 (Fig. 3b). According to Gene Ontology (GO) analysis, upregulated genes, including GAL3, HAP5, PIP2, and SSN2, were associated with the positive regulation of transcription (Fig. 3c and Table 1), while downregulated genes, including CDC14, MKS1, RAP1, GCR2 (encoding a transcriptional activator of glycolysis), HTB1 (encoding histone H2B for transcriptional activation), MED1 (involved in transcriptional regulation), RSC9 [involved in transcriptional repression and the activation of genes regulated by the target rapamycin (TOR) pathway], and PHO80 (encoding cyclin, which regulates nutrient responses in ara2Δ cells, also negatively regulating transcription) (Fig. 3d and Table 2). Notably, upregulated genes, including GAL3, HAP5, PIP2, and SSN2, networked with cellular nutrient response, carbon catabolite regulation and activation of transcription, and positive regulation of transcription (Figs. 4 and S3; Table 1).
Table 1
GO classification of the upregulated genes in ara2Δ cells under oxidative stress
Gene ontology | Associated genes |
Nuclease activity | DAL1, RAD55, REV3, REX4, SPO11, YEN1 |
Endonuclease activity | DAL1, RAD55, SPO11, YEN1 |
Deoxyribonuclease activity | DAL1, RAD55, SPO11, YEN1 |
Endodeoxyribonuclease activity | DAL1, RAD55, SPO11, YEN1 |
Double-strand break repair | MCM10, MMS21, RAD52, RAD55, SPO11 |
DNA replication | LGE1, MCM10, MEC1, RAD30, REV3, RIM4, RTS2 |
Protein kinase regulator activity | CIP1, CLB2, MMS21, TOK1 |
Negative regulation of catalytic activity | CIP1, HSP30, IGD1, MMS21, PTP2 |
Regulation of transferase activity | CIP1, CLB2, MMS21, PTP2, TOK1 |
Negative regulation of phosphorus metabolic process | CIP1, CLB2, MMS21, PTP2 |
Negative regulation of protein metabolic process | CIP1, CLB2, DHH1, MMS21, PTP2 |
Negative regulation of phosphate metabolic process | CIP1, CLB2, MMS21, PTP2 |
Negative regulation of protein modification process | CIP1, CLB2, MMS21, PTP2 |
Regulation of phosphorylation | CIP1, CLB2, MMS21, PTP2, TOK1, TYE7 |
Regulation of kinase activity | CIP1, CLB2, MMS21, PTP2, TOK1 |
regulation of protein phosphorylation | CIP1, CLB2, MMS21, PTP2, TOK1 |
Regulation of protein kinase activity | CIP1, CLB2, MMS21, PTP2, TOK1 |
Regulation of protein serine/threonine kinase activity | CIP1, CLB2, MMS21, PTP2 |
Response to nutrient | GAL3, HAP5, PIP2, SSN2 |
Response to nutrient levels | GAL3, GIS1, HAP5, PIP2, SIP2, SSN2 |
Cellular response to nutrient | GAL3, HAP5, PIP2, SSN2 |
Carbon catabolite regulation of transcription | GAL3, HAP5, PIP2, SSN2 |
Carbon catabolite regulation of transcription from RNA polymerase II promoter | GAL3, HAP5, PIP2, SSN2 |
Carbon catabolite activation of transcription | GAL3, HAP5, PIP2, SSN2 |
Positive regulation of transcription from RNA polymerase II promoter involved in cellular response to chemical stimulus | GAL3, HAP5, PIP2, SSN2 |
Carbon catabolite activation of transcription from RNA polymerase II promoter | GAL3, HAP5, PIP2, SSN2 |
Organelle disassembly | ATG12, ATG20, ATG39, YCL001W-A |
Pre-autophagosomal structure | ATG12, ATG20, ATG38, ATG39 |
Pre-autophagosomal structure membrane | ATG12, ATG20, ATG38, ATG39 |
Anatomical structure formation involved in morphogenesis | DIT1, IME2, RIM4, RRT12, SEF1, SPO11, SPO20, TEP1 |
Meiotic cell cycle | DIT1, IME2, LGE1, MEC1, MPS3, RAD52, RAD55, REC104, RIM4, RRT12, SNF3, SPO11, SPO20, TEP1, ZIP1 |
Meiotic cell cycle process | DIT1, IME2, LGE1, MEC1, MPS3, RAD52, RAD55, REC104, RIM4, RRT12, SNF3, SPO11, SPO20, TEP1, ZIP1 |
Sporulation resulting in formation of a cellular spore | DIT1, IME2, RIM4, RRT12, SEF1, SPO11, SPO20, TEP1 |
Anatomical structure homeostasis | IES3, MEC1, RAD52, TEN1 |
Meiotic nuclear division | IME2, MEC1, MPS3, RAD52, RAD55, REC104, RIM4, SNF3, SPO11, ZIP1 |
Telomere organization | IES3, MEC1, RAD52, TEN1 |
Meiosis I | MEC1, MPS3, RAD52, RAD55, REC104, RIM4, SPO11, ZIP1 |
Telomere maintenance | IES3, MEC1, RAD52, TEN1 |
DNA recombination | IES3, IRC4, MCM10, MEC1, MMS21, RAD52, RAD55, REC104, RIM4, SPO11, ZIP1 |
Reciprocal meiotic recombination | MEC1, RAD52, RAD55, REC104, RIM4, SPO11, ZIP1 |
Recombinational repair | MCM10, MMS21, RAD52, RAD55 |
Mitotic recombination | IES3, IRC4, MEC1, RAD52, RAD55 |
Reciprocal DNA recombination | MEC1, RAD52, RAD55, REC104, RIM4, SPO11, ZIP1 |
Double-strand break repair via homologous recombination | MCM10, MMS21, RAD52, RAD55 |
Zinc ion binding | DAL1, HAL9, MMS21, PIP2, RDR1, RTS2, SEF1, SGF11, TEA1, YER137C, YNR063W, YPR196W |
Vesicle organization | ATG15, ATG20, PRM8, SPO20, YAR028W |
Small nuclear ribonucleoprotein complex | AAR2, IME2, PRP18, SWT21 |
Lipid catabolic process | ATG15, IDP3, PIP2, POT1 |
Cellular lipid catabolic process | ATG15, IDP3, PIP2, POT1 |
Fatty acid metabolic process | IDP3, PIP2, POT1, YAT1 |
Monosaccharide metabolic process | DOG1, FBP1, GAL3, IGD1, PCK1 |
Single-organism carbohydrate catabolic process | FBP1, IGD1, MAL32, TYE7 |
Hexose metabolic process | DOG1, FBP1, GAL3, IGD1, PCK1 |
Glucose metabolic process | DOG1, FBP1, IGD1, PCK1 |
Anatomical structure formation involved in morphogenesis | DIT1, IME2, RIM4, RRT12, SEF1, SPO11, SPO20, TEP1 |
Cellular component assembly involved in morphogenesis | DIT1, RRT12, SPO20, TEP1 |
Carbohydrate transmembrane transporter activity | HXT6, HXT8, SNF3, STL1 |
Secondary active transmembrane transporter activity | AGP2, HXT6, HXT8, SNF3, STL1 |
Carbohydrate transport | HXT6, HXT8, SNF3, STL1 |
Sugar transmembrane transporter activity | HXT6, HXT8, SNF3, STL1 |
Symporter activity | HXT6, HXT8, SNF3, STL1 |
Monosaccharide transmembrane transporter activity | HXT6, HXT8, SNF3, STL1 |
Monosaccharide transport | HXT6, HXT8, SNF3, STL1 |
Carbohydrate transmembrane transport | HXT6, HXT8, SNF3, STL1 |
Cation:sugar symporter activity | HXT6, HXT8, SNF3, STL1 |
Solute:cation symporter activity | HXT6, HXT8, SNF3, STL1 |
Hexose transport | HXT6, HXT8, SNF3, STL1 |
Sugar:proton symporter activity | HXT6, HXT8, SNF3, STL1 |
Solute:proton symporter activity | HXT6, HXT8, SNF3, STL1 |
Hexose transmembrane transport | HXT6, HXT8, SNF3, STL1 |
Glucose transmembrane transporter activity | HXT6, HXT8, SNF3, STL1 |
Glucose transport | HXT6, HXT8, SNF3, STL1 |
DNA biosynthetic process | RAD30, RAD52, REV3, TEN1 |
Chromosome, telomeric region | IES3, MCM10, MPS3, TEN1, YPL216W |
Chromatin silencing at telomere | IES3, MCM10, MPS3, YPL216W |
Table 2
GO classification of the downregulated genes in ara2Δ cells under oxidative stress
Gene ontology | Associated genes |
Negative regulation of transcription from RNA polymerase II promoter | CDC14, GCR2, HTB1, MED1, MKS1, PHO80, RAP1, RSC9 |
Coenzyme metabolic process | ADH5, BNA4, CAT5, ECM31, GCR2, GPD1, PGK1, RAP1, SOL2, SOL4, URH1, YAH1 |
Cofactor biosynthetic process | BNA4, CAT5, ECM31, HEM1, RNR4, URH1, YAH1 |
Nucleobase-containing small molecule metabolic process | ADH5, AIF1, AMD1, BNA4, CDD1, COB, GCR2, GPD1, OLI1, PGK1, RAP1, RNR4, SDH4, SOL2, SOL4, URH1 |
Pyridine-containing compound metabolic process | ADH5, BNA4, GCR2, GPD1, PGK1, RAP1, SOL2, SOL4, URH1 |
Ribose phosphate metabolic process | ADH5, AMD1, COB, GCR2, OLI1, PGK1, RAP1, SDH4, SOL2, SOL4 |
Oxidoreduction coenzyme metabolic process | ADH5, BNA4, CAT5, GCR2, GPD1, PGK1, RAP1, SOL2, SOL4, URH1, YAH1 |
Nucleoside phosphate metabolic process | ADH5, AIF1, AMD1, BNA4, COB, GCR2, GPD1, OLI1, PGK1, RAP1, RNR4, SDH4, SOL2, SOL4, URH1 |
Nucleotide metabolic process | ADH5, AIF1, AMD1, BNA4, COB, GCR2, GPD1, OLI1, PGK1, RAP1, RNR4, SDH4, SOL2, SOL4, URH1 |
Ribonucleoside metabolic process | ADH5, CDD1, COB, GCR2, OLI1, PGK1, RAP1, SDH4, URH1 |
Nucleoside monophosphate metabolic process | ADH5, AMD1, COB, GCR2, OLI1, PGK1, RAP1, SDH4 |
Nucleoside triphosphate metabolic process | ADH5, COB, GCR2, OLI1, PGK1, RAP1, SDH4 |
Purine nucleoside metabolic process | ADH5, COB, GCR2, OLI1, PGK1, RAP1, SDH4, URH1 |
Pyridine nucleotide metabolic process | ADH5, BNA4, GCR2, GPD1, PGK1, RAP1, SOL2, SOL4, URH1 |
Monocarboxylic acid metabolic process | ADH5, BNA4, CAT2, ECM31, GCR2, HST4, IFA38, OAR1, PGK1, PXA1, RAP1 |
Purine nucleoside monophosphate metabolic process | ADH5, AMD1, COB, GCR2, OLI1, PGK1, RAP1, SDH4 |
Purine nucleoside triphosphate metabolic process | ADH5, AMD1, COB, GCR2, OLI1, PGK1, RAP1, SDH4 |
Ribonucleoside monophosphate metabolic process | ADH5, AMD1, COB, GCR2, OLI1, PGK1, RAP1, SDH4 |
Ribonucleoside triphosphate metabolic process | ADH5, COB, GCR2, OLI1, PGK1, RAP1, SDH4 |
Purine ribonucleotide metabolic process | ADH5, AMD1, COB, GCR2, OLI1, PGK1, RAP1, SDH4 |
Nicotinamide nucleotide metabolic process | ADH5, BNA4, GCR2, GPD1, PGK1, RAP1, SOL2, SOL4, URH1 |
Purine ribonucleoside monophosphate metabolic process | ADH5, AMD1, COB, GCR2, OLI1, PGK1, RAP1, SDH4 |
Purine ribonucleoside triphosphate metabolic process | ADH5, COB, GCR2, OLI1, PGK1, RAP1, SDH4 |
ATP metabolic process | ADH5, COB, GCR2, OLI1, PGK1, RAP1, SDH4 |
Sister chromatid segregation | CDC14, DAD1, DMA1, ECO1, ELG1, MCM22, RAD61, SPO13, TUB3 |
Mitotic nuclear division | ACM1, ARP10, CDC14, CDC24, DAD1, DMA1, ECO1, ELG1, MCM22, RAD61, SPC24, TUB3 |
Mitotic sister chromatid segregation | CDC14, DAD1, DMA1, ECO1, ELG1, MCM22, RAD61, TUB3 |
Spindle | CDC14, DAD1, LDB18, SLD7, SPO13, TUB3 |
Microtubule cytoskeleton | ARP10, CDC14, DAD1, HYM1, LDB18, SLD7, SPO13, TUB3 |
Negative regulation of protein metabolic process | ACM1, EBS1, IGO2, PBI2, SIZ1, WHI4 |
Negative regulation of cellular protein metabolic process | ACM1, EBS1, IGO2, PBI2, SIZ1, WHI4 |
Protein localization to vacuole | ATG19, COS12, HOM3, MON1, SNX3, TRE2, VAM7 |
Establishment of protein localization to vacuole | ATG19, COS12, HOM3, MON1, SNX3, TRE2, VAM7 |
Protein targeting to vacuole | ATG19, COS12, HOM3, MON1, SNX3, TRE2, VAM7 |
Aromatic compound catabolic process | BNA4, CBC2, CDD1, EBS1, HBS1, IPK1, POP3, SWT1, URH1, YCL001W-B |
Cellular nitrogen compound catabolic process | BNA4, CBC2, CDD1, EBS1, HBS1, IPK1, POP3, SWT1, URH1, YCL001W-B |
Heterocycle catabolic process | BNA4, CBC2, CDD1, EBS1, HBS1, IPK1, POP3, SWT1, URH1, YCL001W-B |
Organic cyclic compound catabolic process | BNA4, CBC2, CDD1, EBS1, HBS1, IPK1, PHO13, POP3, SWT1, URH1, YCL001W-B |
Nucleobase-containing compound catabolic process | CBC2, CDD1, EBS1, HBS1, IPK1, POP3, SWT1, URH1, YCL001W-B |
mRNA catabolic process | CBC2, EBS1, HBS1, IPK1, POP3, SWT1, YCL001W-B |
Nuclear-transcribed mRNA catabolic process | CBC2, EBS1, HBS1, IPK1, POP3, SWT1, YCL001W-B |
Transcription factor activity, RNA polymerase II core promoter proximal region sequence-specific binding | CDC14, CRZ1, MSN2, MSS11, RAP1, SIP4 |
Oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor | ADH5, ADH7, ARA2, GPD1, IFA38, OAR1, YPL113C |
Transferase activity, transferring acyl groups other than amino-acyl groups | CAT2, ECO1, ERF2, HEM1, OAR1, TGL3 |
Small ribosomal subunit | MRP2, MRPS12, RPS17A, RPS1B, RPS20, RPS27B, RPS4B |
Cytoplasmic translation | RPL21A, RPL25, RPL33B, RPL39, RPL8A, RPM2, RPS17A, RPS1B, RPS20, RPS27B, RPS4B |
Cytosolic ribosome | ANB1, RPL21A, RPL25, RPL33B, RPL39, RPL8A, RPS17A, RPS1B, RPS20, RPS27B, RPS4B |
Telomere organization | EBS1, ECO1, ELG1, RAD57, RAP1, STN1 |
Storage vacuole | CPS1, CSI2, ERS1, HOM3, KOG1, MON1, PBI2, UGA4, VAM3, VAM7 |
Lytic vacuole | CPS1, CSI2, ERS1, HOM3, KOG1, MON1, PBI2, UGA4, VAM3, VAM7 |
Cellular response to oxidative stress | ALO1, GRX4, MSN2, RSC9, TRX3, UGA2 |
Energy derivation by oxidation of organic compounds | ADH5, AIF1, BI3, COB, FRD1, MAM33, OAR1, SDH4, SLS1 |
Cellular respiration | AIF1, BI3, COB, FRD1, MAM33, OAR1, SDH4, SLS1 |
Aerobic respiration | AIF1, COB, MAM33, OAR1, SDH4, SLS1 |
Organelle fusion | ENV7, FUS2, PBI2, PHO80, VAM3, VAM7 |
Moreover, ara2Δ cells upregulated a wide range of zinc ion binding-associated genes, including HAL9, PIP2, RTS2, SEF1, TEA1, YPR196W, DAL1 (encoding allantoinase), MMS21 (encoding SUMO E3 ligase), SGF11 (encoding an integral subunit of the SAGA histone acetyltransferase complex), and YNR063W (PUL4; encoding a putative zinc-cluster TF, under oxidative stress); WT cells did not induce these genes (Fig. 3f). Therefore, activated TF genes in ara2Δ cells were predominantly involved in zinc ion binding.
Furthermore, ara2Δ cells induced the expression of multiple protein kinase-associated genes under oxidative stress. The following genes were upregulated: RCK1, encoding a protein kinase involved in oxidative stress; CIP1, encoding a cyclin-dependent kinase inhibitor activated by environmental stress; SIP1, one of the three β-subunits of the Snf1 kinase complex; PCK1, encoding phosphoenolpyruvate carboxykinase as a key enzyme of gluconeogenesis; IME2, encoding a Ser/Thr protein kinase involved meiosis activation; SMK1, encoding middle sporulation-specific MAPK; ATG38, encoding the homodimeric subunit of the autophagy-specific Ptdlns-3-kinase complex; ADK1, encoding an adenylate kinase required for purine metabolism; MEC1, encoding a Ser/Thr protein kinase essential for DNA replication, DNA repair, and telomere maintenance; and PRR2, encoding a Ser/Thr protein kinase. In contrast, the following genes were downregulated: WSC3, associated with a sensor-transducer of the stress-activated PKC1-MAPK signaling pathway; ENV7, encoding a vacuolar membrane protein kinase; PGK1, encoding a 3-phosphoglycerate kinase involved in glycolysis and gluconeogenesis; YPK3, encoding AGC kinase as a Ser/Thr protein kinase; CAK1, encoding the cyclin-dependent kinase-activating kinase involved in the meiotic cell cycle; PCL1, encoding a cyclin-dependent Ser/Thr protein kinase regulator; IPK1, encoding an inositol pentakisphosphate 2-kinase involved in inositol phosphate biosynthesis; YFH1, encoding a putative kinase with similarity to the PRK/URK/PANK kinase subfamily; HOM3, encoding an aspartate kinase involved in Met, Thr, and homoSer biosynthesis; and SPS1, encoding an STE-family GCKIII protein kinase required for prospore membrane closure (Fig. 3f; Table 1 and 2). In addition, the upregulated genes—including CIP1; CLB2, encoding a B-type cyclin in cell cycle progression; PTP2, encoding a phosphotyrosine phosphatase; and TOK1, related to potassium ion homeostasis via the potassium channel—were connected via the regulation of protein phosphorylation, transferase, protein kinase, and Ser/Thr protein kinase activity, as well as the negative regulation of the metabolism of protein, phosphorous, and phosphate, and protein modification (Figs. 4 and S3; Table 1). Finally, CIP1, MMS21, TOK, HSP30 (encoding a negative regulator of the stress-responsive protein H+-ATPase Pma1p), and IGD1 (encoding a cytoplasmic enzyme inhibitor that downregulates glycogen catabolism), were upregulated in ara2Δ cells upon oxidative stress, which has also been implicated in catalytic activity regulation (Figs. 4 and S3; Table 1).
In contrast to protein kinase regulation, ara2Δ cells decreased the expression of signal network-related genes under oxidative stress, excluding SNF3 which encodes a plasma membrane (PM) low glucose sensor. Related genes were as follows: SSY1, a component of the SPS PM AA sensor system, HYM1, encoding a component of the RAM signaling network involved in cellular morphogenesis, FRQ1, encoding an N-myristoylated calcium-binding protein involved, as an enzyme activator, in signal transduction, and SPC1, a subunit of the signal peptidase complex involved in signal peptide processing and protein targeting to the ER (Fig. 3h). Altogether, these results suggest that increased sensitivity in eAsA-deficient ara2Δ cells partially leads to the downregulation of stress-responsive TFs, protein kinases, and signal networks, activation of genes involved in protein kinase-mediated meiosis, and the negative regulation of catalytic activity, genome stability, and autophagy, under H2O2 stress.
Gene expression network analysis of DNA metabolism, chromosome, chromatin, and telomere-related genes in ara2Δ cells under oxidative stress
ARA2 gene-deleted ara2Δ cells generated changes in the expression of genes involved in processes related to the DNA, chromosomes, and chromatin. Upregulated genes were as follows: MEC1; RIM4; RTS2; MMS21; RAD30, encoding DNA polymerase; REV3, as a catalytic subunit of DNA polymerase zeta; IES3, encoding a subunit of the chromatin remodeling complex; RAD52, encoding a protein involved in homologous recombination; RAD55, encoding a protein that stimulates strand exchange; LGE1, encoding a protein involved in histone H2B ubiquitination; MCM10, encoding a chromatin-associated protein; SPO21, encoding a putative type IIB topoisomerase involved in meiotic-DSB formation; IRC4 and ZIP1, both involved in recombination; REC104, encoding a protein involved in meiotic recombination; TEN1, encoding a protein that regulates telomeric length; MPS3, encoding a nuclear envelope protein; YPL216W, related to chromosome and telomeric regions; NSL1, encoding a component of the MIND kinetochore complex required for accurate chromosome segregation; YEN1, encoding a crossover junction resolvase involved in DNA repair; and NAT4, an N α-acetyl-transferase (Fig. 5a). These genes were associated with DNA replication and recombination, double-strand (ds) DNA repair, meiotic nuclear division, reciprocal meiotic recombination, and recombination repair (Figs. 4 and S3; Table 1). Genes corresponding to each of these categories are shown in Fig. S3. In contrast, ara2Δ cells downregulated the expression of genes involved in chromatid segregation, including CDC14; DAD1, encoding a subunit of the Dam complex; DMA1, encoding an ubiquitin (Ub) protein ligase involved in septin ring assembly and organization; ECO1, encoding an acetyltransferase required for the establishment of sister chromatid cohesion; ELG1, a subunit of the alternative replication factor C complex; MCM22, encoding an outer kinetochore protein and components of the Ctf3 subcomplex; RAD61, a subunit of the complex that inhibits sister chromatid cohesion; SPO13, encoding a meiotic regulator involved in maintaining sister chromatid cohesion; TUB3, encoding α-tubulin; ACM1, encoding a pseudosubstrate inhibitor of APC/C; ARP10, a component of the dynactin complex; and SPC24, involved in chromosome segregation. All downregulated genes were implicated in sister chromatid segregation, mitotic nuclear division, and mitotic sister chromatid segregation (Table 2). The following were also downregulated in the ara2Δ cells: HTB1, encoding a core histone protein required for chromatin assembly and chromosome function; RNR4, involved in dNTP synthesis; EUC1, encoding a sequence-specific DNA-binding protein; SLD7, encoding a protein involved in chromosomal replication; SET5, encoding methyltransferase; NHP6A, encoding a DNA and nucleosome-binding protein involved in chromatin remodeling; and CDC24, encoding a guanidine nucleotide exchange factor (Fig. 5a).
Mutant ara2Δ cells increased the expression of genes involved in small nuclear ribonucleoprotein complexes, including IME2, AAR2, encoding a protein related to spliceosomal tri-snRNP assembly, PRP18, encoding both a splicing factor and a component of snRNP U5, and SWT21, encoding a protein involved in mRNA splicing (Figs. 4, 5b, and S3; Table 1). Furthermore, ara2Δ cells exhibited changes in the expression of various telomere-associated genes; upregulating the expression of TEN1, IES3, MEC1, RAD52, MPS3, and YPL216W, and downregulating ECO1, ELG1, RAP1, EBS1, RAD57, encoding a protein that stimulates strand exchange, and STN1, encoding a telomere end-binding and capping protein. Genes linked to telomere maintenance and organization were found to change as a result of oxidative stress (Figs. 4, 5c, and S3; Table 1 and 2). Furthermore, ara2Δ cells accelerated the expression of nuclease activity-related genes, including RAD55, REV3, YEN1, MCM10, MMS21, REX4, encoding an RNA exonuclease, SPO11, encoding a protein that binds to the ends of dsDNA breaks, and AI4 and AI3, both of which encode endonuclease (Fig. 4, 5d, and S3; Table 1). Taken together, these results suggest that eAsA-deficient ara2Δ cells activate the expression of genes involved in DNA metabolism, including DNA replication and recombination, small nuclear proteins, and nuclease activity, while decreasing the expression of those related to chromatin and chromosome functionality; indicating that eAsA plays a vital role in the function and assembly of chromatids, chromosomes, and telomeres.
Gene expression network analysis of cell cycle-related genes in ara2Δ cells under oxidative stress
The eAsA-deficient ara2Δ cells showed an increased expression of cell cycle-related genes, including IME2, MEC1, MPS3, RAD52, RAD55, REC104, RIM4, SNF3, SPO11, ZIP1, and DIT1, each encoding a sporulation-specific enzyme required for spore wall maturation; LGE1, encoding a protein involved in histone H2B ubiquitination; RRT12, encoding Ser-type peptidases involved in ascospore wall assembly; SPO20, encoding a meiosis-specific subunit of the t-SNARE complex; and TEP1, involved in normal sporulation. All upregulated genes were connected with meiosis I, meiotic cell cycle, meiotic cell cycle progression, and mitotic recombination (Figs. 4 and S3). In addition, the following cell-cycle-related genes were upregulated, including CLB2, encoding B-type cyclin-involved cell cycle progression; KIP2, encoding a kinesin-related motor protein involved in mitotic spindle positioning; SPO19, encoding a meiosis-specific prospore protein; FAR7, encoding a protein involved in the recovery from pheromone-induced cell cycle arrest; and IRC4, involved in the meiosis recombination center (Fig. S3). In contrast, the following genes were downregulated: CDC14, SPO13, CDC24, and FDO1, each encoding a protein involved in the directionality of mating-type switching; HOP2, encoding a meiosis-specific protein that localizes to chromosomes; MEI4, encoding a meiosis-specific protein involved in the formation of ds breaks during meiotic recombination; IGO2, encoding a protein required for G0 program initiation; and GMC1, encoding a protein involved in meiotic progression (Fig. S3).
Furthermore, ara2Δ cells upregulated many sporulation-related genes, including SMK1, SPO19, TIP1, DIT1, RIM4, SEF1, SPO11, SPO20, and SPS4, each encoding a lipid droplet protein implicated in sporulation; these proteins networked with sporulation, resulting in the formation of cellular spores and anatomical structures involved in morphogenesis (Figs. 4 and S3; Table 1). In contrast, the following genes were downregulated: SPO24, encoding a small (67 AAs) protein related to sporulation, and SPS2, encoding a protein involved in ascospore wall assembly (Fig. S4). Additionally, ara2Δ cells upregulated the expression of anatomical structure homeostasis-associated genes, including IES3, MEC1, RAD52, and TEN1 (Figs. 4 and S3; Table 1). Taken together, these results indicate that, regardless of the downregulation of some genes, eAsA deficiency enhances meiosis-mediated cell cycle arrest, sporulation-related processes, and anatomical structure homeostasis, under oxidative stress.
Identification of other upregulated genes in ara2Δ cells under oxidative stress
Mutant ara2Δ cells also upregulated the expression of SNF3, HXT6, encoding a high-affinity glucose transporter, HXT8, encoding a protein with unknown function and similarity to hexose transporters, and STL1, encoding a PM glycerol proton symporter; these genes networked with a wide range of GO categories, including carbohydrate-, sugar-, monosaccharide-, hexose-, glucose-, and secondary active-transmembrane transporter activity, symporter activity of carbon::sugar, solute::cation, sugar::proton, and solute::proton, and carbohydrate, glucose, hexose, and monosaccharide transport (Figs. 4 and S3; Table 1). These results indicate that ara2Δ cells facilitate the transport of glucose-derived compounds into cells.
Gene expression profiling of protein metabolic processes in ara2Δ cells under oxidative stress
Under H2O2-induced oxidative stress, ara2Δ cells altered the expression of genes involved in the negative regulation of protein metabolism. The following genes were upregulated: CIP1, CLB2, DHH1, MMS21, and PTP2, and downregulated: ACM1, EBS1, IGO2, WHI4, and PBI2, encoding inhibitors of vacuolar proteinase B, and SIZ1, encoding a SUMO ligase (Figs. 4 and 6a; Table 1 and 2). Furthermore, ara2Δ cells downregulated most of the genes associated with protein synthesis-related events, including EBS1, SDL1, and ANB1, encoding translational elongation factor eIF-5A (Fig. 6b). Furthermore, ara2Δ cells also downregulated the expression of genes related to protein localization and targeting to vacuoles, including HOM3, ATG19, encoding a receptor protein for cytoplasm-to-vacuole targeting, COS12, encoding an endosomal protein involved in the turnover of PM proteins, MON1, encoding a subunit of the heterodimeric guanidine nucleotide exchange factor, SUX3, encoding a sorting nexin for late-Golgi enzymes, TRE2, encoding a transferrin-like protein, and VAM7, encoding a vacuolar SNARE protein (Fig. 6c and Table 2). Transferase activity-related genes were also largely downregulated in ara2Δ cells, including ECO1, HEM1, encoding 5-aminolevulinate synthase, CAT2, encoding carnitine acetyl-CoA transferase, ERF2, encoding a subunit of palmitoyltransferase, OAR1, encoding Mt 3-oxoacyl-(acyl-carrier-protein) reductase, and TGL3, encoding bifunctional triacylglycerol (TAG) lipase and lypophosphophatidylethanomine (LPE) acyltransferase (Fig. 6d and Table 2).
In ara2Δ cells, the expression of multiple proteostasis-related genes was altered under H2O2 stress; the following genes were upregulated (HSP30, HSP26, and HSP42, each encoding a small heat shock protein), SSA4, a HSP70 family A, PMP1, encoding a regulatory subunit of PM H+-ATPase Pma1p, BTN2, encoding SNARE- and chaperone-binding proteins, and MRH1, encoding a membrane protein related to Hsp30 and downregulated: NAS6, encoding a 19S regulatory particle assembly-chaperone, MPD1, encoding a protein disulfide isomerase, JID1, encoding a Hsp40 co-chaperone, TAH1, encoding a protein that interacts with Hsp90 (Hsp82 and Hsc82), HBS1, a Hsp70 subfamily B suppressor, OST6, encoding a protein disulfide oxidoreductase, and BTT1, encoding the heterodimeric nascent polypeptide-associated complex β3 subunit (Fig. 6e). When considering proteolysis and protein degradation, ara2Δ cells upregulated the expression of various genes under oxidative stress, including MMS21, RRT12, ALY1, encoding a ubiquitin protein ligase-binding protein (or α-arrestin), TRE1, encoding a transferrin receptor-like protein that regulates ubiquitination and vacuolar degradation, and CUL3, encoding a ubiquitin-protein ligase. In contrast, ara2Δ cells downregulated the following protein degradation-related genes: PBI2, IPA1, encoding a protein associated with proteasomal degradation, NFI1 and SIZ1, both of which encode a SUMO E3 ligase, CPS1, encoding vacuolar carboxypeptidase S, which regulates proteolytic enzymes, MIY2, encoding the evolutionally-conserved K48-specific deubiquitinase involved in recycling Ub from Ub-conjugated proteins destined for proteolysis, KOG1, encoding a subunit of the TORC1 complex binding to Ub, ACM1, encoding an inhibitor of Ub-protein transferase that negatively regulates Ub protein ligase activity, and ARL3, a Golgi-to-PM protein transporter (Fig. 6f). Hence, these results indicate that eAsA deficiency facilitates proteasomal degradation following imbalances in chaperone activity, as the genes relating to chaperone protein co-factors were downregulated, even when those encoding small HSPs were upregulated, during oxidative stress. This indicates that eAsA deficiency decreases protein synthesis by reducing the expression of genes implicated in translational elongation factors.
Expression of genes associated with redox and ion homeostasis and cellular stress responses to H 2 O 2 -associated stress
Mutant ara2Δ cells altered the expression of a variety of redox state-associated genes under H2O2 stress; the following genes were upregulated: YKL107W, encoding an NADH-dependent aldehyde reductase; SPG4, encoding a protein required for heat shock-induced oxidative stress; CYC3, encoding cytochrome c heme lyase; ALR2, predicting a probable Mg2+ transporter; ARN2, encoding a siderophore transmembrane transporter involved in iron homeostasis through siderophore-iron chelation; FRE2, encoding ferric and cupric reductase reducing siderophore-bound iron and oxidized copper; COS111, encoding a protein required for resistance to the antifungal drug ciclopirox olamine; and TOK1 and ECM27, involved in potassium and calcium homeostasis, respectively (Fig. 7a), while the following were downregulated: PHO80; ALO1, encoding D–arabino–1,4–lactone oxidase, which catalyze the final step in eAsA biosynthesis; HAL1, encoding a cytoplasmic protein involved in halotolerance; GRX4, encoding GSH-dependent oxidoreductase and GSH S-transferase (commonly known as glutaredoxin (Grx) isoform 4); ARR2, encoding an arsenate reductase required for arsenate resistance; TRX3, encoding Mt thioredoxin (Trx); COB, encoding a ubiquinol-cytochrome-c reductase; UGA2, encoding a succinate semialdehyde dehydrogenase involved in oxidative stress responses; AIF1, encoding a Mt cell death effector and an apoptosis-inducing factor; COA2, encoding a protein involved in Mt cytochrome c oxidase assembly; ADH7 and ADH5, both encoding NAD(P)H-dependent alcohol dehydrogenases involved in aldehyde tolerance; FRD1, encoding a fumarate reductase involved in the anoxia response; YAH1, encoding ferredoxin of the Mt matrix required for cellular iron-sulfur protein formation and ubiquinone and heme a biosynthesis; and YBR284W, encoding a metallo-dependent hydrolase (Fig. 7a). In particular, genes involved in the cellular response to oxidative stress, including ALO1, GRX4, MSN2, TRX3, UGA4 [encoding gamma-aminobutyrate (GABA) permease], and RSC9 (encoding a component of the RSC chromatin remodeling complex), were downregulated in the H2O2-treated ara2Δ cells, compared with the WT cells (Figs. 7b, 8, and S5; Table 2). Additionally, oxidoreductase-related genes, including ADH5, ADH7, ARA2, OAR1, GPD1 [encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GAPDH)], IFA38 (encoding microsomal β-keto-reductase), and YPL113C (encoding glyoxylate reductase), were also downregulated under oxidative stress (Figs. 7c, 8, and S5; Table 2). Taken together, these results suggest that eAsA deficiency increased the expression of genes related to the homeostasis of ions such as iron, copper, potassium, and calcium; also indicating that eAsA deficiency further destabilizes redox homeostasis following the downregulation of genes associated with antioxidant enzymes, cellular response systems, and oxidoreductase, particularly in the mitochondria.
Furthermore, H2O2-exposed ara2Δ cells upregulated multiple nutrient response-associated genes, including GAL3, GIS1, HAP5, PIP2, SIP2, encoding one of three alternate β-subunits of the Snf1 kinase complex, and SSN2, encoding an RNA polymerase II mediator complex subunit essential for transcriptional regulation (Fig. 7d and Table 1). In particular, GAL3, GIS1, HAP5, PIP2, and SSN2 were associated with nutrient- or stress-responsive TFs, while SIP2 was associated with protein kinases. Furthermore, GAL3, HAP5, PIP2, and SSN2 networked with a broad range of GO categories, as noted above (Figs. 4 and S3). Thus, these results suggest that eAsA-deficient ara2Δ cells reinforce nutrient uptake by activating TF genes under oxidative stress.
Gene expression network analysis of ara2Δ cell metabolic processes under oxidative stress
H2O2-treated ara2Δ cells regulated the expression of a wide range of genes associated with metabolism, such as glycolysis, lipid, and FA metabolism, and respiratory metabolic processes. The following genes were upregulated: IGD1, DOG1, encoding 2-deoxyglucose-6-phosphate (2-DG-6) phosphatase, IDP3, encoding peroxisomal NADP-dependent isocitrate dehydrogenase (ICDH), TKL2, encoding a transketolase involved in the pentose-phosphate pathway (PPP), RSF1, encoding a protein required for respiratory growth, FBP1, encoding fructose-1,6-bisphosphatase, as related to gluconeogenesis and the metabolism of ROS, JLP1, encoding Fe2+-dependent sulfonate/α-ketoglutarate dioxygenase, POT1, encoding 3-ketoacyl-CoA thiolase, which is involved in FA β-oxidation, and AAD14, encoding aryl-alcohol dehydrogenase, which is involved in cellular aldehyde metabolism; the following genes were downregulated: GPD1, CAT2, OAR1, SDH4, encoding succinate dehydrogenase (SDH), SOL4, encoding 6-phosphogluconolactonase, and PHO13, encoding a conserved phosphatase that act as a metabolite repair enzyme (Fig. 7e; Table 1 and 2). Furthermore, ara2Δ cells increased the expression of glucose and hexose metabolism-related genes, including DOG1, FBP1, IGD1, and PCK1, each of which networked with monosaccharide, hexose, and glucose metabolism, and single-organism carbohydrate catabolism (Figs. 4 and S3).
Moreover, ara2Δ cells upregulated the expression of lipid and FA metabolism-related genes, including PCK1, PIP2, TEP1, POT1, ATG15, encoding phospholipase, and YAT1, encoding Mt carnitine O-palmitoyltransferase, each of which formed a network with lipid catabolism, lipid modification, cellular lipid catabolism, and FA metabolism (Figs. 4, 7g, and S3; Table 1). In contrast, ara2Δ cells downregulated the expression of genes involved in respiration metabolism, including ADH5, AIF1, BI3, COB, FRD1, OAR1, SDH4, MAM33, encoding a specific translational activator for Mt COX1 mRNA, and SLS1, encoding a Mt membrane protein that may facilitate mRNA delivery to membrane-bound translation machinery; each of these genes networked with energy derivation by oxidation of organic compounds, cellular respiration, and aerobic respiration (Figs. 7h, 8, and S5; Table 2).
Furthermore, ara2Δ cells downregulated stress-responsive TFs, including CDC14, CRZ1, MSN2, MSS11, and SIP4, as well as RAP1, which is involved in chromatin silencing, establishment of protein localization to chromatin, and telomere maintenance under H2O2-induced oxidative stress (Figs. 7i, 8, and S5; Table 2). In contrast, genes associated with the negative regulation of transcription, including CDC14, GCR2, HTB1, MED1, MKS1, PHO80, RAP1, and RSC9, were downregulated in ara2Δ cells under the same conditions (Figs. 8 and S5; Table 2). Altogether, these results indicate that eAsA-deficiency in ara2Δ cells activate energy-generating systems (ATP and NADPH), including glycolysis, PPP (TKL2 and IDP3), and FA metabolism, such as β-oxidation in the cytoplasm and peroxisomes, but not respiratory metabolism in mitochondria, after the limited expression of predominantly stress-responsive TFs. Furthermore, these results indicate that eAsA depletion inactivates glycogen synthesis and intracellular glycerol accumulation following activation of IGD1, which encodes an enzyme inhibitor of glycogen catabolism, and downregulation of GPD1, which encodes GAPDH, a key enzyme in glycerol synthesis during oxidative stress.
Gene expression network analysis of purine metabolism-associated genes in ara2Δ cells under oxidative stress
eAsA-lacking ara2Δ cells downregulated the expression of various purine and pyrimidine metabolism-related genes, including ADH5, GCR2, GPD1, PGK1, RAP1, SOL2, SOL4, COB, YAH1, AIF1, RNR4, SDH4, CAT2, IFA38, OAR1, RNA4, encoding a pre-mRNA splicing factor, URH1, encoding a uridine nucleosidase involved in pyrimidine extermination, AMD1, encoding a cytoplasmic AMP deaminase involved in the regulation of intracellular purine (adenine, guanine, and inosine) nucleotide pools, OLI1, encoding F0-ATP synthase subunit c, CDD1, encoding a cytidine deaminase which catalyzes the modification of cytidine to uridine, ECM31, encoding the Mt 3-methyl-2-oxobutanoate hydroxymethyltransferase involved in pantothenate biosynthesis, HST4, encoding NAD+-dependent protein deacetylase, PXA1, encoding an ATPase integral to the peroxisomal membrane, which catalyzes the transmembrane movement of long chain FAs, and URA10, encoding orotate phosphoribosyltransferase isozymes that catalyzes the fifth enzymatic step in the de novo biosynthesis of pyrimidines (Fig. 9a). Additionally, ara2Δ cells downregulated the expression of genes associated with cofactors in biosynthetic processes, including BNA4, ECM31, HEM1, RNR4, URH1, YAH1, and CAT5, encoding a monooxygenase required for ubiquinone (coenzyme Q) biosynthesis (Fig. 9b). Downregulated genes created a network with oxidoreduction coenzyme metabolism, coenzyme metabolism, cofactor biosynthesis, and a wide range of purine and pyrimidine metabolic processes, including nucleobase-containing small molecules, pyridine-containing compounds, ribose and nucleoside phosphates, nucleotides, ribonucleosides, nucleotide mono- and triphosphates, purine and pyridine nucleosides, monocarboxylic acid, purine nucleoside mono- and triphosphates, ribonucleoside mono- and triphosphates, purine ribonucleotides, nicotinamide nucleotides, purine ribonucleoside mono- and triphosphates, and ATP (Figs. 8 and S5; Table 2).Taken together, these results suggest that eAsA deficiency provokes the severely limited metabolism of purine and pyrimidine, as well as downregulates genes implicated in the synthesis of cofactors and coenzymes during H2O2-induced oxidative stress.
Gene expression network analysis of aromatic and cyclic compound processes, biosynthetic metabolites, and transporters in ara2Δ cells under oxidative stress
Mutant ara2Δ cells downregulated multiple genes associated with aromatic and cyclic compounds, including BNA4, CDD1, EBS1, HBS1, IPK1, PHO13, SWT1, URH1, CBC2 (encoding an RNA cap-binding protein), POP3 (a processing precursor to RNA), and YCL001W-B (present in a region duplicated between chromosome XIV and III) (Fig. 9c). These downregulated genes networked with the catabolism of aromatic compounds, cellular nitrogen compounds, heterocyclic and organic cyclic compounds, nucleobase-containing compounds, and mRNA (Figs. 8 and S5; Table 2).
Furthermore, ara2Δ cells altered the expression of metabolic synthesis-associated genes in H2O2-induced oxidative stress; upregulating RMA1, encoding dihydrofolate synthetase; ATP10, encoding ATP synthase; STR3, encoding a cystathionine β-lyase involved in methionine biosynthesis; and TAM41, encoding a Mt phosphatidate cytidyltransferase required for cardiolipin biosynthesis, and downregulating URA10 and BNA4, involved in the de novo biosynthesis of pyrimidine and NAD, respectively; HEM1, ECM31, GPD1, IFA38, and OLI1, related to the biosynthesis of heme, pantothenic acid, glycerol, sphingolipid, and ATP, respectively; SRT1, forming the dehydrodolichyl diphosphate synthase complex involved in dolichol biosynthesis; ILV3, encoding dihydroxyacid dehydratase, which catalyzes the third step of a common pathway and leads to biosynthesis of branched-chain AAs; HIS5, encoding histidinol-phosphate aminotransferase, which catalyzes the seventh step in histidine biosynthesis; CAT5 and KEI1, involved in the biosynthesis of ubiquinone and inositol phosphorylceramide, respectively; SCS3, encoding a protein required for normal ER membrane biosynthesis; and TRP3, encoding an indole-3-glycerol-phosphate synthase involved in tryptophan biosynthesis (Fig. 9d).
Furthermore, ara2Δ cells upregulated various transporters-, symporters-, and permease-associated genes, including HXT8, STL1, HXT6, EBS1, TCA17, encoding a component of transport protein particle complex II, RSB1, encoding a sphingoid long-chain base efflux transporter, AGP2, a PM regulator of polyamine and carnitine transport, ATO2, encoding a transmembrane protein involved in the export of ammonia, YCT1, encoding a high-affinity Cys-specific transporter, MAL11, encoding a high-affinity maltose transporter, and APL6, encoding the β3-like subunit of the AP-3 complex, which functions to transport alkaline phosphatase from Golgi bodies to vacuoles, also downregulating PXA1, EBS1, UGA4, FLC1, encoding a flavin adenosine dinucleotide transporter required for the uptake of FAD into the ER, VBA3, encoding the permease of basic AAs in the vacuolar membrane and ER, IMA1, encoding an isomaltose required for isomaltose utilization, and MMP1, encoding high-affinity S-methylmethionine, which is required for using S-methylmethionine as a sulfur source (Fig. 9e). As noted above, upregulated genes, including HXT6, HXT8, SNF3, and STL1, networked with a wide range of GO categories (Figs. 4 and S3). Together, these results indicate that eAsA deficiency in ara2Δ cells promotes the uptake of molecules, such as glucose and hexose, and the efflux of ammonia, while also inhibiting the intracellular synthesis and transport of stress-responsive metabolites, such as glycerol, sphingolipid, ubiquinone, heme, ATP, NAD, FAD, and an especially in the ER, AA biosynthesis, such as His and Trp, de novo pyrimidine biosynthesis, and translation control.
Gene expression network analysis of organelles in ara2Δ cells under oxidative stress
Mutant ara2Δ cells had altered expression of a broad range of organelle-related genes when under H2O2-induced oxidative stress; the following genes were upregulated: MPS3, TMA20, encoding an unknown function associated with ribosomes, MPS2, encoding an essential membrane protein localized at the nuclear envelope and spindle pole bodies (SPBs), UTP4, encoding a subunit of the U3-containing 90S pre-ribosome and SSU processome complex, SAC7, encoding a GTPase activator involved in signal transduction, action skeleton organization, and cell wall (CW) organization, SPC42, encoding a cytoskeleton protein involved in SPB duplication, NVJ1, encoding a nuclear envelope protein, and AIM4, encoding a protein reportedly associated with the nuclear pore complex, and downregulated: SFP1, regulating the transcription of ribosomal protein and biogenesis genes, RSC37, encoding a protein associated with the large mitoribosomal subunit, RPS27B, encoding a protein component of the small (40S) ribosomal subunit, RPL33B, encoding the ribosomal 60S subunit protein L33B, RPS1B and RPS4B, both encoding a ribosomal protein of the small (40S) subunit involved in the maturation of small subunit rRNA and translation, MRPS12, encoding a component of the small subunit of the Mt ribosome, VAM3, encoding the syntaxin-like vacuolar t-SNARE, which functions with Vam7p in vacuolar protein trafficking, VAM7, encoding a vacuolar SNARE protein, RPL39, RPL8A, and RPL21A, encoding ribosomal 60S subunit proteins L39, L8A, and L21A, respectively, ZEO1, encoding a PM peripheral membrane protein for CW organization, MRP2, encoding a component of the small subunit of the Mt ribosome, RPS17A, encoding a component of the cytosolic small (40S) ribosomal subunit, TUB3, encoding α-tubulin, YDR106W, encoding a component of the dynactin complex, as an actin-related protein, YDR034W-B, predicting the tail-anchored PM protein, DAD1, encoding a microtubule-related protein, and ARC18, encoding a Mt protein of the Arp2/3 complex involved in actin cortical organization (Fig. 10a). Additionally, ara2Δ cells downregulated telomere organization-associated genes, including EBS1, ECO1, ELG1, RAD57, RAP1, and STN1 (Figs. 8 and S5; Table 2). In summary, eAsA-deficient ara2Δ cells upregulated the expression of genes related to the nuclear envelope and SPBs, while downregulating those associated with telomere organization, ribosomes, vacuoles, cytoskeleton, tubulin, and actin.
Furthermore, ara2Δ cells exhibited increased expression of majority of vesicle organization-associated genes, including ATG15, SPO20, PRM8, encoding a pheromone-regulated membrane protein, and YAR028W, encoding an integral membrane protein (Figs. 4, 10c, and S3; Table 1). Autophagy-related genes were also upregulated in ara2Δ cells, namely, ATG15, ATG20, MON1, ATG12, encoding an Ub-like modifier involved in autophagy, ATG29, encoding an autophagy-specific protein, ATG38, encoding a subunit of the phosphatidylinositol 3-kinase complex involved in macroautophagy, and ATG39, encoding a protein involved in autophagic degradation of the nucleus and ER; these genes formed a network with pre-autophagosomal structure, pre-autophagosomal structure membrane, and organelle disassembly (Figs. 4, 10c and S3; Table 1).
Moreover, ara2Δ cells downregulated a majority of ribosome-associated genes, including MRP2, MRPS12, RPS17A, RPS1B, RPS20, RPS27B, RPS4B, RPL21A, RPL33B, RPL39, RPL8A, RPL25, encoding ribosomal 60S subunit protein L25, and RPM2, encoding a protein subunit of Mt RNase P, a promoter of transcription; these genes formed a network with cytoplasmic translation, cytosolic ribosomes, and small ribosomal subunits, when exposed to H2O2-induced oxidative stress (Figs. 8d, 10d, and S5; Table 2). As noted above, downregulated genes, ACM1, EBS1, COS2, PBI2, SIZ2, and WHI4, associated negatively with cellular protein metabolism regulation (Figs. 6a, 8, and S5; Table 2). In addition, certain downregulated genes, including ATG19, COS12, HOM3, MON1, SNX3, TRE2, and VAM7, were involved in protein targeting to vacuoles, protein localization, and the establishment of protein localization to vacuoles (Figs. 8 and S5; Table 2). Next, downregulated genes, including CAT2, ECO1, ERF2, HEM1, OAR1, and TGL3, were implicated in transferase activity (Figs. 8 and S5; Table 2).
Moreover, ara2Δ cells exhibited downregulated expression of various vacuole-related genes, including CPS1, HOM3, KOG1, MON1, PBI2, UGA4, VAM3, VAM7, CSI2, encoding an unknown protein localized to vacuoles, and ERS1, encoding a protein involved in cystine transport under oxidative stress; these genes created a network with storage, lytic, and fungal-type vacuoles (Figs. 8, 10e, and S5; Table 2). ara2Δ cells also exhibited downregulated expression in microtubule cytoskeleton-associated genes, including ARP10, CDC14, DAD1, HYM1, SLD7, GPO13, TUB3, and LDB18, encoding a component of the dynactin complex (Fig. 10f); these downregulated genes formed a network with microtubule cytoskeleton, spindles, and sister chromatid segregation (Figs. 8 and S5; Table 2). Moreover, ara2Δ cells downregulated organelle fusion-associated genes, including ENV7, PBI2, PHO80, VAM3, VAM7, and FUS2, encoding a cell fusion regulator (Figs. 8, 10g, and S5; Table 2). In contrast, ara2Δ cells exhibited altered hypothetical protein expression (HPs; indicating an unknown protein and ambigous open reading frames (ORFs) under oxidative stress). As compared to WT cells, ara2Δ cells up- and downregulated 111 and 64 genes, respectively, encoding HPs. Genes with a greater than 10-fold change are shown in Table 3. Of these differentially expressed genes, those up- and downregulated accounted for approximately 30% and 23%, respectively (Table S2). Taken together, these results suggest that eAsA-deficiency in ara2Δ cells results in the downregulated expression of organelle-associated genes, including vacuoles, ribosomes, cytoskeleton, spindles, organelle fusion, and telomere organization, following the downregulation of positive and negative TFs and the negative regulation of protein metabolism; this indicates that differentially expressed genes encoding HPs can, in part, participate in cellular responses to oxidative stress, the mechanism for which remains to be elucidated. A schematic diagram of the ara2Δ cells sensitivity mechanism to oxidative stress is shown in Fig. 11.
Table 3
Identification of 10–fold changed gene encoding hypothetical protein in ara2Δ cells under oxidative stress
Gene name | Ratio (ara2Δ/WT) | Description |
YGR190C | 24.678 | Dubious open reading frame |
YLR458W | 23.574 | Dubious open reading frame |
YAL064W | 22.545 | Protein of unknown function |
YFL015W-A | 21.516 | Dubious open reading frame |
YMR013W-A | 12.425 | Dubious open reading frame |
YHR212C | 10.527 | Dubious open reading frame |
YCL048W | 0.089 | Protein of unknown function |
YKL162C | 0.076 | Putative protein of unknown function |
YDL023C | 0.064 | Dubious open reading frame |
YJL150W | 0.064 | Dubious open reading frame |
YKR075W-A | 0.063 | Dubious open reading frame |
YNL269W | 0.050 | Protein of unknown function |
YJL136W-A | 0.048 | Protein of unknown function |
YBR224W | 0.048 | Dubious open reading frame |
YOR345C | 0.042 | Dubious open reading frame |
* WT, wild-type yeast cells; ara2Δ, ARA2-deleted yeast cells. |