An Ultradian Oscillator Mediates Longevity


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========================================== An Ultradian Oscillator Mediates Longevity ==========================================

Abstract

The lifespan extension phenomena by dietary restriction remains a mystery. Using a systems biology approach employing GenDR, interaction networks and gene expression data we investigated the obscure role of autophagy in mediating the longevity effect of dietary restriction. We found evidence for a crucial role of ultradian oscillation in lifespan prolongation.

Authors

Daniel Wuttke1; Fusheng Tang2; Joao Pedro de Magalhaes1

1 Integrative Genomics of Ageing Group, Institute of Integrative Biology, University of Liverpool, Liverpool UK

2 Department of Biology, University of Arkansas, Little Rock, Arkansas 72204-1099, USA

Introduction

Autophagy ("eat oneself") is a tightly regulated catabolic process, which degrades cellular components by using lysosomal machinery and is important for cell growth, development and homeostasis by maintaining a balance between synthesis, degradation and subsequent recycling of cellular macromolecules. Autophagy occurs at low baseline levels in all cells and ensures homeostatic turnover of long-lived proteins and organelles [4 in (Morselli et al., 2009)]. Autophagy is upregulated well beyond basal levels to mobilize intracellular nutrients, as it is the major mechanism by which a starving for nutrients (carbon, nitrogen, sulphur and various amino acids) or a stressed (e.g. ER stress, hypoxia, growth factor withdrawal, etc.) cell reallocates nutrients from ancillary processes to more important ones [2,3 in (Morselli et al., 2009)].

The magnitude of autophagosome formation is tightly regulated by intracellular and extracellular amino acid concentrations and ATP levels via signalling pathways including TOR (Eskelinen and Saftig, 2009).

An evolutionary conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation. mTOR signalling is inhibited during autophagy initiation, but reactivated by prolonged starvation. mTOR reactivation is autophagy-dependent. mTOR then initiates the autophagic lysosme reformation (ALR). Rab7 is a key regulator of this process. Rab7 residues on autolysosomes and mediates autophagasome-lysosme fusion. Rab7 must dissociate from tubules before reformation can proceed. Overexpression of a constitutively active Rab7, which permanently associated with the membrane, abrogates ALR, resulting in enlarged and long-lasting autolysosomes. ALR inhibition by rapamycin blocks dissociation of Rab7 from the distended autolysosomes it produced. Thus, mTOR might regulate ALR through Rab7.

Autophagy is required for the lifespan extension by DR, many genetic manipulations (e.g. in eat-2, p53, TOR and insulin/IGF1 signalling as well as pnc-1 and sirtuins) and pharmacological interventions (resveratrol, rapamycin and spermidine). However, mitochondrial mutants and reduced protein synthesis (e.g. by mutation in elongation factors or rsks-1) appear not to require autophagy for lifespan extension. Subjecting long-lived mitochondria mutants clk-1 and isp-1 to bec-1 or vps-34 RNAi during adulthood has no effect on lifespan (Hansen et al., 2008). Inhibiting protein synthesis under ad libitum (AL) extends lifespan in the absence of autophagy. Autophagy is required specifically for longevity pathways that are fully integrated with and regulated by environmental signals that reflect availability of food, like the insulin/IGF1 pathway and the response to DR (Hansen et al., 2008).

Surprisingly, although autophagy seems to be a very important catabolic process with anti-ageing effect, not all forms of autophagy are actually necessary for lifespan extension by DR. The intravacular disintegration of autophagy body membranes and vacuole-vacuole fusion machineries (ergosterol, Nyv1, Ypt7, etc.) is absolutely indispensible for lifespan extension. Secretophagy is the only DR-essential form of autophagy (Tang et al., 2008).

Results

Previously we showed that DR-essential genes are conserved on the molecular level and likely to interact with each other more than expected by chance. We established a database of DR-essential genes and their conserved orthologs and identified further DR-essential genes implicated in vacuolar/endosomal trafficking (Wuttke et al. 2012).

These conserved genes can be used as seed genes to identify new DR-essential genes. For example, mammalian Rab7 is required for the maturation of mTORC1 (Flinn et al., 2010). The yeast homolog of Rab7 (Ypt7) shows a negative genetic interaction with Tor1 (Costanzo et al., 2010), suggesting a collaborative interaction between Ypt7 and Tor1. Although genes interacting with Rab7 or Ypt7 may not be conserved, the mechanisms of TORC1 activation may be conserved. Genes associated with Rab7 in mammalian cells or Ypt7 in yeast cells may be DR-essential genes.

Endocytosis genes are differentially expressed upon DR

Genes which were more than two-fold transcriptional differentially expressed upon DR were examined using the DAVID bioinformatics resource [Table: KEGG pathways enriched in DR-differentially expressed genes; Table: KEGG pathways enriched in DR-induced und suppressed genes]. Enriched KEGG (Kyoto of Enyclopdia of Genes and Genomes) pathways for all differentially expressed genes [Table: KEGG pathways enriched in DR-differentially expressed genes] and the pathways specifically enriched for up- and down-regulated genes (Table: ) give a simplified overview of the affected process. "Endocytosis" was found to be among the upregulated terms [Table: KEGG pathways enriched in DR-induced und suppressed genes]. Genes annotated with "Endocytosis" (81 genes) were checked for differentially expression. By an 1.5-fold change cut-off, 42 genes (>50% of all) exhibit differentially transcription. 25 were up- and 17 genes were downregulated. Among the upregulated genes were YPT7 (1.5-fold) and GTS1 (2-fold).

GTS1 is a potential DR-essential transcriptional regulator

We used a gene-regulatory network to identify the transcription factors which had a high specificity for DR-differentially expressed genes [Table: Transcription factors regulating DR-induced genes; Table: Transcription factors regulating DR-suppressed genes]. Transcription factors which were reported to be activated upon DR such as Gis1, Hot1, Mig1, Hsf1, Hap4, Msn2 controlled DR-induced genes. Thus, verifying that this approach is correct and can identify relevant regulation.

GTS1 also acts as a transcriptional regulator [Table: Transcription factors regulating DR-induced genes]. 26 of the 62 target genes of GTS1 changed their expression more than 2-fold upon DR. By a cut-off about 1.5-fold change it is even 43 target genes of GTS1 with differentially expression upon DR. GTS1 regulates DR-essential and vacuolar-associated genes with increased expression such as DR-essential YDL180W (2.3-fold) and HSP12 (3.6-fold) as well as autophagy genes ATG11 (3.2-fold), ATG19 (3.6-fold) and ATG34 (4.5-fold). It also regulates the expression of DR-essential HXT17 and upregulates the yeast Glycogen synthase kinase 3 (YGK3) as well as YHR138C (5.3-fold) which is involved in vacuole fusion (Xu et al., 1998).

GTS1 transcription is controlled by several transcription factors of which all of them exhibit either a significant increased expression (> 2-fold) or a trend to increased expression [Table: Differentially expression of transcription factors regulating GTS1], which supports the finding that GTS1 transcription is likely be enhanced upon DR.

Ultradian genes are primarily upregulated by DR

Numerous of the DR-affected processes such as DNA replication and nitrogen metabolism, especially the sulfhydryl containing amino acid (methionine and cysteine) are reminiscent to the processes affected by the ultradian clock (Klevecz et al., 2004; Murray et al., 2007). Indeed, 43 out of the 69 strongest ultradian genes were differentially expressed upon DR (p-value < 0.05). The enriched KEGG pathways of only the strongest ultradian oscillating genes indicate that they primarly affect sulur metabolism [Table: KEGG pathways for highly ultradian differentially expressed genes]. Among them DR upregulates 3-times as many genes (43 out of 74; p-value < 0.0005) as it downregulates (11 out of 74; p-value = 0.06) also with a much higher magnitude (by an 2-fold change cut-off) [Table: Differentially expression of highly ultradian genes upon DR]. A likely nexus could be cysteine and methionine metabolism as it is shared by the DR and ultradian signature.

ATG1 is upstream of GTS1 and potentially coregulated

As genes which are clustered at the same genomic position are likely to be coregulated and/or functional associated (Al-Shahrour et al., 2010; Woo and Li, 2011) we investigated the genomic neighbourhood of DR-essential and differentially expressed genes loci. Downstream to GTS1 on the same strand is ATG1 which is 1.6-fold upregulated [Figure: GTS1 loci from SGD; Figure: GTS1 loci from UCSC with transcription factor binding sites]. Upstream of GTS1 on the opposite strand is MND1 which was 3.7-fold upregulated. Downstream of MND1 (Meiotic Nuclear Divisions) is STR3 (Sulfur TRansfer) encoding peroxisomal cystationine beta-lyase, which convertes cystathionine into homocysteine and may be regulated by Gto1 (Glutathione Transferase Omega-like). GTO1 deletion increased hibernating lifespan at low temperature (Postma et al., 2009).

ATG1 encodes a protein Ser/Thr kinase required for vesicle formation in autophagy and cytoplasm-to-vacuole targeting (Cvt) pathway. It is structurally required for phagophore assembly site formation. During autophagy it forms a complex with ATG13 and ATG17. Most of ATG1 transcriptional regulators were upregulated [Table: Transcription factor regulating ATG1]. Although ATG1 is not DR-essential, this close association to GTS1 implies a relationship between Atg1 and Gts1 and opens the possibility that certain aspects of autophagy are oscillating.

GTS1 transcription is regulated by regions located in its two upstream located orfs (Tonozuka et al., 2001). It is possible that this regulation also extends to ATG1 [Figure: GTS1 loci from UCSC with transcription factor binding sites] and therefore couple their expression.

Rapamycin Resistance/Sensitivity Genes Differentially Expressed upon DR

Rapamycin is assumed to be an anti-ageing drug and the target of rapamycin (TOR) to be at the center of lifespan extension by DR. As it is commonly thought that DR mainly mediates lifespan extension via suppression of TOR, we tested the hypothesis that rapamycin treatment phenocopies the effect of DR.

The cellular target genes and modulators of the DR-mimetic drug rapamycin signalling were recently identified via a transcriptional profiling approach of overexpression mutants which alter rapamcycin resistance (Butcher et al., 2006). We used this data and crosschecked the rapamycin resistance modulating genes with DR-differential genes. Rapamycin is thought to mimic the effect of DR also on the transcriptional level. Therefore, we look at DR-differentially expressed genes. What kind of DR regulated genes impact on rapamycin resistance? Out of 308 overexpression mutants 136 had differentially expression upon DR (p-value < 0.05). Among the 136 overexpression mutants which were either enriched (72) or depleted (64) upon rapamycin treatment, there were 62 genes commonly differentially expressed in the same direction, while 74 genes were expressed in the opposite [Table: DR vs. rapamycin statistics].

Commonly enriched/upregulated genes were related to catabolic processes/autophagy, stress response, protein transport, transcription regulation, metal binding, nitrogen component biosynthetic processes as well as transmembrane proteins [Table: DR vs. rapamycin terms a]. Commonly depleted/downregulated were gene associated to metal ion binding, transmembrane proteins, ubl conjugation, transcription and purine binding [Table: DR vs. rapamycin terms b]. By Rapamycin depleted overexpression mutants and DR upregulated genes share terms associated to non-membrane-bound organelles (ribosome, nucleolus), ATP binding, translation regulation, gene silencing, nitrogen metabolism, mitochondrion, protein transport and endoplasmic reticulum [Table: DR vs. rapamycin terms c]. By Rapamycin enriched overexpression mutants and DR downregulated genes have terms in common related to transmembrane proteins, protein ubiquitination, vacuole, transition ion binding/autophagy, mitochondrion and transcription [Table: DR vs. rapamycin terms d].

TOR1, BMH1 and GSH1 impact on rapamycin resistance/sensitivity

Genes which by mutation abolishe the DR-lifespan extension (DR-essential genes) are potential crucial regulators of its underlying mechanisms. We looked if their overexpression affects rapamycin sensitivity. Out of 89 DR-essential genes and orthologs 5 (p-value = 0.17) were among the rapamcyin resistance/sensitive overexpression mutants [Table: Rapamycin resistant DR-essential]. 14-3-3/BMH1 and MTOR/TOR1 overexpression mutants were enriched by rapamycin treatment, while overexpression of Gamma glutamylcysteine synthetase GCLC/GSH1 and stress response transcription factor MSN4 were depleted by rapamycin treatment. Overexpression of a potential DR-essential aquaporin gene (aqp-1) ortholog (YFL054C) was also depleted by rapamycin treatment. GSH1 is downregulated during ageing, in sip2delta and snf4delta mutants.

Ultradian transcription factors influence rapamycin resistance/sensitivity

Rapamycin has strong effects on ultradian rhythmicity (Murray et al., 2007). Out of 74 highly ultradian genes 7 genes (p-value = 0.04) exhibit rapamcyin resistance/sensitive as overexpression mutants [Table: Rapamycin resistant ultradian genes]. All but one (cytosolic aldehyde dehydrogenase ALD6) are transcriptional regulators. The enzyme ALD6 locates to the mitochondrial outer membrane upon oxidative stress. Two are stress response transcription factors (MSN4 and YAP1 (H2O2)) and two are transcriptional factors involved in regulation of methionine metabolism (MET31 and CBF1).

GTS1 targets genes conferring resistance to rapamycin are vacuole-related

Out of 62 clock gene GTS1's target genes 8 (p-value = 0.006) were overexpression mutants affecting rapamcyin resistance/sensitive [Table: Rapamycin resistant GTS1 target genes]. Half of them were implicated in catabolic processes (ATG19, MMS21, GSY2 and HSP104) of which besides MMS21 was implicated to the vacuole. Three of these 8 genes were of unknown function. One of them (YGR125W) is also known to be localized to the vacuole. Most of these gene overexpressions (besides two) were enriched by rapamycin treatment.

Differential Expression by TORC1 Inhibition

Rapamycin extended chronological lifespan proportional with increasing concentrations from 100 pg/mL to 1 ng/mL (Powers et al., 2006).

Gene expression profiles of different strains treated with the TORC1 inhibitors rapamycin or caffeine were used (Reinke et al., 2006). Rapamycin triggers translocation of Gln3 and Msn2 to the nucleus and induces the expression of several genes subjected to nitrogen catabolic repression (GAT1, MEP1, and GLN1) and stress-response element driven promoters (Gonzalez et al., 2009).

Rapamycin (BY4741 2 microgram/mL for 1h) mediates transcriptional repression of ribosomal protein genes. Mediator and Maf1 function in parallel pathways to negatively regulate ribosomal protein mRNA and tRNA synthesis (Willis et al., 2008).

DR and rapamycin commonly and differentially affect the nucleolus

What are the similarities and differences in the transcriptional signatures of DR and rapamycin? DR and rapamycin commonly most strongly affect genes related to the ribonucleoprotein complex as well as nucleolus. Upregulated genes common to DR and rapamycin treatment were enriched for heat shock response [Table: DR vs. rapamycin terms e]. Commonly downregulated genes by DR and rapamycin were enriched for translation and nucleolus [Table: DR vs. rapamycin terms b]. DR upregulated and rapamycin downregulated genes are enriched for nitrogen metabolism. DR downregulated and rapamycin upregulated genes were enriched for nucleolus.

TORC1 inhibition upregulates GTS1 a subset of its target genes

TORC1 inhibition experiments consistently upregulated GTS1 as well as some of its targets genes such as ATG19, GSY1, YFR039C and YEL073C. SSA1 increases in mRNA expression level during ageing. This could result in progressively increased inhibition of Gts1. Of the Gts1 target genes, YEL073C is induced (5.4-fold) during ageing.

SEO1 is induced during sip2delta ageing (6.1-fold), while it is upregulated in snf4delta (4.9-fold) in comparision to wild-type. SEO1 encodes a putative permease, member if the allantoate transporter subfamiliy of the major facilitator superfamily.

SUC2 is downregulated in sip2delta (4.9-fold) and snf4delta (2.2-fold). SUC2 encodes the invertase a sucrose hydrolysing enzyme. A secreted, glycosylated form of Suc2 is regulated by glucose repression.

YGK3 is the yeast GSK3, which controls Msn2-dependent transcription and of stress responsive genes and protein degradation. YGK3 is upregulated upon DR, while downregulated (3.7-fold) in rapid ageing SIP2 deletion mutants.

NQM1, encoding a transaldolase of unknown function is repressed by Mot1 and induced by alpha-factor and during diaxic shift. Its mRNA is 2-fold upregulated upon DR, sip2delta and in Hap4OE, 4-fold in hxk2delta and dramatically increased upon pharamacological TORC1 inhibition (200ng/mL for 1 h).

CRF1 (Co-Repressor with FHL1) encodes a transcriptional corepressor involved in repression of ribosomal protein gene transcription via the TOR signalling which promotes nuclear Crf1 accumulation. Its mRNA is 3-fold upregulated upon DR, 2-fold in hap4OE and 4-fold in hxk4delta.

Ultradian Genes

MET28 is upregulated by DR (11-fold) and TORC1 inhibition. Interestingly, it is 7.4-fold lower in sip2dela and induced during ageing in both sip2delta and snf4deta.

Drug-Detoxification

We investigated the rapamycin induced differential expression of genes related to drug detoxification. BSD2 is upregulated by TORC1 inhibition. Although OPT2 is strongly downregulated upon DR, TORC1 inhibition induce its expression. ECM38, Gamma-glutamyltranspeptidase, the major glutathione-degrading enzyme involved in detoxification of electrophilic xenobiotics is, downregulated by DR, while upregulated by TORC1 inhibition. DR and TORC1 inhibiton upregulated GLO1.

Progeric sip2delta and long-lived snf4delta

sip2delta upregulates DR-essential NDE2, MDH2 and ATG1 during ageing.

During ageing snf4 upregulates COX5B (hypoxy? Paper) and MEP2 (circadian) and downregulates GUT2, FLO1 and FLO9. sip2 underexpress GSH1 and MEP2, while FLO1 and FLO9 as well as copper transporter CTR1 and CTR2 were all overexpressed.

Gts1 is a transcriptional coactivator of Sfl1 which represses FLO1.

FKH1 is downregulated in snf4delta (young vs. old) and DR (slightly).

DR and ageing induce gluconeogenesis

Both DR as well as aging idunce genes related to gluconeogensis. It probably makes much more sense to look at which time in the metabolic rhythm of a cell a certain process occurs. TORC1 is presumably only active in the oxidative phase of the ultradian oscillation. Glycolysis occurs at the reductive recharging phase. It would be intersting to determine at wich phase gluconeogenesis occurs.

Rejuvenation and DR-lifespan extension share common mechanism

Sporulation is a process of surviving for extended periods of time in unfavourable conditions. Sporulation is initiated by nutrient deficiency. During sporulation rejuvenation occurs. It is possible that DR also activates rejuvenation controlled processes. I.e. DR induces a light form of rejuvenation, although not sufficient to stop ageing. It makes sense that evolution come up with single mechanisms that it reuses several times (e.g. gene duplications, protein domains, etc.).

The top transcription factor regulating DR-induced genes are UME1, IME1, and UME6. UME1 (Unscheduled Meiotic gene Expression) was slightly downregulated (-1.3-fold), is a negative regulator of meiosis, which is required for repression of a subset of meiotic genes during vegetative growth and requires the binding of histone deaceylase Rpd3 for its activity. RPD3 deletion extends lifespan under AL but not DR. UME1 deletion increased rapamycin resistance. IME1 (Inducer of Meiosis) which was slightly upregulated (+1.43) is the master regulator of meiosis that is active only during meiotic events. It activates transcription of early meiotic genes through interaction with Ume6 and is degraded by the 26S proteosome following phosphorylation by Ime2. UME6 which was upregulated (+1.62-fold) is a key transcriptional regulator of early meiotic genes, such as SPO11 (+2.61-fold), SPO13 (+2.18-fold) and IME2 (+7.4-fold), via binding to upstream regulatory sequence URS1 (5'-AGCCGCCGA-3') and couples metabolic responses to nutritional cues with initiation and progression of meiosis. It forms a complex with Ime1 as well as with Sin3-Rpd3. UME6 deletion mutant had decreased endocytosis as well as resistance to rapamycin and caffeine, but increased heat sensitivity. IME2 is a serine/threonine protein kinase participating in triggering meiosis. It associates with Ime1 and mediates its stability, while IME2 expression is positively regulated by Ime1. IME2 activates Ndt80. NDT80 was 2.5-fold upregulated, while its antagonist SUM1 which was 1.7-fold downregulated. Most of the transcription factors known to regulate NDT80 were upregulated supporting its induction by DR. Strikingly NDT80 is suppressed during ageing 30-fold (need to check again). GTS1 and HSP12 might be transcriptional targets of NDT80, although their expression pattern does not follow those of the middle meiotic genes. After sporulation, which resets lifespan, GTS1 was 3.5-fold upregulated. UME6 is one of the highly ultradian genes. Sporulation also downregulated BMH1. This 14-3-3 protein binds several transcription factors such as Gal4, Gat1, Gcr2, Gln3, Gts1, Hot1, Mig1, Msn2, Sfl1, Ume6 and Xbp1 [21 in (van Heusden, 2009)]. Interestingly after sporulation TOR1 was greatly upregulated.

GTS1 overexpression induces meiotic genes and SIR2

Genes which are differentially expressed upon GTS1 overexpression were eximined with DAVID. The most strongly 100 upregulated genes were enriched for "sexual reproduction" / "sporulation" and "intrinsic to membrane". Strikingly SIR2 was the 7th most strongly upregulated gene.

Ndt80 ageing-related targets are non-canonical middle meiotic genes

Ndt80 induces the expression of Yeast casein kinase YCK3 (+2-fold), encoding a palmitoylated, vacuolar membrane-localized casein kinase I isoform, which negatively regulates vacuole fusion during stress. Note, vacuole fusion is required for DR-lifespan extension.

Ndt80 has several proposed target genes known to be related to ageing and DR, however their expression pattern is different from that of canonical middle meiotic genes.

A major role of Ndt80 is to start the synthesis of new membranes since cells after meiosis I are already rejuvenated. ERG25 encodes a C-4 methyl sterol oxidase, was proposed to be an Ndt80 target gene (Doniger et al., 2005) and indeed during the time course of sporulation it was 2-3 fold upregulated.

Gsh2 operates in the same pathway as DR-essential Gsh1 and is a known Ndt80 target gene.

SIR1 is greatly downregulated by Ndt80 overexpression. SIR1 deletion diminishes the periodic fluctuations in NADH levels.

GTS1 is downregulated in aged cells

Ageing downregulates retrotranspons, telomere maintaince, ribosomal assembly, s-adenosylmethionine and sporulation.

GTS1 is 50-fold downregulated in aged cells and placed on number 4th of the strongest ageing-suppressed genes.

Gametogenesis (sporulation). Ectopic NDt80 synthesis in vegetative cells induces transcription of its target genes. NDT80 transciption is dependent on Ime1, which activates early sporulation genes (Chu and Herskowitz, 1998).

Conclusion

Sulphur-containing amino acid metabolism (GSH1, MET31 and CBF1) and stress response seem to be a nexus of rapamycin/DR and the ultradian oscillation. TOR may inhibit Gts1 via Bmh1 phosphorylation. MSN2/4 may transcriptional regulate GTS1. GTS1 appears to transcriptional target catabolic processes associated to the vacuole. Catabolic and anabolic reactions are separated in time. Oscillations of different scales are driven by cycles of oxidative and reductive phases generated by redox cycling of intracellular thiols. In both yeast and mammalian cells mitochondria behave as networks of coupled oscillators producing ROS as signalling molecules with scale-free dynamics (Aon et al., 2008). Interestingly, rapamycin and another small molecule LY-83583, which suppresses growth inhibition by rapamycin appear to converge on affecting mitochondria. The transcriptional profiling approach of rapamcycin resistance overexpression mutants (Butcher et al., 2006) identified GUF1 is a guanylate cyclase inhibitor with poor specificity of unknown function, as a potential hub. GUF1 overexpression as well as deletion renders cells hypersensitive to rapamycin. Exact levels of Guf1 are critical determinants of rapamycin sensitivity. Similar to the effect of GTS1 overexpression deletion on lifespan. GUF1 also genetically interacts with another potential clock gene Timeless/TOF1 which is evolutionarily conserved replication fork-associated factor.

Among the proteins exhibiting abundance changes upon rapamycin treatment, almost 90% of them demonstrated homodirectional transcriptomic changes under conditions of heat and oxidative stress. Activation of heat/oxidative stress responses phenocopied TOR inhibition. Strains like hsf1-R206S, F256D, ssa1-3 and ssa2-2 which are constitutively activated for Hsf1 inhibited rapamycin resistance. Constitutive activation of other regulators of heat/oxidative stress responses (Msn2/4 and Hyr1, did not inhibit TOR signalling. Activated Hsf1 inhibits rapamycin resistance and TOR signalling via elevated expression of specific target genes (Bandhakavi et al., 2008).

It is here hypothesised that during ageing there is a progressive change in the oscillation of gene expression and metabolism, resulting finally in the loss of homeostasis. Short-lived mutants upregulate genes in the detrimental oxidative phase, while long-lived mutant overexpress reductive phase genes. DR induces an alternative oscillatory behaviour. Rejuvenation resets the oscillation back to the beginning.

A possible approach to investigate this would be to separate ultradian genes in three phases (1. oxidative, 2. reductive/recharging, 3. reductive/building) and look for enrichment of genes associated long-lived and short-lived mutants as well as genes differential expressed during aging or upon DR. Comparsion should be done on the level of genes or only terms.

GTS1 interacts with endocytosis associated protein YAP1802 involved in clatherin cage assembly, which binds Pan1 and clathrin. Its null mutant has abnormal silencing. YAP1802 also interacts with ESCRT-I subunit (MVB12) required to stabilize oligomers of the ESCRT-I core complex, which is involved in ubiquitin-dependent sorting of proteins into the endosomes. Its deletion increases rapamycin sensitivity.

The s-adenosylmethionine synthetase activity was commonly differential expressed in yeast, worm, fly and mammals (Wuttke et al. 2012).

Several key enzymes of gluconeogenesis are degraded in the vacuole via the vacuolar import and degradation (Vid) pathway upon refeeding after long-term starvation (> 3 days). TORC1 components associate with these enzymes and excessive Tor1 inhibits their degradation, while TOR1 deletion had little effect on enzyme degradation. Upon glucose addition Tor1 and Tco89 dissociate from these enzymes. Tor1 and Tco89 were in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole. TORC1 traffics to and from the vacuole. Vacuoles become enlarged and twisted after rapamycin treatment. TORC1 cycles between plasma membrane and vacuole to maintain the size of vacuole. Endocytosis results in an increased influx and an expansion of vacuole membrane (Alibhoy and Chiang, 2010).

GTR1 (GTP binding protein Resemblance) is predominantly localized to the cytoplasm and a negative regulator of the Ran/Tc4 GTPase cycle. It is a component of the GSE(required for proper sorting of amino acid permease Gap1)/EGO complex and involved in phosphate transport as well as telomeric silencing. Human RRAGA and RRAGB are the functional homologs of GTR1 (52% sequence similarity), which when expressed in yeast can rescue GTR1 mutation (Hirose et al., 1998). Its homolog raga-1 in nematode was implicated in the age-related decline in motor performance (Schreiber et al., 2010). Ras-like GTPases, GTR1 and GTR2, are involved in epigenetic control of gene expression (in TOR signalling). GTR1 and GTR2 are molecular switches in TOR signalling and genetically interact with INO80 (a chromatin remodeller that associates to actin and modifies stress gene transcription). Gtr2 interacts physical with both Rvb1 and Rvb2, localize to chromatin and could activate transcription. Gtr1 and Gtr2 are involved in chromatin silencing in the vicinity of telomeres. Gtr1 and Gtr2 were required to repress nitrogen catabolite-repressed genes, which are repressed by TOR signalling (Sekiguchi et al., 2008).

SLM4 (Synthetic Lethal with Mss4) is a component of the EGO complex (involved in microautophagy) and the GSE complex. MSS4 is a phosphatidylinositol-4-phosphate 5-kinase participating in actin cytoskeleton organisation and morphogenesis. Slm proteins are involved in endocytosis (Kamble et al., 2011). SLM4 negatively interacts with TOR1, which means that they function in distinct but parallel pathways (EGOC and TORC, respectively) in a given process (regulation of microautophagy or maybe endocytosis). SLM4's positive interaction with VPS30 could indicate that they operate in different pathways (GSE complex and PI3K Complex II, respectively) on the same process (vacuolar protein sorting) in opposite fashion.

Gluconeogenesis

The effect of pharamacological inhibition of TORC1 by rapamycin or caffeine on transcription levels of gluconeogenesis related genes is in Supplemental table 2.

If DR upregulates gluconeonegenesis and ageing as well as rapid ageing mutants do it too, so what is it all about?

Up-regulated gluconeogenesis enzymes may sequester components of TORC1 and actually downregulate TORC1. Since overexpression of TOR1 or deletion of TCO89 inhibits the degradation of gluconeogenesis enzymes after glucose-refeeding, the interaction between gluconeogenesis enzymes and components of TORC1 likely brings components of TORC1 to the vacuole import vesicles (Vid) and separate TORC1 from its normal functioning membranes.

Recent studies showed that part of Tor1 is localized to the vacuolar membrane (Aronova et al., 2007) and the shape of vacuolar morphology controls the activity of TORC1 (Binda et al., 2009; Dubouloz et al., 2005). The vacuolar localization is thus a reinforcing criterion for our predicted DR-essential proteins.

Role of Endocytosis in DR

Interestingly, numerous autophagy-related DR-essential genes such as bec-1 or vps-34 have also roles in vesicle trafficking and endocytosis. In fact, among the conserved DR-essential interactions was VPS34/vps-34/PI3K59F/Pik3c3 which synthesize phosphatidylinositol 3-phosphate and forms membrane associated signal transduction complex to regulate protein sorting. The mammalian class III phosphatidylinositol 3-kinase complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Distinct PI3K-III sub-complex can confer functional specificity. A specific sub-complex containing VPS34, Beclin 1, UVRAG and BIF-1 regulates both endocytic receptor degradation and cytokinesis, whereas ATG14L (a PI3K-III subunit complex involved in autophagy) is not required (Thoresen et al., 2010).

We found that DR alters numerous genes related endocytosis on transcript level. Among them was the DR-essential gene YPT7. Ypt7, a small Ras-like GTPase which localizes to the vacuolar membrane, is required for docking and fusion of endosomal vesicles to vacuolar membranes as well as homotypic fusion events between vacuolar compartments.

We identified three novel DR-essential and vacuolar-related genes when deleted prolonged lifespan and cancelled out the effect of DR. These genes are primarily implicated in drug detoxification (OPT2), transition metal ion homeostasis (FRE6) and endosomal-vacuolar trafficking of plasma membrane proteins (RCR2).

Opt2 maintains vacuolar morphology and function in the formation of mature vacuoles via vesicle fusion. OPT2 mutants have several small vesicles instead of a large vacuole and are sensitive to various toxic agents, such as rapamycin, polyamines and divalent metal ions which are normally detoxified (via endocytosis and ABC transporters) in the vacuole (Aouida et al., 2009). opt2Delta causes similar vacuolar morphology as in vam7Delta [Heider and Barnekow 2008 in (Aouida et al., 2009)]. OPT2 interacts genetically with TOR1 (synthetic growth defect), its gene product interacts physical with Sur4 (sphingolipid synthesis), and its mRNA expression is dramatically downregulated upon DR. The defect in vacuolar sequestration of polyamines could be responsible for its lifespan extension under AL and the unresponsiveness to lifespan-prolongation by DR. OPT2 mutants exhibit normal uptake but are defective in vacuolar sequestration of polyamines. Opt2 may be involved in a process that leads to the proper sequestration of polyamines into the vacuoles.

Polyamines can also enter the cell via fluid endocytosis [Aouida et al., 2005 in (Aouida et al., 2009)].

DR -| OPT2 -> Vesicle fusion -> polyamine sequestration in vacuole -| Longevity DR = Glucose down -> OPT2 down -> Vesicle fusion down -> Many small vesicles -> [polyamine]vacuole down -> [polyamine]cytosol down -> Secretophagy -> Longevity up

Fre6 is responsible for the reduction of iron and copper inside the vacuolar, which is required for their efflux (Singh et al., 2007). FRE6 has negative genetic interactions with both VPS30 (vacuolar protein sorting) and TCO89 (TORC1). FRE6 deletion could impair the metal ion efflux and subsequently decrease cytosolic iron and cooper level, which mimic the drop normally observed under DR (Sharma et al., 2010). As response FET3 expression is induced and its activity reduces the plasma membrane coenzyme Q pools. This allows Pga3 to rise the cytosolic NAD/NADH ratio which is associated with increased longevity (Jimenez-Hidalgo et al., 2009).

fre6Delta -> vacuolar Fe3+ & Cu2+ up -> cytosolic Fe3+ & Cu2+ down -> FET3 up -> plasma membrane CoQ/CoQH2 up -> Pga3 up -> cytosolic NAD/NADH up -> Longevity

RCR2 (Resistance to Congo Red) is localized to vacuolar structures and endosome-like vesicles. RCR2 together with vacuolar proteins Ssh4 and Rcr1 function in the endosomal-vacuolar trafficking pathway, affect events that determine whether plasma membrane proteins are degraded or routed to the plasma membrane. Rcr2 is similar to Rcr1 and is primarily localized to structures associated with the vacuole and also to endosome-like vesicles. Downregulation of amino acid permeases and other transporters in the plasma membrane involves their endocytotic removal and degradation in the vacuole, which requires Rsp5-dependent ubiquitination, an event often modulated by phosphorylation. Overexpression of SSH4, RCR2, or RCR1 increases General Amino acid Permease Gap1 and tryptophane permease Tat2 levels (Kota et al., 2007). Rcr2 interaction partners are enriched for DNA repair (e.g. TOF1), response to heat and carbohydrate metabolism. Notably, Rcr2 interacts physically with Ypt7 (Ito et al., 2001) and has a genetic interaction (synthetic growth defect) with SIR2 (Liu et al., 2010).

Ultradian Rhythms at the Center of Lifespan Prolongation

What is the role of endocytosis related processes in the longevity effect by DR? We found that DR upregulates GTS1 as well as its target genes (including DR-essential genes such as HSP12, YDL180W and ATG11). In line with this discovery, another study found that GTS1 and its target genes were downregulated by switching from DR to AL, upon glucose excess (van den Brink et al., 2008).

GTS1 was initially identified as having partially sequence homology (Gly-Thr repeats) to period in Drosophila (Mitsui et al., 1994), but as the repeat was translated as Ala-Gln repeats, it is more similar to the Gln-rich domain in rhythmicity-related protein Clock [21 in(Liu et al., 2002) as well as Mistui et al., 19994; Yaguchi et al., 1997 in (Yaguchi et al., 2007) and King et la., 1997; Saleem et al., 2001 in (Xu and Tsurugi, 2007)].

Gts1 impacts on glycolytic (2-4 min), respiratory (40 min) and energy-metabolism (4h) oscillation.

GTS1 mRNA and protein level are rhythmic and it facilitates the self-organization of energy metabolism oscillation. GTS1 encodes a clock protein that contains a zinc finger and an Arf-GAP domain in the N-terminus, an UBA domain in the center and a long- Gln-rich region forming a coiled-coil in the C-terminus (Tab. X+3). Gts1 regulates ultradian rhythm, cell size, cell cycle, lifespan, heat tolerance (heat shock resistance) and multi-drug transport as well as timing of budding and sporulation in a gene-dose dependent manner [Mitsui et al., 1994; Yaguchi et al., 1996 in (Yaguchi et al., 2007)]. All being clock-regulated in other organisms [reviews: Hall, 1990 Dunlap 1993 in (Yaguchi et al., 2007)]. It functions both as GTPase Activating Protein (GAP) and transcription factor that localizes both to endocytic patches and the nucleus.

GTS1 encodes an Arf GTPase Activating Protein (GAP) and transcription factor that localizes both to endocytic patches and the nucleus, and affects cell size, lifespan and the capacity of heat tolerance as well as timing of budding in a gene-dose dependent manner.

In cortical actin patches Gts1 function in fluid-phase endocytosis and membrane trafficking related to the formation of the central vacuole (Yaguchi et al., 2007). Gts1 regulates the glucose-repressible ADP-ribosylation factor 3 (Arf3) to modulate plasma membrane PtdIns(4,5)P2 levels to facilitate endocytosis (Smaczynska-de et al., 2008). Multiple pathways including that of Arf3 and Gts1 regulate endocytic coat disassembly (Toret et al., 2008). Gts1 may serve to link endocytosis to the metabolic oscillator and growth machinery (Smaczynska-de et al., 2008).

As endocytosis is a major process by which cells take up nutrients and it involves vesicle trafficking and the vacuole, it is not surprising that major nutrient sensors like TOR are localized on endocytic vesicles and vacuole/lysosome. Extracellular levels of glucose and inorganic phosphate were higher in gts1Delta and their intracellular levels were lower, indicating that their uptake were restricted in gts1Delta [Xu and Tsurugi, 2007 in (Yaguchi et al., 2007)]. Attenuation of energy-metabolism oscillation in gts1Delta is caused in part by a decrease in the endocytic uptake activity of gts1Delta.

Both inactivation or overexpression of GTS1 shortened lifespan (Yaguchi et al., 1996) and were accompanied with increased level of copper in both mutants. In either GTS1 deletion or overexpression mutant, the imbalanced homeostasis of copper induced an accumulation of ROS which caused inactivation of SODs further increasing ROS levels (Abudugupur et al., 2003). As DR essential protein Fre6 regulates vacuolar copper efflux there might a fascinating connection. Also, GTS1 deletion reduces NAD amplitude in energy metabolism oscillations (Xu and Tsurugi, 2007): fre6 -> vacuolar Fe3+ & Cu2+ up -> cytosolic Iron/copper down -> FET3 up -> plasma membrane CoQ/CoQH2 up -> Pga3 up -> cytosolic NAD/NADH up -> Higher oscillatory amplitudes -> Longevity

However, we did not found evidence for upregulation of neither FET3 nor PGA3 on the mRNA upon DR (although they might be upregulated on protein and/or activity level). Therefore, an alternative possibility is that fre6-mediated reduced cytosolic copper level increase the oscillatory homeostasis of ROS levels (Abudugupur et al., 2003): fre6 -> vacuolar Fe3+ & Cu2+ up -> cytosolic Iron/copper down -> Oscillatory homeostasis up -> ROS down

GTS1 regulates transcription of DR-essential gene HSP12. Deletion of GTS1 or HSP12 impact on heat resistance. Further, HSP12 deletion decreased resistance to Congo Red. Resistance to Congo Red 2 (RCR2) is DR-essential.

Gts1 is linked to the DR signalling web. PKA regulates Gts1 via direct or indirect phosphorylation (Yaguchi et al., 2000). TOR and PKA may regulate Gts1 via phosphorylation of 14-3-3 protein Bmh1 (Kakiuchi et al., 2007) indirectly (Wang et al., 2009a). Bmh1 (-1.61-fold) might similar to Ssa1 (-4.76-fold) and Ssa2 (-1.1-fold) negatively regulate Gts1 subcellular localization (Sanada et al., 2011b), as Ssa1 and Ssa2 double deletion increased heat tolerance and Gts1 nuclear import (Sanada et al., 2011b) and Bmh1 deletion decreased ROS levels and heat-shock element-driven transcription activity (Wang et al., 2009a). The phosphorylated Bmh1 increase with ageing and this may negatively impact on homeostasis (Fig. 10).

Further, Gts1 binds Snf1 kinase (AMPK homolog) subunits (Yaguchi and Tsurugi, 2003) as well as to some ABC transporters (Kawabata et al., 1999) and modulates their activity. As ABC transporters are implicated in ageing (Jones, 2008) and mediate drug detoxification (which is tightly associated to vacuole) there might be a connection to Opt2. Gts1 controls the oscillation of glutathione levels and therefore to oscillation in stress resistance.

What is the role of polyamines in ageing and its modulation by DR? DR-essential ATG15 (encoding vacuolar lipase) mRNA was found to be upregulated upon DR as well as by spermidine supplementation, which increased longevity in yeast, nematode, flies and mammalian cell culture (Eisenberg et al., 2009). Thus, spermidine might work like DR in inducing secretophagy, which is also enhances other forms of autophagy. Note Gts1 regulates autophagy related genes which exhibit upregulation on the transcript level upon DR (e.g. ATG11, ATG19, ATG34) and is upstream of ATG1.

Interestingly, overexpression of vacuolar aspartyl protease (PEP4) extended lifespan by increasing cytosolic polyamine and S-adenosylmethionine (SAM) levels (Carmona-Gutierrez et al., 2011). Although PEP4 is not DR-essential (Tang et al., 2008) it might be relevant for lifespan extension as S-adenosylmethionine synthetases in yeast and worm are downregulated upon DR on transcript level. SAM is a crucial metabolite in cysteine and methionine metabolism which we found may be a potential nexus of the ultradian and DR transcriptional signature. It needs to be stressed that restriction of methionine is sufficient to extend lifespan in yeast, fly, mice and rats.

Gts1 oscillatory protein levels are regulated by ubiquitination (Saito et al., 2002). Gts1 has a UBA domains of Class 4, which mediates ubiquitin-binding (Raasi et al., 2005). A conserved ubiquitination pathway is DR-essential (Carrano et al., 2009). Numerous autophagy-related genes harbour ubiquitin binding domains. p62, and NBR1 also contain UBA domain. Ubiqination mediates selective forms of autophagy (Johansen and Lamark, 2011). RSP5 the ortholog of DR-essential wwp-1 (Carrano et al., 2009) is implicated in multi-vacuole body sorting, heat shock response, transcription and endocytosis.

Gts1 might be in the intersection of endocytosis and lifespan regulation by DR [Figure: Gts1 is at the centre of the DR-signalling and endocytosis intersection].

Is there any evidence that DR alters the ultradian rhythmicity? Rapamycin has profound effect on the ultradian cycle and appears to lengthening the reductive phase (Murray et al., 2007). Gts1 mediates the mechanism that communicates external conditions, such as temperature or nutrients to the central oscillating loop (Adams et al., 2003).

By lengthening the reductive phase and shortening the oxidative phase of the metabolic cycle, many processes associated to lifespan extension (e.g. autophagy, heat shock proteins, mitochondrial biogenesis, and histone production) will be enhanced, whereas processes which are considered to be detrimental (e.g. ribosomal biogenesis and translation) will be suppressed [Figure: The ultradian cycle as a potential nexus of ageing and longevity]. Finally, we propose that alteration in the ultradian oscillations is the driving force of ageing by decreasing homeostasis which results in all its pleiotropic outcomes.

Are ultradian rhythms also altered in long-lived mutants and by pharmacological lifespan extending interventions besides rapamycin, (i.e. spermidine, resveratrol, etc.)? How does ageing impact on ultradian rhythmicity. What causes alteration in ultradian oscillation during ageing? What is the role of chromatin in this process? Insights might stem by studying the intersections of yeast transcriptional signatures. Also shortest-pathway analysis might identify connection between DR-essential genes.

There must be factors which are responsible for the observed differentially expression upon DR. We tried to identify the most crucial ones by linking the functional terms with DR-essential interactors.

Does GTS1 deletion/overexpression cancel out DR or Ndt80-induced lifespan extension? Can Ndt80 induction rejuvenate chronological aged cells? Can repeated or constant Ndt80-induction immortalize cells? What are the functional homologs of Ndt80?

Both endocytosis and autophagy transport cargoes (most of them are proteins) into vacuoles for degradation, which allows the recycling of amino acids and other building blocks for cell's essential metabolism. DR limits nutrients from the environment. The decrease of nutrient in the environment will make endocytosis a futile cycle; cells just recycle the membrane transporters. So DR should down-regulate endocytosis and up-regulate autophagy. Although endocytosis and autophagy do not have to be exculsive, they both are controlled by TORC1. Active TORC1 stimulates endocytosis [MacGurn et al. 2011. Cell 147:1104] and represses autophagy. This, the downregulation of TORC1 under DR is expected to down-regulate endocytosis and up-regulate authophagy.

Amino acid starvation up-regulates both endocytosis and MVB (multi-vesicular body) pathways [Jones et al., 2012 Traffic 13:468 endocytosis starvation]. The later steps of endocytosis use the MVB pathway. Most ESCRT sub-units are up-regulated by DR (Vps27, Hse1, etc.) [Bast 2011 Current Opinion in Cell Biol 23:452].

Methods

A twofold change cut-off was chosen for the data. p-values for the chance of finding an overlap of a two sets of genes were calculated with the hypergeometric test.

Annotations

Genes association to process were identified by combining annotation of different sources: SGD, GO and KEGG.

Gene Expression Profile

By an 2-fold cut-off, out of 5716 probed genes 2587 (45.26%) were differentially expressed, with 1413 genes (24.72%) upregulated and 1174 genes (20.54%) downregulated.

Transcription factor Identification

Transcription factor - target gene interactions were retrieved from YEASTRACT (http://www.yeastract.com/). Transcription factors interacting with DR-essential genes were identified and ordered by their binominal p-value of the ratio of regulated genes with more than 2-fold differentially expression upon DR, relative to the total number of genes regulated by the same transcription factor.

Functional Enrichment

Tables of enriched KEGG pathways were retrieved from DAVID.

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Figures

Figure: GTS1 loci from SGD

Figure: GTS1 loci from UCSC with transcription factor binding sites

Figure: Gts1 is at the centre of the DR-signalling and endocytosis intersection

Figure: The ultradian cycle as a potential nexus of ageing and longevity

Tables

Table: KEGG pathways enriched in DR-differentially expressed genes", data='''

Term Genes p-Value Benjamin Effectors Ribosome 83 3.73E-6 3.24E-4 RPL31A Pentose phosphate pathway 24 2.3E-4 0.02
Glycine, serine and threonine metabolism 17 0.033 0.947
Nitrogen metabolism 11 0.036 0.959 GLN1 Cysteine and methionine metabolism 20 0.048 0.986 SAM1 SAM2 Purine metabolism 52 0.054 0.992 ADE4 DNA replication 20 0.072 0.999
Arginine and proline metabolism 19 0.099 1.0

Table: KEGG pathways enriched in DR-induced und suppressed genes", data='''

Term Genes p-Value Benjamin Effectors Regulators Spliceosome 24 6.95E-5 0.005
Methane metabolism 5 0.018 0.723 SAM1 SAM2
Glyoxylate and dicarboxylate metabolism 7 0.028 0.865
Endocytosis 11 0.038 0.937 YPT7 GTS1
Citrate cycle (TCA cycle) 11 0.038 0.937 PCK1 Hap4 Pyruvate metabolism 11 0.038 0.937 LAT1
Pentose phosphate pathway 9 0.083 0.998
Ribosome 81 5.05E-17 4.19E-15
DNA replication 20 2.1E-4 0.017
Purine metabolism 42 0.001 0.104
Aminoacyl-tRNA biosynthesis 22 0.002 0.158 GLN1 Gat1 Gln3 Gcn4 Glycine, serine and threonine metabolism 15 0.003 0.195 SAM1 SAM2
Mismatch repair 12 0.013 0.653
Cysteine and methionine metabolism 16 0.014 0.7
Nitrogen metabolism 9 0.021 0.831
Pentose phosphate pathway 15 0.025 0.881 ADE4 Gcn4 O-Mannosyl glycan biosynthesis 8 0.071 0.998
Selenoamino acid metabolism 10 0.096 1.0 RPL31A Sfp1

Table: KEGG pathways for highly ultradian differentially expressed genes", data='''

Term Genes p-Value Benjamin Sulfur metabolism 6 1.22E-6 3.78E-5 Selenoamino acid metabolism 5 2.31E-4 0.007 Tryptophan metabolism 4 0.002 0.067 Cysteine and methionine metabolism 4 0.013 0.324 Propanoate metabolism 3 0.014 0.364 Fatty acid metabolism 3 0.034 0.653 Glycolysis / Gluconeogenesis 4 0.045 0.763 Glycine, serine and threonine metabolism 3 0.059 0.847 MAPK signaling pathway 4 0.067 0.884 Arachidonic acid metabolism 2 0.084 0.934

Table Differentially expression of highly ultradian genes upon DR", data='''

mRNA Change ADH2 +43.4 CTA1 +28.83 SIP4 +25.71 YGR067C +25.68 ACS1 +24.02 CAT8 +23.76 MET28 +10.97 ARG82 +6.12 MET2 +5.36 ARG80 +5.36 UGA3 +5.07 CTT1 +4.53 CAD1 +4.33 PRX1 +4.32 GPX1 +4.32 SUT1 +4.03 XBP1 +3.89 INO4 +3.83 ROX1 +3.72 YAP1 +3.71 MET4 +3.66 GRX2 +3.57 YRR1 +3.15 HSF1 +2.94 MSN2 +2.89 MET30 +2.88 GLN3 +2.76 SUL1 +2.75 GRX1 +2.62 MSN1 +2.16 MET3 +1.98 MET31 +1.93 GAT1 +1.81 ALD6 +1.71 SUL2 +1.7 UME6 +1.62 ARG81 +1.5 MET10 +1.48 MET14 +1.45 ACA1 -1.63 MET16 -1.65 WTM1 -1.67 GCN4 -1.83 GLY1 -2.46 YHP1 -2.61 RET1 -2.84 CYS4 -3.38 GPX2 -3.68 MGA1 -3.84 STP4 -4.25 CYS3 -4.48 RPI1 -4.94 MET17 -5.11 ALD5 -5.87

Table: Endocytosis-associated genes differentially expressed upon DR", data='''

mRNA Change YAP1802 +4.58 YPT53 +4.05 PKH1 +3.84 DOA4 +3.60 SNC1 +3.32 BRE4 +3.26 AKR2 +2.84 VPS33 +2.56 SNC2 +2.33 OSH7 +2.32 YCK1 +2.30 HES1 +2.25 BSP1 +2.20 LSB5 +2.12 GTS1 +2.01 YAP1801 +1.96 MYO3 +1.90 WHI2 +1.84 INP51 +1.83 OSH3 +1.76 ENT2 +1.74 ARF3 +1.73 NEO1 +1.68 LAS17 +1.64 YPT7 +1.54 SCD5 -1.50 GVP36 -1.76 YPK1 -1.77 MON2 -1.85 INP53 -1.89 RVS161 -2.22 CDC50 -2.35 MYO5 -2.43 RCY1 -2.47 KES1 -3.53 DNF1 -3.86 DNF2 -4.26 SVL3 -4.68 COS10 -4.95 THR4 -6.07 DRS2 -8.43 LDB17 -8.83

Table: Transcription factors regulating DR-induced genes.", data='''

Factor Total Specific Ratio Factor fold-change Ume1 4 3 0.75 -1.3 Gis1 191 97 0.51 +2.18 Hot1 70 32 0.46 +5.63 Ime1 17 7 0.41 +1.43 Mig1 239 93 0.39 +4.23 Xbp1 179 67 0.37 +3.89 Ume6 238 89 0.37 +1.62 Cst6 193 68 0.35 +2.76 Hsf1 571 196 0.34 +2.94 Sko1 335 113 0.34 -1.8 Yrr1 92 31 0.34 +3.15 Adr1 443 146 0.33 +5.27 Sok2 1034 318 0.31 -1.13 Hac1 213 65 0.31 +1.23 Mth1 69 21 0.3 +2.85 Nrg1 399 118 0.3 +2.11 Cdc14 75 22 0.29 +3.18 Cbf1 331 97 0.29 -1.03 Gzf3 147 43 0.29 +1.96 Ste12 1364 397 0.29 -2.07 Pho4 372 107 0.29 +1.96 Rdr1 7 2 0.29 -1.16 Hap1 189 54 0.29 +1.33 Haa1 14 4 0.29 +1.36 Hap4 426 121 0.28 -3.83 Msn4 503 142 0.28 -1.31 Ecm22 270 76 0.28 +1.87 Hap3 185 51 0.28 +1.33 Msn2 908 249 0.27 +2.89 Hap2 196 53 0.27 +2.41 Rgt1 63 17 0.27 +1.14 Dal82 167 45 0.27 +1.37 Skn7 419 109 0.26 +1.37 Stp2 341 88 0.26 +4.74 Gts1 62 16 0.26 +2.01 Tec1 537 138 0.26 -1.15 Hap5 195 50 0.26 +1.56 Stp1 232 59 0.25 +2.92 Gln3 177 45 0.25 +2.76 Rtg3 218 55 0.25 +1.17 Ino4 635 160 0.25 +3.83 Spt2 20 5 0.25 +1.09 Pdr1 653 163 0.25 +2.08 Mot3 133 33 0.25 +7.29 Rfx1 198 49 0.25 +1.51 Rtg1 126 31 0.25 -1.53 Gcr2 187 46 0.25 +3.6 Upc2 208 51 0.25 -5.15 Fhl1 863 210 0.24 +1.0 Kar4 29 7 0.24 -3.45 Hcm1 249 60 0.24 -1.88

Table: Transcription factors regulating DR-suppressed genes", data='''

Factor Total Specific Ratio Factor fold-change Rpn10 1 1 1.0 -1.02 Hpc2 1 1 1.0 +1.12 Elp6 1 1 1.0 -1.39 Dig2 1 1 1.0 +1.89 Rdr1 7 3 0.43 -1.16 Rtg2 5 2 0.4 -1.35 Mga2 27 10 0.37 +3.11 Ifh1 308 114 0.37 +2.44 Swi6 177 60 0.34 -3.42 Mot3 133 45 0.34 +7.29 Pdr3 546 176 0.32 +1.81 Bas1 137 44 0.32 +1.05 Mss11 67 21 0.31 -1.03 Rgt1 63 19 0.3 +1.14 Stb1 41 12 0.29 +1.19 Sfp1 2183 637 0.29 +4.48 Gcn4 574 155 0.27 -1.83 Opi1 26 7 0.27 +2.97 Swi4 583 156 0.27 -1.64 Stb5 339 88 0.26 +1.29 Yap1 1824 473 0.26 +3.71 Ppr1 27 7 0.26 -1.08 Ndt80 35 9 0.26 +2.65 Ume1 4 1 0.25 -1.3 Msn1 44 11 0.25 +2.16 Rpn4 1023 254 0.25 +3.81 Ash1 114 28 0.25 -2.11 Upc2 208 50 0.24 -5.15 Met31 121 29 0.24 +1.93 Ecm22 270 64 0.24 +1.87 Fhl1 863 203 0.24 +1.0 Phd1 520 121 0.23 +1.08 Pho4 372 86 0.23 +1.96 Rim101 208 48 0.23 +1.77 Hir1 65 15 0.23 -1.4 Dal81 249 57 0.23 -1.09 Ino2 163 37 0.23 -1.3 Spt23 53 12 0.23 +3.49 Gln3 177 40 0.23 +2.76 Tec1 537 121 0.23 -1.15 Rap1 1226 276 0.23 +1.12 Pdr1 653 147 0.23 +2.08 Ixr1 120 27 0.23 +2.37 Hac1 213 47 0.22 +1.23 Ino4 635 137 0.22 +3.83 Haa1 14 3 0.21 +1.36 Gis2 19 4 0.21 -2.2 Rfx1 198 41 0.21 +1.51 Msn2 908 188 0.21 +2.89 Cup9 93 19 0.2 +2.31 Gcr2 187 38 0.2 +3.6

Table: Differentially expression of transcription factors regulating GTS1.", data='''

mRNA Change Gene Name Implication PUT3 +5.68 Proline UTilization Proline utilization REB1 +2.94 RNA polymerase I Enhancer Binding protein Enhances RNA pol I & II MSN2 +2.89 Multicopy suppressor of SNF1 mutation Stress response AFT1 +2.35 Activator of Ferrous Transport Metal ion utilization DAL82 +1.37 Degradation of Allantoin Positive regulator of allophanate inducible genes CIN5 +1.33 Chromosome INstability Pleiotropic drug resistance AFT2 +1.23 Activator of Fe (iron) Transcription Metal ion utilization RAP1 +1.12 Repressor Activator Protein Telomere maintenance, silencing and high level transcription

Table: Transcription factor regulating ATG1", data='''

mRNA Change Description STP2 +4.74 Transcription factor, activated by proteolytic processing in response to signals from the SPS sensor system for external amino acids; activates transcription of amino acid permease genes MIG1 +4.23 Transcription factor involved in glucose repression; sequence specific DNA binding protein containing two Cys2His2 zinc finger motifs; regulated by the SNF1 kinase and the GLC7 phosphatase RPN4 +3.81 Transcription factor that stimulates expression of proteasome genes; Rpn4p levels are in turn regulated by the 26S proteasome in a negative feedback control mechanism; RPN4 is transcriptionally regulated by various stress responses YAP1 +3.71 Basic leucine zipper (bZIP) transcription factor required for oxidative stress tolerance; activated by H2O2 through the multistep formation of disulfide bonds and transit from the cytoplasm to the nucleus; mediates resistance to cadmium MET4 +3.66 Leucine-zipper transcriptional activator, responsible for the regulation of the sulfur amino acid pathway, requires different combinations of the auxiliary factors Cbf1p, Met28p, Met31p and Met32p HSF1 +2.94 Trimeric heat shock transcription factor, activates multiple genes in response to stresses that include hyperthermia; recognizes variable heat shock elements (HSEs) consisting of inverted NGAAN repeats; posttranslationally regulated ARG81 +1.5 Zinc-finger transcription factor of the Zn(2)-Cys(6) binuclear cluster domain type, involved in the regulation of arginine-responsive genes; acts with Arg80p and Arg82p RAP1 +1.12 DNA-binding protein involved in either activation or repression of transcription, depending on binding site context; also binds telomere sequences and plays a role in telomeric position effect (silencing) and telomere structure DAL81 -1.09 Positive regulator of genes in multiple nitrogen degradation pathways; contains DNA binding domain but does not appear to bind the dodecanucleotide sequence present in the promoter region of many genes involved in allantoin catabolism GCN4 -1.83 Basic leucine zipper (bZIP) transcriptional activator of amino acid biosynthetic genes in response to amino acid starvation; expression is tightly regulated at both the transcriptional and translational levels

Table: DR vs. rapamycin statistics", data='''

a, Genes differentially expressed upon DR and exhibting rapamycin resistance/sensitivity as overexpression mutants. Rapamycin enriched or depleted overexpression mutants DR up- or downregulated genes #Genes Up Up 35 Down Down 27 Up Down 37 Down Up 37

b, Genes differentially expressed upon DR and rapamycine or caffeine treatment by an 2-fold cut-off.
Strain Regime Incubation time Delta Up (common opposite) Down (common opposite) BY4741 DR 2587 1413 1174 W303a 1 ng/mL 30 min 95(38) 52(11 8) 43(10 9) W303a 5 ng/mL 30 min 230(111) 104(23 18) 126(56 15) W303a 200 ng/mL 30 min 1219(586) 537(154 76) 682(297 60) S288c 200 ng/mL 30 min 1580(733) 881(290 83) 699(300 61) Sigma 2000 200 ng/mL 30 min 1498(666) 714(203 81) 784(297 86) BY4741 200 ng/mL 60 min 1415(662) 672(223 64) 743(288 87) BY4741 ? ng/mL 30 min 4(3) 3(2 0) 1(1 0) W303a 0.3 mM 30 min 7(4) 4(1 1) 3(0 2) W303a 1 mM 30 min 28(16) 22(5 6) 6(2 3) W303a 3 mM 30 min 456(215) 269(63 58) 187(73 22) W303a 6 mM 30 min 1430(618) 405(100 65) 1025(343 111) W303a 9 mM 30 min 1526(680) 752(212 91) 774(299 80) S288c 9 mM 30 min 1589(717) 900(290 78) 689(293 57) Sigma 2000 9 mM 30 min 1595(702) 763(212 70) 832(316 104)

c, Genes differentially expressed upon DR and rapamycine or caffeine treatment without a cut-off.
BY4741 DR 5713 3116 2597 W303a 1 ng/mL 30 min 5133(4470) 2543(1109 1144) 2590(1044 1182) W303a 5 ng/mL 30 min 4852(4285) 2365(1139 984) 2487(1152 1017) W303a 200 ng/mL 30 min 5611(4915) 2840(1521 1006) 2771(1323 1075) S288c 200 ng/mL 30 min 5410(4793) 3088(1665 1081) 2322(1205 852) Sigma 2000 200 ng/mL 30 min 5033(4488) 2596(1398 957) 2437(1211 930) BY4741 200 ng/mL 60 min 5215(4706) 2773(1557 961) 2442(1235 962) BY4741 ? ng/mL 30 min 5716(5713) 3605(2130 1491) 2111(1120 999) W303a 0.3 mM 30 min 5270(4643) 2597(1078 1286) 2673(956 1332) W303a 1 mM 30 min 5359(4716) 2593(1169 1137) 2766(1126 1292) W303a 3 mM 30 min 4852(4294) 2301(1149 884) 2551(1226 1044) W303a 6 mM 30 min 5282(4669) 1894(972 678) 3388(1564 1465) W303a 9 mM 30 min 5334(4688) 2636(1354 934) 2698(1300 1110) S288c 9 mM 30 min 5545(4890) 3031(1676 994) 2514(1324 908) Sigma 2000 9 mM 30 min 5229(4658) 2629(1421 934) 2600(1319 994)

d, Genes differentially expressed upon DR and rapamycine or caffeine treatment by an 1.5-fold cut-off.
BY4741 DR 3735 2061 1674 W303a 1 ng/mL 30 min 774(415) 341(94 85) 433(109 127) W303a 5 ng/mL 30 min 707(435) 307(105 68) 400(191 73) W303a 200 ng/mL 30 min 2380(1411) 1191(455 236) 1189(518 203) S288c 200 ng/mL 30 min 2736(1637) 1616(661 277) 1120(522 178) Sigma 2000 200 ng/mL 30 min 2654(1568) 1382(538 261) 1272(524 248) BY4741 200 ng/mL 60 min 2498(1500) 1304(543 218) 1194(507 233) BY4741 ? ng/mL 30 min 35(30) 34(25 4) 1(1 0) W303a 0.3 mM 30 min 55(29) 20(4 7) 35(4 14) W303a 1 mM 30 min 202(120) 116(27 41) 86(21 31) W303a 3 mM 30 min 1263(752) 650(225 159) 613(252 117) W303a 6 mM 30 min 2665(1556) 808(284 183) 1857(646 444) W303a 9 mM 30 min 2736(1592) 1376(505 263) 1360(539 287) S288c 9 mM 30 min 2816(1681) 1595(674 240) 1221(559 209) Sigma 2000 9 mM 30 min 2795(1672) 1409(547 248) 1386(589 289)

Table: DR vs. rapamycin terms", data='''

a, DR differential and rapamycin differential.

Cluster Score Term Count % p-Value Benjamini 1 25.55 ribonucleoprotein complex 164 28.37 4.17E-46 1.22E-43 1 25.55 cytosolic ribosome 80 13.84 1.22E-45 3.56E-43 1 25.55 cytosolic part 80 13.84 1.18E-34 3.44E-32 1 25.55 protein biosynthesis 93 16.09 1.58E-34 4.91E-32 1 25.55 ribonucleoprotein 96 16.61 1.79E-33 5.56E-31 1 25.55 ribosome 70 12.11 1.26E-32 3.91E-30 1 25.55 ribosome 99 17.13 1.24E-30 3.61E-28 1 25.55 sce03010:Ribosome 71 12.28 5.42E-30 3.36E-28 1 25.55 ribosomal subunit 76 13.15 1.75E-28 5.11E-26 1 25.55 ribosomal protein 71 12.28 2.30E-26 7.14E-24 1 25.55 cytosolic large ribosomal subunit 43 7.44 4.60E-24 1.34E-21 1 25.55 cytosol 103 17.82 2.82E-22 8.23E-20 1 25.55 structural constituent of ribosome 72 12.46 4.67E-22 2.73E-19 1 25.55 cytosolic small ribosomal subunit 33 5.71 1.23E-20 3.59E-18 1 25.55 large ribosomal subunit 43 7.44 2.59E-15 7.46E-13 1 25.55 translation 134 23.18 2.90E-15 3.64E-12 1 25.55 cytosol 30 5.19 5.59E-13 1.74E-10 1 25.55 small ribosomal subunit 33 5.71 9.18E-13 2.68E-10 1 25.55 structural molecule activity 78 13.49 2.06E-11 1.20E-08 2 24.95 ribonucleoprotein complex 164 28.37 4.17E-46 1.22E-43 2 24.95 ribosome biogenesis 127 21.97 1.25E-41 1.58E-38 2 24.95 preribosome 69 11.94 3.14E-38 9.16E-36 2 24.95 nucleolus 99 17.13 2.30E-37 6.71E-35 2 24.95 ribonucleoprotein complex biogenesis 130 22.49 2.61E-37 3.29E-34 2 24.95 rRNA processing 93 16.09 1.04E-32 1.31E-29 2 24.95 non-membrane-bounded organelle 211 36.51 1.79E-32 5.21E-30 2 24.95 intracellular non-membrane-bounded organelle 211 36.51 1.79E-32 5.21E-30 2 24.95 rRNA metabolic process 93 16.09 3.18E-31 4.01E-28 2 24.95 ribosome biogenesis 66 11.42 1.56E-30 4.84E-28 2 24.95 ncRNA processing 104 17.99 3.77E-27 4.75E-24 2 24.95 ncRNA metabolic process 112 19.38 8.66E-26 1.09E-22 2 24.95 rrna processing 60 10.38 7.67E-24 2.38E-21 2 24.95 nuclear lumen 110 19.03 4.73E-17 1.38E-14 2 24.95 RNA processing 113 19.55 5.37E-16 7.01E-13 2 24.95 intracellular organelle lumen 124 21.45 8.00E-11 2.34E-08 2 24.95 organelle lumen 124 21.45 8.00E-11 2.34E-08 2 24.95 RNA binding 98 16.96 1.05E-09 6.12E-07 2 24.95 membrane-enclosed lumen 125 21.63 1.61E-09 4.71E-07 3 21.68 regulation of translation 69 11.94 1.37E-23 1.73E-20 3 21.68 posttranscriptional regulation of gene expression 70 12.11 1.82E-22 2.30E-19 3 21.68 regulation of cellular protein metabolic process 70 12.11 3.66E-21 4.61E-18 4 12.07 preribosome 69 11.94 3.14E-38 9.16E-36 4 12.07 ribosome biogenesis 66 11.42 1.56E-30 4.84E-28 4 12.07 90S preribosome 43 7.44 9.00E-24 2.63E-21 4 12.07 maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 39 6.75 7.01E-17 1.40E-13 4 12.07 maturation of SSU-rRNA 39 6.75 1.88E-16 2.80E-13 4 12.07 small-subunit processome 25 4.33 4.74E-14 1.38E-11 4 12.07 maturation of 5.8S rRNA 29 5.02 3.86E-11 4.87E-08 4 12.07 maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 29 5.02 3.86E-11 4.87E-08 4 12.07 cleavages during rRNA processing 23 3.98 3.24E-08 4.08E-05 4 12.07 endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 18 3.11 5.16E-08 6.51E-05 4 12.07 endonucleolytic cleavage of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 18 3.11 1.30E-07 1.64E-04 4 12.07 endonucleolytic cleavages during rRNA processing 18 3.11 1.30E-07 1.64E-04 5 8.56 ribosome assembly 39 6.75 1.86E-20 2.34E-17 5 8.56 ribosomal subunit assembly 30 5.19 1.95E-15 2.52E-12 5 8.56 ribosomal large subunit biogenesis 33 5.71 4.19E-14 5.28E-11 5 8.56 ribonucleoprotein complex assembly 42 7.27 3.35E-13 4.23E-10 5 8.56 ribosomal large subunit assembly 22 3.81 7.70E-12 9.72E-09 6 6.76 amine biosynthetic process 41 7.09 1.41E-09 1.78E-06 6 6.76 cellular amino acid biosynthetic process 39 6.75 3.01E-09 3.80E-06 6 6.76 nitrogen compound biosynthetic process 67 11.59 8.85E-09 1.12E-05 6 6.76 amino-acid biosynthesis 28 4.84 4.81E-08 1.50E-05 6 6.76 carboxylic acid biosynthetic process 40 6.92 1.33E-06 0.002 6 6.76 organic acid biosynthetic process 40 6.92 1.33E-06 0.002 7 3.76 nucleoside monophosphate biosynthetic process 17 2.94 1.76E-08 2.23E-05 7 3.76 ribonucleoside monophosphate biosynthetic process 16 2.77 2.76E-08 3.48E-05 7 3.76 ribonucleoside monophosphate metabolic process 16 2.77 5.00E-08 6.31E-05 7 3.76 nucleoside monophosphate metabolic process 17 2.94 8.84E-08 1.12E-04 7 3.76 purine biosynthesis 12 2.08 1.02E-07 3.19E-05 7 3.76 purine nucleotide biosynthesis 9 1.56 5.01E-07 1.56E-04 7 3.76 purine nucleoside monophosphate biosynthetic process 12 2.08 3.26E-06 0.004 7 3.76 purine nucleoside monophosphate metabolic process 12 2.08 5.67E-06 0.007 7 3.76 purine ribonucleoside monophosphate biosynthetic process 11 1.9 1.61E-05 0.02 7 3.76 purine ribonucleoside monophosphate metabolic process 11 1.9 2.69E-05 0.033 7 3.76 sce00230:Purine metabolism 27 4.67 5.06E-04 0.031 8 3.36 Initiation factor 12 2.08 2.04E-05 0.006 9 3.01 glutamine family amino acid biosynthetic process 13 2.25 8.71E-06 0.011 10 2.7 nucleobase metabolic process 14 2.42 3.36E-05 0.041 11 2.42 pentosyltransferase 7 1.21 3.29E-05 0.01 14 2.19 short sequence motif:Q motif 11 1.9 2.84E-05 0.029 16 1.84 DNA-directed RNA polymerase I complex 9 1.56 3.26E-05 0.009 16 1.84 sce00230:Purine metabolism 27 4.67 5.06E-04 0.031 22 1.49 purine nucleotide biosynthesis 9 1.56 5.01E-07 1.56E-04

b, DR down and rapmycin down. Cluster Score Term Count % p-Value Benjamini 1 62.42 ribonucleoprotein complex 146 50.52 6.66E-75 1.35E-72 1 62.42 intracellular non-membrane-bounded organelle 175 60.55 2.45E-61 4.97E-59 1 62.42 non-membrane-bounded organelle 175 60.55 2.45E-61 4.97E-59 1 62.42 ribosome biogenesis 112 38.75 3.36E-61 2.75E-58 1 62.42 ribonucleoprotein complex biogenesis 114 39.45 6.04E-57 4.94E-54 2 40.94 ribonucleoprotein complex 146 50.52 6.66E-75 1.35E-72 2 40.94 cytosolic ribosome 80 27.68 1.07E-67 2.17E-65 2 40.94 protein biosynthesis 92 31.83 1.91E-61 3.84E-59 2 40.94 ribonucleoprotein 91 31.49 1.78E-56 3.57E-54 2 40.94 cytosolic part 79 27.34 1.51E-54 3.06E-52 2 40.94 ribosome 69 23.88 2.30E-52 4.61E-50 2 40.94 sce03010:Ribosome 71 24.57 6.61E-49 2.51E-47 2 40.94 ribosome 92 31.83 8.46E-49 1.72E-46 2 40.94 ribosomal subunit 76 26.3 6.45E-48 1.31E-45 2 40.94 ribosomal protein 71 24.57 5.33E-47 1.07E-44 2 40.94 structural constituent of ribosome 72 24.91 9.62E-40 3.07E-37 2 40.94 cytosolic large ribosomal subunit 43 14.88 6.49E-35 1.32E-32 2 40.94 cytosol 85 29.41 2.80E-31 5.69E-29 2 40.94 cytosolic small ribosomal subunit 33 11.42 7.14E-29 1.45E-26 2 40.94 large ribosomal subunit 43 14.88 2.00E-25 4.05E-23 2 40.94 translation 104 35.99 4.68E-25 3.83E-22 2 40.94 structural molecule activity 73 25.26 3.43E-24 1.09E-21 2 40.94 cytoplasm 137 47.4 2.64E-23 5.31E-21 2 40.94 small ribosomal subunit 33 11.42 1.97E-20 4.00E-18 2 40.94 cytosol 29 10.03 4.78E-20 9.62E-18 3 36.05 regulation of translation 66 22.84 3.12E-38 2.55E-35 3 36.05 posttranscriptional regulation of gene expression 66 22.84 3.13E-36 2.56E-33 3 36.05 regulation of cellular protein metabolic process 67 23.18 7.26E-36 5.94E-33 4 33.91 ribosome biogenesis 112 38.75 3.36E-61 2.75E-58 4 33.91 ribonucleoprotein complex biogenesis 114 39.45 6.04E-57 4.94E-54 4 33.91 preribosome 64 22.15 1.35E-49 2.75E-47 4 33.91 rRNA processing 80 27.68 1.29E-43 1.06E-40 4 33.91 nucleolus 81 28.03 1.84E-43 3.73E-41 4 33.91 ribosome biogenesis 60 20.76 1.67E-42 3.35E-40 4 33.91 rRNA metabolic process 80 27.68 3.00E-42 2.45E-39 4 33.91 ncRNA metabolic process 96 33.22 5.95E-40 4.87E-37 4 33.91 ncRNA processing 88 30.45 6.66E-39 5.45E-36 4 33.91 rrna processing 50 17.3 1.10E-29 2.21E-27 4 33.91 RNA processing 94 32.53 4.17E-28 3.41E-25 4 33.91 nuclear lumen 87 30.1 2.16E-24 4.38E-22 4 33.91 organelle lumen 90 31.14 3.28E-15 6.76E-13 4 33.91 intracellular organelle lumen 90 31.14 3.28E-15 6.76E-13 4 33.91 membrane-enclosed lumen 90 31.14 1.04E-13 2.12E-11 4 33.91 nucleus 119 41.18 1.70E-09 3.41E-07 5 13.88 preribosome 64 22.15 1.35E-49 2.75E-47 5 13.88 90S preribosome 41 14.19 7.61E-32 1.55E-29 5 13.88 maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 35 12.11 4.13E-22 3.38E-19 5 13.88 maturation of SSU-rRNA 35 12.11 1.05E-21 8.56E-19 5 13.88 small-subunit processome 22 7.61 3.81E-16 6.76E-14 5 13.88 maturation of 5.8S rRNA 24 8.3 5.69E-13 4.66E-10 5 13.88 maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 24 8.3 5.69E-13 4.66E-10 5 13.88 endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 16 5.54 4.54E-10 3.72E-07 5 13.88 endonucleolytic cleavage of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 16 5.54 1.07E-09 8.74E-07 5 13.88 endonucleolytic cleavages during rRNA processing 16 5.54 1.07E-09 8.74E-07 5 13.88 cleavages during rRNA processing 18 6.23 8.54E-09 6.98E-06 5 13.88 endonucleolytic cleavage in 5'-ETS of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 10 3.46 5.70E-06 0.005 5 13.88 endonucleolytic cleavage to generate mature 5'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 10 3.46 1.14E-05 0.009 5 13.88 rRNA 5'-end processing 10 3.46 1.58E-05 0.013 5 13.88 ncRNA 5'-end processing 10 3.46 1.58E-05 0.013 5 13.88 RNA 5'-end processing 10 3.46 2.15E-05 0.017 6 13.52 ribosome assembly 37 12.8 5.31E-28 4.34E-25 6 13.52 ribosomal subunit assembly 28 9.69 1.49E-20 1.21E-17 6 13.52 ribonucleoprotein complex assembly 39 13.49 2.04E-20 1.67E-17 6 13.52 ribosomal large subunit biogenesis 31 10.73 4.49E-20 3.67E-17 6 13.52 ribosomal large subunit assembly 20 6.92 9.27E-15 7.54E-12 6 13.52 cellular macromolecular complex assembly 44 15.22 6.72E-09 5.49E-06 6 13.52 cellular macromolecular complex subunit organization 47 16.26 6.41E-06 0.005 6 13.52 macromolecular complex assembly 44 15.22 1.13E-05 0.009 7 6.08 Initiation factor 12 4.15 1.90E-08 3.82E-06 7 6.08 translation initiation factor activity 13 4.5 5.19E-07 1.66E-04 7 6.08 translation factor activity, nucleic acid binding 15 5.19 4.57E-06 0.001 7 6.08 translational initiation 12 4.15 1.10E-05 0.009 8 3.39 ribonucleoside monophosphate biosynthetic process 13 4.5 1.41E-08 1.16E-05 8 3.39 ribonucleoside monophosphate metabolic process 13 4.5 2.24E-08 1.83E-05 8 3.39 nucleoside monophosphate biosynthetic process 13 4.5 5.29E-08 4.33E-05 8 3.39 nucleoside monophosphate metabolic process 13 4.5 1.68E-07 1.38E-04 8 3.39 sce00230:Purine metabolism 19 6.57 7.16E-04 0.027 9 3.33 nuclear transport 25 8.65 1.32E-06 0.001 9 3.33 nucleocytoplasmic transport 25 8.65 1.32E-06 0.001 9 3.33 nuclear export 22 7.61 3.05E-06 0.002 10 3.21 nuclear transport 25 8.65 1.32E-06 0.001 10 3.21 nucleocytoplasmic transport 25 8.65 1.32E-06 0.001 10 3.21 nuclear export 22 7.61 3.05E-06 0.002 11 3.02 short sequence motif:DEAD box 9 3.11 3.82E-06 0.002 11 3.02 short sequence motif:Q motif 9 3.11 7.80E-06 0.005 11 3.02 RNA helicase activity 12 4.15 2.16E-05 0.007 11 3.02 ATP-dependent RNA helicase activity 11 3.81 2.25E-05 0.007 11 3.02 RNA-dependent ATPase activity 11 3.81 2.25E-05 0.007 11 3.02 IPR014014:RNA helicase, DEAD-box type, Q motif 9 3.11 2.62E-05 0.013 11 3.02 P-loop 21 7.27 3.50E-05 0.007 11 3.02 IPR000629:RNA helicase, ATP-dependent, DEAD-box, conserved site 9 3.11 5.23E-05 0.025 11 3.02 nucleotide binding 18 6.23 1.43E-04 0.028 11 3.02 DNA replication, recombination, and repair / Transcription / Translation, ribosomal structure and biogenesis 5 1.73 0.002 0.038 12 2.77 RNA modification 21 7.27 5.96E-06 0.005 12 2.77 RNA methylation 9 3.11 1.85E-05 0.015 12 2.77 binding site:S-adenosyl-L-methionine 7 2.42 8.70E-05 0.05 17 1.66 IPR000836:Phosphoribosyltransferase 7 2.42 2.46E-05 0.012 18 1.55 nucleotide-binding 55 19.03 9.51E-05 0.019 19 1.55 DNA-directed RNA polymerase I complex 7 2.42 8.72E-05 0.018 19 1.55 sce00230:Purine metabolism 19 6.57 7.16E-04 0.027

c, DR down and rapamycin up Cluster Score Term Count % p-Value Benjamini 1 15.36 nitrogen compound biosynthetic process 31 40.79 6.97E-19 2.94E-16 1 15.36 amine biosynthetic process 23 30.26 2.76E-18 1.17E-15 1 15.36 cellular amino acid biosynthetic process 21 27.63 2.73E-16 9.37E-14 1 15.36 amino-acid biosynthesis 17 22.37 7.97E-15 1.06E-12 1 15.36 organic acid biosynthetic process 21 27.63 4.17E-14 1.76E-11 1 15.36 carboxylic acid biosynthetic process 21 27.63 4.17E-14 1.76E-11 2 4.38 heterocycle biosynthetic process 10 13.16 1.50E-06 6.31E-04 2 4.38 Histidine biosynthesis 5 6.58 2.04E-06 2.69E-04 2 4.38 histidine metabolic process 5 6.58 1.00E-05 0.004 2 4.38 histidine biosynthetic process 5 6.58 1.00E-05 0.004 2 4.38 histidine family amino acid biosynthetic process 5 6.58 1.00E-05 0.004 2 4.38 histidine family amino acid metabolic process 5 6.58 1.00E-05 0.004 2 4.38 Amino acid transport and metabolism 6 7.89 8.88E-04 0.008 3 2.91 glutamine family amino acid metabolic process 9 11.84 1.38E-06 5.84E-04 3 2.91 glutamine family amino acid biosynthetic process 6 7.89 3.26E-05 0.014 4 2.78 pyridoxal phosphate 6 7.89 1.95E-04 0.025 5 2.58 biogenic amine biosynthetic process 5 6.58 2.13E-05 0.009 5 2.58 cellular amino acid derivative biosynthetic process 6 7.89 2.23E-05 0.009 6 2.45 aspartate family amino acid biosynthetic process 7 9.21 5.99E-05 0.025 7 2.43 glycoprotein 19 25 4.18E-06 5.52E-04 7 2.43 glycosylation site:N-linked (GlcNAc...) 15 19.74 1.85E-04 0.035 8 2.32 lyase 8 10.53 2.09E-05 0.003 9 2.15 purine biosynthesis 6 7.89 1.91E-06 2.51E-04 9 2.15 purine nucleotide biosynthesis 4 5.26 2.33E-04 0.03

d, DR up and rapamycin down. Cluster Score Term Count % p-Value Benjamini 1 4.7 nucleolus 18 30 4.59E-11 5.78E-09 1 4.7 nuclear lumen 21 35 8.98E-09 1.13E-06 1 4.7 membrane-enclosed lumen 23 38.33 4.24E-07 5.34E-05 1 4.7 organelle lumen 22 36.67 8.29E-07 1.05E-04 1 4.7 intracellular organelle lumen 22 36.67 8.29E-07 1.05E-04 1 4.7 ncRNA processing 14 23.33 4.51E-06 0.002 1 4.7 rRNA processing 12 20 5.74E-06 0.002 1 4.7 rRNA metabolic process 12 20 8.21E-06 0.003 1 4.7 RNA processing 16 26.67 2.36E-05 0.009 1 4.7 ncRNA metabolic process 14 23.33 2.59E-05 0.01 1 4.7 ribosome biogenesis 13 21.67 4.16E-05 0.016 1 4.7 rrna processing 9 15 7.29E-05 0.006 1 4.7 intracellular non-membrane-bounded organelle 22 36.67 2.71E-04 0.034 1 4.7 non-membrane-bounded organelle 22 36.67 2.71E-04 0.034 1 4.7 nucleus 28 46.67 5.17E-04 0.042 2 1.64 sce03018:RNA degradation 4 6.67 0.002 0.01

e, DR up and rapamycin up. Cluster Score Term Count % p-Value Benjamini 1 10.72 cellular response to heat 28 18.18 1.54E-14 9.75E-12 1 10.72 response to temperature stimulus 30 19.48 2.71E-14 1.71E-11 1 10.72 response to heat 28 18.18 2.79E-13 1.76E-10 1 10.72 response to abiotic stimulus 31 20.13 6.53E-10 4.13E-07 1 10.72 cellular response to stress 31 20.13 3.44E-05 0.022 2 3.52 organic acid catabolic process 9 5.84 4.88E-05 0.03 2 3.52 carboxylic acid catabolic process 9 5.84 4.88E-05 0.03 3 3.18 peroxisome 9 5.84 3.00E-05 0.006 3 3.18 peroxisome 9 5.84 6.15E-05 0.008 3 3.18 microbody 9 5.84 6.15E-05 0.008 8 1.52 protein catabolic process 23 14.94 7.83E-05 0.048

Table: Differential Expression of rapamcyin enriched/depleted overexpression mutants", data='''

a, Overexpressed genes enriched or depleted by rapamycin treatment and differentially expressed upon DR. Orf Gene_symbol Rapamycin DR Classification YAL030W SNC1 -2.2 3.32
YBL024W NCL1 7.4 -2.58
YBL060W YEL1 -3.3 4.8
YBR121C GRS1 4.2 -4.78
YBR165W UBS1 -2.1 4.95
YBR283C SSH1 2.3 -2.02
YBR284W -3.2 3.08
YBR290W BSD2 4.6 2.65
YCL011C GBP2 -2.8 2.07
YCL014W BUD3 -2.3 -3.12
YCR007C -2.2 7.76
YDL051W LHP1 3.3 2.46
YDL063C 2.5 -4.07
YDL153C SAS10 2.3 -5.73
YDL186W -3.3 2.93
YDL199C -2.4 3.24
YDL240W LRG1 -2.2 -2.74
YDR043C NRG1 -2.3 2.11
YDR075W PPH3 3.8 2.58
YDR309C GIC2 -4.5 -16.36
YDR313C PIB1 -2.3 2.78
YDR528W HLR1 7 4.94
YEL019C MMS21 2.1 2.84
YER101C AST2 -2.6 2.93
YER171W RAD3 -2.1 -2.64
YFL023W BUD27 -4.9 6.13
YFL030W AGX1 3.2 12.33
YFL054C -2.5 3.8 DEO DEP YGL008C PMA1 -2.2 -5.49
YGL027C CWH41 -2.7 -8.54
YGL056C SDS23 3 4.13
YGL245W GUS1 2.2 -2.24
YGL250W RMR1 -2.3 5.09
YGR103W NOP7 2.4 -6.31
YGR122W 2 2.02
YGR123C PPT1 -3.2 -6.24
YGR130C 2 2.84
YGR131W FHN1 -2.3 5.16
YGR157W CHO2 -3.1 -2.54
YGR224W AZR1 -3.1 3.62
YGR287C IMA1 7 -2.15
YHL011C PRS3 2.3 -4.25
YHR004C NEM1 2.2 2.77
YHR208W BAT1 2.4 -5.19
YIL061C SNP1 2.8 2.04
YIL097W FYV10 2.5 2.58
YIL109C SEC24 5.3 -2.09
YIL123W SIM1 -2.1 -6.58
YJL033W HCA4 2.9 -3.89
YJL045W -2.3 4.57
YJL051W IRC8 -2.5 -2.37
YJL082W IML2 24.4 -4.15
YJL221C IMA4 9.8 -3.49
YKL059C MPE1 -2.3 5.12
YKL091C 5.7 3.1
YKL174C TPO5 -5.6 -2.42
YKL178C STE3 -2.9 -8.31
YKL185W ASH1 -3 -2.11
YKR008W RSC4 2.1 2.67
YKR059W TIF1 3.2 -2.86
YLR135W SLX4 6.2 2.27
YLR150W STM1 5.7 -4.7
YLR153C ACS2 2.5 -2.9
YLR157C ASP3-2 2.1 6.35
YLR158C ASP3-3 2.5 6.35
YLR160C ASP3-4 2.4 6.35
YLR224W -2.9 4.33
YLR243W -3.9 -3.17
YLR297W 3.6 2.35
YLR346C 2.7 4.16
YLR377C FBP1 2.1 33.63
YLR392C ART10 -3.3 2.3
YLR410W VIP1 -2.3 -2.57
YML007W YAP1 5.4 3.71
YML047C PRM6 -4.4 -2.18
YML106W URA5 2.2 -3.84
YML109W ZDS2 13.6 -2.9
YML126C ERG13 2.5 -3.47
YML130C ERO1 -2.3 -3.87
YML132W COS3 -2 9.86
YMR006C PLB2 3.4 -5.74
YMR034C -4.4 2.24
YMR046C 2.2 2.26
YMR079W SEC14 4.1 -3.94
YMR102C 14.5 -3.33
YMR212C EFR3 -3.1 -2.46
YMR319C FET4 -5.1 -4.49
YNL026W SAM50 -2.2 -2.45
YNL076W MKS1 -2 2.22
YNL090W RHO2 -2.6 -2.16
YNL115C -2.4 3.65
YNL199C GCR2 -4 3.6
YNL264C PDR17 2.9 -2.99
YNR021W 2.1 -2
YNR051C BRE5 6.9 -4.57
YOL022C TSR4 2.6 -2.09
YOL055C THI20 2.1 2.12
YOL056W GPM3 2.5 -2.7
YOL082W ATG19 5.3 3.55
YOL090W MSH2 2.9 -7.96
YOL097C WRS1 2.8 -18.98
YOL103W ITR2 -3.3 -2.72
YOL126C MDH2 3.9 2.51
YOL139C CDC33 2 -3.97
YOR051C ETT1 2.3 -6.86
YOR064C YNG1 -2.1 7.03
YOR134W BAG7 -2.9 2.9
YOR136W IDH2 2.1 -2.16
YOR152C 2.3 5.13
YOR173W DCS2 2.2 2.66
YOR204W DED1 -2.1 -2.38
YOR220W RCN2 3.2 2.22
YOR228C -2 2.56
YOR237W HES1 -3.1 2.25
YOR303W CPA1 3.3 10.81
YOR323C PRO2 2.3 -7.1
YOR348C PUT4 -3.1 100.69
YOR384W FRE5 -8.1 9.8
YPL069C BTS1 2.4 2.12
YPL093W NOG1 2.9 -2.69
YPL106C SSE1 11.4 -2.58
YPL133C RDS2 2 3.14
YPL137C GIP3 -2.3 3
YPL189W GUP2 -2 2.33
YPL207W TYW1 -2.9 -3.55
YPL227C ALG5 -4 -2.97
YPL246C RBD2 -2.8 -2.19
YPL258C THI21 2.2 3.03
YPR009W SUT2 -3.8 -5.61
YPR019W MCM4 4.5 -2.64
YPR029C APL4 -2.7 2.46
YPR040W TIP41 2.4 3.46
YPR078C -2.2 9.53
YPR151C SUE1 -2 26.09
YPR153W 2.1 2.67
YPR193C HPA2 -2.1 3.26

b, Overexpressed genes enriched by rapamycin treatment and upregulated upon DR.
Orf Gene_symbol Rapamycin DR Classification YAL030W SNC1 -2.2 3.32
YBL024W NCL1 7.4 -2.58
YBL060W YEL1 -3.3 4.8
YBR121C GRS1 4.2 -4.78
YBR165W UBS1 -2.1 4.95
YBR283C SSH1 2.3 -2.02
YBR284W -3.2 3.08
YBR290W BSD2 4.6 2.65
YCL011C GBP2 -2.8 2.07
YCL014W BUD3 -2.3 -3.12
YCR007C -2.2 7.76
YDL051W LHP1 3.3 2.46
YDL063C 2.5 -4.07
YDL153C SAS10 2.3 -5.73
YDL186W -3.3 2.93
YDL199C -2.4 3.24
YDL240W LRG1 -2.2 -2.74
YDR043C NRG1 -2.3 2.11
YDR075W PPH3 3.8 2.58
YDR309C GIC2 -4.5 -16.36
YDR313C PIB1 -2.3 2.78
YDR528W HLR1 7 4.94
YEL019C MMS21 2.1 2.84
YER101C AST2 -2.6 2.93
YER171W RAD3 -2.1 -2.64
YFL023W BUD27 -4.9 6.13
YFL030W AGX1 3.2 12.33
YFL054C -2.5 3.8 DEO DEP YGL008C PMA1 -2.2 -5.49
YGL027C CWH41 -2.7 -8.54
YGL056C SDS23 3 4.13
YGL245W GUS1 2.2 -2.24
YGL250W RMR1 -2.3 5.09
YGR103W NOP7 2.4 -6.31
YGR122W 2 2.02
YGR123C PPT1 -3.2 -6.24
YGR130C 2 2.84
YGR131W FHN1 -2.3 5.16
YGR157W CHO2 -3.1 -2.54
YGR224W AZR1 -3.1 3.62
YGR287C IMA1 7 -2.15
YHL011C PRS3 2.3 -4.25
YHR004C NEM1 2.2 2.77
YHR208W BAT1 2.4 -5.19
YIL061C SNP1 2.8 2.04
YIL097W FYV10 2.5 2.58
YIL109C SEC24 5.3 -2.09
YIL123W SIM1 -2.1 -6.58
YJL033W HCA4 2.9 -3.89
YJL045W -2.3 4.57
YJL051W IRC8 -2.5 -2.37
YJL082W IML2 24.4 -4.15
YJL221C IMA4 9.8 -3.49
YKL059C MPE1 -2.3 5.12
YKL091C 5.7 3.1
YKL174C TPO5 -5.6 -2.42
YKL178C STE3 -2.9 -8.31
YKL185W ASH1 -3 -2.11
YKR008W RSC4 2.1 2.67
YKR059W TIF1 3.2 -2.86
YLR135W SLX4 6.2 2.27
YLR150W STM1 5.7 -4.7
YLR153C ACS2 2.5 -2.9
YLR157C ASP3-2 2.1 6.35
YLR158C ASP3-3 2.5 6.35
YLR160C ASP3-4 2.4 6.35
YLR224W -2.9 4.33
YLR243W -3.9 -3.17
YLR297W 3.6 2.35
YLR346C 2.7 4.16
YLR377C FBP1 2.1 33.63
YLR392C ART10 -3.3 2.3
YLR410W VIP1 -2.3 -2.57
YML007W YAP1 5.4 3.71
YML047C PRM6 -4.4 -2.18
YML106W URA5 2.2 -3.84
YML109W ZDS2 13.6 -2.9
YML126C ERG13 2.5 -3.47
YML130C ERO1 -2.3 -3.87
YML132W COS3 -2 9.86
YMR006C PLB2 3.4 -5.74
YMR034C -4.4 2.24
YMR046C 2.2 2.26
YMR079W SEC14 4.1 -3.94
YMR102C 14.5 -3.33
YMR212C EFR3 -3.1 -2.46
YMR319C FET4 -5.1 -4.49
YNL026W SAM50 -2.2 -2.45
YNL076W MKS1 -2 2.22
YNL090W RHO2 -2.6 -2.16
YNL115C -2.4 3.65
YNL199C GCR2 -4 3.6
YNL264C PDR17 2.9 -2.99
YNR021W 2.1 -2
YNR051C BRE5 6.9 -4.57
YOL022C TSR4 2.6 -2.09
YOL055C THI20 2.1 2.12
YOL056W GPM3 2.5 -2.7
YOL082W ATG19 5.3 3.55
YOL090W MSH2 2.9 -7.96
YOL097C WRS1 2.8 -18.98
YOL103W ITR2 -3.3 -2.72
YOL126C MDH2 3.9 2.51
YOL139C CDC33 2 -3.97
YOR051C ETT1 2.3 -6.86
YOR064C YNG1 -2.1 7.03
YOR134W BAG7 -2.9 2.9
YOR136W IDH2 2.1 -2.16
YOR152C 2.3 5.13
YOR173W DCS2 2.2 2.66
YOR204W DED1 -2.1 -2.38
YOR220W RCN2 3.2 2.22
YOR228C -2 2.56
YOR237W HES1 -3.1 2.25
YOR303W CPA1 3.3 10.81
YOR323C PRO2 2.3 -7.1
YOR348C PUT4 -3.1 100.69
YOR384W FRE5 -8.1 9.8
YPL069C BTS1 2.4 2.12
YPL093W NOG1 2.9 -2.69
YPL106C SSE1 11.4 -2.58
YPL133C RDS2 2 3.14
YPL137C GIP3 -2.3 3
YPL189W GUP2 -2 2.33
YPL207W TYW1 -2.9 -3.55
YPL227C ALG5 -4 -2.97
YPL246C RBD2 -2.8 -2.19
YPL258C THI21 2.2 3.03
YPR009W SUT2 -3.8 -5.61
YPR019W MCM4 4.5 -2.64
YPR029C APL4 -2.7 2.46
YPR040W TIP41 2.4 3.46
YPR078C -2.2 9.53
YPR151C SUE1 -2 26.09
YPR153W 2.1 2.67

c, Overexpressed genes depleted by rapamycin treatment and downregulated upon DR.
Orf Gene_symbol Rapamycin DR Classification YOR384W FRE5 2.8 -18.98
YKL174C TPO5 -4.5 -16.36
YMR319C FET4 -2.7 -8.54
YFL023W BUD27 -2.9 -8.31
YDR309C GIC2 2.9 -7.96
YML047C PRM6 2.3 -7.1
YMR034C 2.3 -6.86
YNL199C GCR2 -2.1 -6.58
YPL227C ALG5 2.4 -6.31
YLR243W -3.2 -6.24
YPR009W SUT2 3.4 -5.74
YBL060W YEL1 2.3 -5.73
YDL186W -3.8 -5.61
YLR392C ART10 -2.2 -5.49
YOL103W ITR2 2.4 -5.19
YBR284W 4.2 -4.78
YGR123C PPT1 5.7 -4.7
YGR157W CHO2 6.9 -4.57
YGR224W AZR1 -5.1 -4.49
YMR212C EFR3 2.3 -4.25
YOR237W HES1 24.4 -4.15
YOR348C PUT4 2.5 -4.07
YKL185W ASH1 2 -3.97
YKL178C STE3 4.1 -3.94
YLR224W 2.9 -3.89
YOR134W BAG7 -2.3 -3.87
YPL207W TYW1 2.2 -3.84
YCL011C GBP2 -2.9 -3.55
YPL246C RBD2 9.8 -3.49
YGL027C CWH41 2.5 -3.47
YPR029C APL4 14.5 -3.33
YER101C AST2 -3.9 -3.17
YNL090W RHO2 -2.3 -3.12
YFL054C 2.9 -2.99 DEO DEP YJL051W IRC8 -4 -2.97
YDL199C 2.5 -2.9
YNL115C 13.6 -2.9
YCL014W BUD3 3.2 -2.86
YDR043C NRG1 -2.2 -2.74
YDR313C PIB1 -3.3 -2.72
YGL250W RMR1 2.5 -2.7
YGR131W FHN1 2.9 -2.69
YJL045W -2.1 -2.64
YKL059C MPE1 4.5 -2.64
YLR410W VIP1 7.4 -2.58
YML130C ERO1 11.4 -2.58
YPL137C GIP3 -2.3 -2.57
YAL030W SNC1 -3.1 -2.54
YCR007C -3.1 -2.46
YDL240W LRG1 -2.2 -2.45
YGL008C PMA1 -5.6 -2.42
YNL026W SAM50 -2.1 -2.38
YPR078C -2.5 -2.37
YBR165W UBS1 2.2 -2.24
YER171W RAD3 -2.8 -2.19
YIL123W SIM1 -4.4 -2.18
YOR064C YNG1 -2.6 -2.16
YOR204W DED1 2.1 -2.16
YPR193C HPA2 7 -2.15
YML132W COS3 -3 -2.11

d, Overexpressed genes enriched by rapamycin treatment and downregulated upon DR.
Orf Gene_symbol Rapamycin DR Classification YAL030W SNC1 -2.2 3.32
YBL024W NCL1 7.4 -2.58
YBL060W YEL1 -3.3 4.8
YBR121C GRS1 4.2 -4.78
YBR165W UBS1 -2.1 4.95
YBR283C SSH1 2.3 -2.02
YBR284W -3.2 3.08
YBR290W BSD2 4.6 2.65
YCL011C GBP2 -2.8 2.07
YCL014W BUD3 -2.3 -3.12
YCR007C -2.2 7.76
YDL051W LHP1 3.3 2.46
YDL063C 2.5 -4.07
YDL153C SAS10 2.3 -5.73
YDL186W -3.3 2.93
YDL199C -2.4 3.24
YDL240W LRG1 -2.2 -2.74
YDR043C NRG1 -2.3 2.11
YDR075W PPH3 3.8 2.58
YDR309C GIC2 -4.5 -16.36
YDR313C PIB1 -2.3 2.78
YDR528W HLR1 7 4.94
YEL019C MMS21 2.1 2.84
YER101C AST2 -2.6 2.93
YER171W RAD3 -2.1 -2.64
YFL023W BUD27 -4.9 6.13
YFL030W AGX1 3.2 12.33
YFL054C -2.5 3.8 DEO DEP YGL008C PMA1 -2.2 -5.49
YGL027C CWH41 -2.7 -8.54
YGL056C SDS23 3 4.13
YGL245W GUS1 2.2 -2.24
YGL250W RMR1 -2.3 5.09
YGR103W NOP7 2.4 -6.31
YGR122W 2 2.02
YGR123C PPT1 -3.2 -6.24
YGR130C 2 2.84
YGR131W FHN1 -2.3 5.16
YGR157W CHO2 -3.1 -2.54
YGR224W AZR1 -3.1 3.62
YGR287C IMA1 7 -2.15
YHL011C PRS3 2.3 -4.25
YHR004C NEM1 2.2 2.77
YHR208W BAT1 2.4 -5.19
YIL061C SNP1 2.8 2.04
YIL097W FYV10 2.5 2.58
YIL109C SEC24 5.3 -2.09
YIL123W SIM1 -2.1 -6.58
YJL033W HCA4 2.9 -3.89
YJL045W -2.3 4.57
YJL051W IRC8 -2.5 -2.37
YJL082W IML2 24.4 -4.15
YJL221C IMA4 9.8 -3.49
YKL059C MPE1 -2.3 5.12
YKL091C 5.7 3.1
YKL174C TPO5 -5.6 -2.42
YKL178C STE3 -2.9 -8.31
YKL185W ASH1 -3 -2.11
YKR008W RSC4 2.1 2.67
YKR059W TIF1 3.2 -2.86
YLR135W SLX4 6.2 2.27
YLR150W STM1 5.7 -4.7
YLR153C ACS2 2.5 -2.9
YLR157C ASP3-2 2.1 6.35
YLR158C ASP3-3 2.5 6.35
YLR160C ASP3-4 2.4 6.35
YLR224W -2.9 4.33
YLR243W -3.9 -3.17
YLR297W 3.6 2.35
YLR346C 2.7 4.16
YLR377C FBP1 2.1 33.63
YLR392C ART10 -3.3 2.3
YLR410W VIP1 -2.3 -2.57
YML007W YAP1 5.4 3.71
YML047C PRM6 -4.4 -2.18
YML106W URA5 2.2 -3.84
YML109W ZDS2 13.6 -2.9
YML126C ERG13 2.5 -3.47
YML130C ERO1 -2.3 -3.87
YML132W COS3 -2 9.86
YMR006C PLB2 3.4 -5.74
YMR034C -4.4 2.24
YMR046C 2.2 2.26
YMR079W SEC14 4.1 -3.94
YMR102C 14.5 -3.33
YMR212C EFR3 -3.1 -2.46
YMR319C FET4 -5.1 -4.49
YNL026W SAM50 -2.2 -2.45
YNL076W MKS1 -2 2.22
YNL090W RHO2 -2.6 -2.16
YNL115C -2.4 3.65
YNL199C GCR2 -4 3.6
YNL264C PDR17 2.9 -2.99
YNR021W 2.1 -2
YNR051C BRE5 6.9 -4.57
YOL022C TSR4 2.6 -2.09
YOL055C THI20 2.1 2.12
YOL056W GPM3 2.5 -2.7
YOL082W ATG19 5.3 3.55
YOL090W MSH2 2.9 -7.96
YOL097C WRS1 2.8 -18.98
YOL103W ITR2 -3.3 -2.72
YOL126C MDH2 3.9 2.51
YOL139C CDC33 2 -3.97
YOR051C ETT1 2.3 -6.86
YOR064C YNG1 -2.1 7.03
YOR134W BAG7 -2.9 2.9
YOR136W IDH2 2.1 -2.16
YOR152C 2.3 5.13
YOR173W DCS2 2.2 2.66
YOR204W DED1 -2.1 -2.38
YOR220W RCN2 3.2 2.22
YOR228C -2 2.56
YOR237W HES1 -3.1 2.25
YOR303W CPA1 3.3 10.81
YOR323C PRO2 2.3 -7.1
YOR348C PUT4 -3.1 100.69
YOR384W FRE5 -8.1 9.8
YPL069C BTS1 2.4 2.12
YPL093W NOG1 2.9 -2.69
YPL106C SSE1 11.4 -2.58
YPL133C RDS2 2 3.14
YPL137C GIP3 -2.3 3
YPL189W GUP2 -2 2.33
YPL207W TYW1 -2.9 -3.55
YPL227C ALG5 -4 -2.97
YPL246C RBD2 -2.8 -2.19
YPL258C THI21 2.2 3.03
YPR009W SUT2 -3.8 -5.61
YPR019W MCM4 4.5 -2.64

e, Overexpressed genes depleted by rapamycin treatment and upregulated upon DR.
Orf Gene_symbol Rapamycin DR Classification YAL030W SNC1 -2.2 3.32
YBL024W NCL1 7.4 -2.58
YBL060W YEL1 -3.3 4.8
YBR121C GRS1 4.2 -4.78
YBR165W UBS1 -2.1 4.95
YBR283C SSH1 2.3 -2.02
YBR284W -3.2 3.08
YBR290W BSD2 4.6 2.65
YCL011C GBP2 -2.8 2.07
YCL014W BUD3 -2.3 -3.12
YCR007C -2.2 7.76
YDL051W LHP1 3.3 2.46
YDL063C 2.5 -4.07
YDL153C SAS10 2.3 -5.73
YDL186W -3.3 2.93
YDL199C -2.4 3.24
YDL240W LRG1 -2.2 -2.74
YDR043C NRG1 -2.3 2.11
YDR075W PPH3 3.8 2.58
YDR309C GIC2 -4.5 -16.36
YDR313C PIB1 -2.3 2.78
YDR528W HLR1 7 4.94
YEL019C MMS21 2.1 2.84
YER101C AST2 -2.6 2.93
YER171W RAD3 -2.1 -2.64
YFL023W BUD27 -4.9 6.13
YFL030W AGX1 3.2 12.33
YFL054C -2.5 3.8 DEO DEP YGL008C PMA1 -2.2 -5.49
YGL027C CWH41 -2.7 -8.54
YGL056C SDS23 3 4.13
YGL245W GUS1 2.2 -2.24
YGL250W RMR1 -2.3 5.09
YGR103W NOP7 2.4 -6.31
YGR122W 2 2.02
YGR123C PPT1 -3.2 -6.24
YGR130C 2 2.84
YGR131W FHN1 -2.3 5.16
YGR157W CHO2 -3.1 -2.54
YGR224W AZR1 -3.1 3.62
YGR287C IMA1 7 -2.15
YHL011C PRS3 2.3 -4.25
YHR004C NEM1 2.2 2.77
YHR208W BAT1 2.4 -5.19
YIL061C SNP1 2.8 2.04
YIL097W FYV10 2.5 2.58
YIL109C SEC24 5.3 -2.09
YIL123W SIM1 -2.1 -6.58
YJL033W HCA4 2.9 -3.89
YJL045W -2.3 4.57
YJL051W IRC8 -2.5 -2.37
YJL082W IML2 24.4 -4.15
YJL221C IMA4 9.8 -3.49
YKL059C MPE1 -2.3 5.12
YKL091C 5.7 3.1
YKL174C TPO5 -5.6 -2.42
YKL178C STE3 -2.9 -8.31
YKL185W ASH1 -3 -2.11
YKR008W RSC4 2.1 2.67
YKR059W TIF1 3.2 -2.86
YLR135W SLX4 6.2 2.27
YLR150W STM1 5.7 -4.7
YLR153C ACS2 2.5 -2.9
YLR157C ASP3-2 2.1 6.35
YLR158C ASP3-3 2.5 6.35
YLR160C ASP3-4 2.4 6.35
YLR224W -2.9 4.33
YLR243W -3.9 -3.17
YLR297W 3.6 2.35
YLR346C 2.7 4.16
YLR377C FBP1 2.1 33.63
YLR392C ART10 -3.3 2.3
YLR410W VIP1 -2.3 -2.57
YML007W YAP1 5.4 3.71
YML047C PRM6 -4.4 -2.18
YML106W URA5 2.2 -3.84
YML109W ZDS2 13.6 -2.9
YML126C ERG13 2.5 -3.47
YML130C ERO1 -2.3 -3.87
YML132W COS3 -2 9.86
YMR006C PLB2 3.4 -5.74
YMR034C -4.4 2.24
YMR046C 2.2 2.26
YMR079W SEC14 4.1 -3.94
YMR102C 14.5 -3.33
YMR212C EFR3 -3.1 -2.46
YMR319C FET4 -5.1 -4.49
YNL026W SAM50 -2.2 -2.45
YNL076W MKS1 -2 2.22
YNL090W RHO2 -2.6 -2.16
YNL115C -2.4 3.65
YNL199C GCR2 -4 3.6
YNL264C PDR17 2.9 -2.99
YNR021W 2.1 -2
YNR051C BRE5 6.9 -4.57
YOL022C TSR4 2.6 -2.09
YOL055C THI20 2.1 2.12
YOL056W GPM3 2.5 -2.7
YOL082W ATG19 5.3 3.55
YOL090W MSH2 2.9 -7.96
YOL097C WRS1 2.8 -18.98
YOL103W ITR2 -3.3 -2.72
YOL126C MDH2 3.9 2.51
YOL139C CDC33 2 -3.97
YOR051C ETT1 2.3 -6.86
YOR064C YNG1 -2.1 7.03
YOR134W BAG7 -2.9 2.9
YOR136W IDH2 2.1 -2.16
YOR152C 2.3 5.13
YOR173W DCS2 2.2 2.66
YOR204W DED1 -2.1 -2.38
YOR220W RCN2 3.2 2.22
YOR228C -2 2.56
YOR237W HES1 -3.1 2.25
YOR303W CPA1 3.3 10.81
YOR323C PRO2 2.3 -7.1
YOR348C PUT4 -3.1 100.69
YOR384W FRE5 -8.1 9.8
YPL069C BTS1 2.4 2.12
YPL093W NOG1 2.9 -2.69
YPL106C SSE1 11.4 -2.58
YPL133C RDS2 2 3.14
YPL137C GIP3 -2.3 3
YPL189W GUP2 -2 2.33
YPL207W TYW1 -2.9 -3.55
YPL227C ALG5 -4 -2.97
YPL246C RBD2 -2.8 -2.19
YPL258C THI21 2.2 3.03
YPR009W SUT2 -3.8 -5.61
YPR019W MCM4 4.5 -2.64
YPR029C APL4 -2.7 2.46
YPR040W TIP41 2.4 3.46
YPR078C -2.2 9.53
YPR151C SUE1 -2 26.09
YPR153W 2.1 2.67
YPR193C HPA2 -2.1 3.26

Table: Rapamycin resistant DR-essential", data='''

Classification Orf Gene_symbol Rapamycin DR Description DE YER177W BMH1 6.5 -1.6 14-3-3 protein, major isoform; controls proteome at post-transcriptional level, binds proteins and DNA, involved in regulation of many processes including exocytosis, vesicle transport, Ras/MAPK signaling, and rapamycin-sensitive signaling DEO YFL054C -2.5 3.8 Putative channel-like protein; similar to Fps1p; mediates passive diffusion of glycerol in the presence of ethanol DE YJL101C GSH1 -5.7 -1.2 Gamma glutamylcysteine synthetase catalyzes the first step in glutathione (GSH) biosynthesis; expression induced by oxidants, cadmium, and mercury DE YJR066W TOR1 8.4 1.1 PIK-related protein kinase and rapamycin target; subunit of TORC1, a complex that controls growth in response to nutrients by regulating translation, transcription, ribosome biogenesis, nutrient transport and autophagy; involved in meiosis DE YKL062W MSN4 -2.5 -1.3 Transcriptional activator related to Msn2p; activated in stress conditions, which results in translocation from the cytoplasm to the nucleus; binds DNA at stress response elements of responsive genes, inducing gene expression

Table: Rapamycin resistant ultradian genes", data='''

Orf Gene_symbol Rapamycin DR Description YDR123C INO2 -2 -1.3 Component of the heteromeric Ino2p/Ino4p basic helix-loop-helix transcription activator that binds inositol/choline-responsive elements (ICREs), required for derepression of phospholipid biosynthetic genes in response to inositol depletion YJR060W CBF1 2.3 -1 Helix-loop-helix protein that binds the motif CACRTG, which is present at several sites including MET gene promoters and centromere DNA element I (CDEI); required for nucleosome positioning at this motif; targets Isw1p to DNA YKL062W MSN4 -2.5 -1.3 Transcriptional activator related to Msn2p; activated in stress conditions, which results in translocation from the cytoplasm to the nucleus; binds DNA at stress response elements of responsive genes, inducing gene expression YML007W YAP1 5.4 3.7 Basic leucine zipper (bZIP) transcription factor required for oxidative stress tolerance; activated by H2O2 through the multistep formation of disulfide bonds and transit from the cytoplasm to the nucleus; mediates resistance to cadmium YOR230W WTM1 2 -1.7 Transcriptional modulator involved in regulation of meiosis, silencing, and expression of RNR genes; required for nuclear localization of the ribonucleotide reductase small subunit Rnr2p and Rnr4p; contains WD repeats YPL038W MET31 -2.2 1.9 Zinc-finger DNA-binding protein, involved in transcriptional regulation of the methionine biosynthetic genes, similar to Met32p YPL061W ALD6 3 1.7 Cytosolic aldehyde dehydrogenase, activated by Mg2+ and utilizes NADP+ as the preferred coenzyme; required for conversion of acetaldehyde to acetate; constitutively expressed; locates to the mitochondrial outer surface upon oxidative stress

Table: Rapamycin resistant GTS1 target genes", data='''

Orf Gene_symbol Rapamycin DR Description YDR222W 5 -1.9 Protein of unknown function; green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm in a punctate pattern yes YEL019C MMS21 2.1 2.8 SUMO ligase involved in chromosomal organization and DNA repair; essential subunit of the Mms21-Smc5-Smc6 complex; mutants are sensitive to methyl methanesulfonate and show increased spontaneous mutation and mitotic recombination yes YGR016W 2.2 1.7 Putative protein of unknown function no YGR125W -2.3 -1.4 Putative protein of unknown function; deletion mutant has decreased rapamycin resistance but normal wormannin resistance; green fluorescent protein (GFP)-fusion protein localizes to the vacuole no YLL026W HSP104 6.3 1.1 Heat shock protein that cooperates with Ydj1p (Hsp40) and Ssa1p (Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses including: heat, ethanol, and sodium arsenite; involved in [PSI+] propagation yes YLR258W GSY2 2.4 -1.4 Glycogen synthase, similar to Gsy1p; expression induced by glucose limitation, nitrogen starvation, heat shock, and stationary phase; activity regulated by cAMP-dependent, Snf1p and Pho85p kinases as well as by the Gac1p-Glc7p phosphatase yes YOL082W ATG19 5.3 3.5 Receptor protein specific for the cytoplasm-to-vacuole targeting (Cvt) pathway; delivers cargo proteins aminopeptidase I (Lap4p) and alpha-mannosidase (Ams1p) to the phagophore assembly site for packaging into Cvt vesicles yes YOL130W ALR1 -2.7 -1.1 Plasma membrane Mg(2+) transporter, expression and turnover are regulated by Mg(2+) concentration; overexpression confers increased tolerance to Al(3+) and Ga(3+) ions yes

Table: CLUSTAL 2.1 multiple sequence alignment of yeast Gts1 and its best blast hit in nematode", data='''

sp|P40956|GTS1_YEAST ---MRFRSSSHSLKHVDRELKELINSSENANKCGECGNFYPTWCSVNLGV 47 tr|O18181|O18181_CAEEL MLRGKVDPKKEEQERLQGFLLDMLKEEENK-YCADCQAKTPRWAAWNLGV 49 :. ..... :::: * ::::.. .: * *.: **

sp|P40956|GTS1_YEAST FLCGRCASVHRKVFGSRDDDAFSNVKSLSMDRWTREDIDELVSLGGNKGN 97 tr|O18181|O18181_CAEEL FICIRCAGIHRNLG-----VHISKVRSVNLDSWTPEQVQTMRVMGNEKAR 94 : .::: ::::.:* ::: : :.:*..

sp|P40956|GTS1_YEAST ARFWNPKNVPFPFDGDD---DKAIVEHYIRDKYILGKFRYDEIKPEDFGS 144 tr|O18181|O18181_CAEEL QVYEHDLPAQFRRPTNDQQMEQFIRSKYEQKRYILRDFVYPRVDASQLPK 144 : : . * : :: * .: :.:** . * .:...:: .

sp|P40956|GTS1_YEAST RMDDFDGESDR--FDERNRSRSRSRSHSFYKGGHNRSDYGGSRD--SFQS 190 tr|O18181|O18181_CAEEL SLSQAQKKVGTPVVNIASRGSSSSNGHSTASAAAAAPSLLDFSDPPASTT 194 :.: : : . .: .. * ..** ... .. . * : :

sp|P40956|GTS1_YEAST SGSRYSRQLAELKDMGFG---------------------------DTNKN 213 tr|O18181|O18181_CAEEL PAKKAVNLFDDFDGLSLGPAAPAAAPAALNDDFDDFGSFVSANSQNAQQN 244 ...: . : ::..:.: ::::

sp|P40956|GTS1_YEAST LDALSSAHGNINRAIDYLEKSSSSRN---SVSAAATTSTPPLPRRRATTS 260 tr|O18181|O18181_CAEEL AQSLGGGFADFSSAPTTAAAAPSASSGLDDLTALSSTAPATNGSDQKKTN 294 ::.....::. * :.: . .:: :::... : .*.

sp|P40956|GTS1_YEAST GPQPAIFDG------TNVITP-------------DFTSNSASFVQAKPAV 291 tr|O18181|O18181_CAEEL ADILSLFGPSGGISAPNVVAPGGFAGFGLQAAPAQIPQQASSFHQFGAPA 344 . ::*. .::* ::..::: * ...

sp|P40956|GTS1_YEAST FDGTLQQYYDPATGMIYVDQQQYAMAMQQQQQQQQQLAVAQAQAQAQAQA 341 tr|O18181|O18181_CAEEL PAPQQNDMFGGLSGINFGAPPPSAQMSQIPPMAPPQYFGAAQFGAMSSSG 394 :: :. :*: : * * * * . .:..

sp|P40956|GTS1_YEAST QAQVQAQAQAQAQAQAQAQ---------------------------QIQM 364 tr|O18181|O18181_CAEEL FGGMSMQSKPTPTSPTGSQGFNIPNKSNAFADLALGKVMKTNYGQSAIHH 444 . :. ::. . : : : *:

sp|P40956|GTS1_YEAST QQLQMQQQQQAPLSFQQMSQGGNLPQGYFYTQ------------------ 396 tr|O18181|O18181_CAEEL QAAPISSNRASPMPAQSNTAANNDLFDMFASAPPPVTVNSSSSGLDDLLG 494 * :..:: ::. . : ..* . * :

sp|P40956|GTS1_YEAST - tr|O18181|O18181_CAEEL L 495

Table: GTS1 upstream region", data='''

GTS1 6321257 upstream sequence, from -1000 to -1, size 1000 CTTTACTTCCTGTATCTTTTTTTTTATCAACTCGCTTGAATCATACAATTTTTGCAGTGT CTGATTTTTAAAGCACCAGTAGATGTTAATATTACCACATTTTTCCACCGAAATGACGCC GTCTTCATCAATCATTTGTTGCACTAAATCTTTAACGATCATGGGTGAGATTCCGCATTT TTTAGGTATGCTTTTCTCTAATTCCTTGATGTTGTAGAATGTGTACGTCTCTTGAAAAAA GTTCAGAATCCGGTTCTTTTTCTCTTGGAGTGATACTGTCTGTCTCTTTGGCCCCTAATA ATAAGAAGAGGGTAGCACGCACCCGTCTATAAGTTAGTGTTCAGTCCTGTGTACCATGAT GGTTTTATTAGACATACCATTGTTGAATGTTTATGATACTACACATAAAGTTTCCGCCGT TTAGTCTATATCGCAAAGAATAACGGTGGGACGGGAGATCGCCTTTTATATGCAATTTCG TGATCTGGGCGGTTATTTTACTTTTTTTCGACGCATAAAAAAGCAGGAAAGATGGTAAAT TCGGTTCGTTATTCAAGATTTTTAGAGCCAGAAAGATATTTGAGCGTGCCTTCGTGAGAA GAGGCTTAGGTTGAGACGATGATGCTGAAATTATGGCAAGACACGAATTCTTCTTCAAGT TTCCCTTGTCATAATAAGAAGAGAAAAACATACCTTTATGTCGGTTGCAATTAAGAAGGT TGTATAGAGCATCTCAAATGCTGCTTTTACTTTATTAAACGTTGACCAAATAATACGGCG CTTTTGTTTCGACAACGACACACTATGATGCAAAGCGGATCCGGGTAATCGATAAATGGG GGCACCCGGAAATTATTGTAAGTTGAAGAAAGTTAGTGTTGATCTCTAAATGCTTCACAA AAGGGTGGAAGAAGAACATTTTCTCTTGATCAATCATTAATATAGATGGAGGCAGCGTTC TGTCAATCACAGATTATAGTTTATTTATACTTAGTCAAAA

Table: Gts1 amino acid sequence", data='''

sp|P40956|GTS1_YEAST Protein GTS1 OS=Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GN=GTS1 PE=1 SV=2 NES _
MRFRSSSHSLKHVDRELKELINSSENANKCGECGNFYPTWCSVNLGVFLCGRCASVHRKVFGSRDDDAFSNVKSLSMDRWTREDIDELVSLGGNKGNARFWNPKNVPFPFDGDDDKAIVEHYIRDKYILGKFRYDEIKPEDFGSRMDDFDGESDRFDERNRSRSRSRSHSFYKGGHNRSDYGGSRDSFQSSGSRYSRQLAELKDMGFGDTNKNLDALSSAHGNINRAIDYLEKSSSSRNSVSAAATTSTPPLPRRRATTSGPQPAIFDGTNVITPDFTSNSASFVQAKPAVFDGTLQQYYDPATGMIYVDQQQYAMAMQQQQQQQQQLAVAQAQAQAQAQAQAQVQAQAQAQAQAQAQAQQIQMQQLQMQQQQQAPLSFQQMSQGGNLPQGYFYTQ

NES (nuclear export signal) Arf GAP (Arf3) Zn finger NLS (nuclear localisation signal - recognized by importin ?) UBA (ubiquitin associated domain) Coiled-coil QA-rich (transactivation - associates to transcriptional machinery)

Table: Enriched Clusters for highly Ultradian Genes", data='''

Cluster Score Term Count % p-Value Benjamin 1 24.19 transcription factor activity 36 48.65 9.66E-35 1.26E-32 1 24.19 dna-binding 42 56.76 1.71E-32 1.73E-30 1 24.19 sequence-specific DNA binding 39 52.7 6.48E-29 8.42E-27 1 24.19 transcription regulation 42 56.76 3.13E-28 3.16E-26 1 24.19 transcription regulator activity 43 58.11 4.10E-27 5.33E-25 1 24.19 Transcription 42 56.76 8.79E-27 8.87E-25 1 24.19 DNA binding 46 62.16 1.96E-22 2.54E-20 1 24.19 transcription 43 58.11 4.38E-22 1.80E-19 1 24.19 regulation of transcription 45 60.81 2.44E-21 1.00E-18 1 24.19 regulation of transcription, DNA-dependent 39 52.7 5.21E-21 2.14E-18 1 24.19 regulation of RNA metabolic process 39 52.7 1.05E-20 4.30E-18 1 24.19 nucleus 49 66.22 3.35E-13 3.38E-11 2 10.02 Cysteine biosynthesis 11 14.86 1.18E-18 1.19E-16 2 10.02 cysteine biosynthetic process 11 14.86 1.68E-17 6.92E-15 2 10.02 cysteine metabolic process 11 14.86 7.37E-16 3.19E-13 2 10.02 sulfur amino acid metabolic process 15 20.27 1.44E-15 5.93E-13 2 10.02 serine family amino acid biosynthetic process 12 16.22 7.18E-15 2.97E-12 2 10.02 sulfur amino acid biosynthetic process 13 17.57 5.83E-14 2.40E-11 2 10.02 serine family amino acid metabolic process 13 17.57 1.20E-13 4.94E-11 2 10.02 methionine biosynthesis 10 13.51 7.33E-12 7.40E-10 2 10.02 sulfur metabolic process 16 21.62 7.38E-12 3.03E-09 2 10.02 sulfur compound biosynthetic process 14 18.92 1.28E-11 5.28E-09 2 10.02 methionine biosynthetic process 11 14.86 1.31E-11 5.37E-09 2 10.02 methionine metabolic process 11 14.86 1.56E-10 6.42E-08 2 10.02 organic acid biosynthetic process 18 24.32 2.35E-10 9.68E-08 2 10.02 carboxylic acid biosynthetic process 18 24.32 2.35E-10 9.68E-08 2 10.02 cellular amino acid biosynthetic process 16 21.62 5.44E-10 2.23E-07 2 10.02 amino-acid biosynthesis 13 17.57 5.80E-10 5.86E-08 2 10.02 aspartate family amino acid metabolic process 12 16.22 1.12E-09 4.62E-07 2 10.02 amine biosynthetic process 16 21.62 1.24E-09 5.11E-07 2 10.02 aspartate family amino acid biosynthetic process 11 14.86 2.07E-09 8.51E-07 2 10.02 response to drug 14 18.92 2.00E-08 8.21E-06 2 10.02 sce00920:Sulfur metabolism 6 8.11 1.22E-06 3.78E-05 2 10.02 sulfate assimilation 5 6.76 8.63E-06 0.004 2 10.02 nitrogen compound biosynthetic process 17 22.97 1.03E-05 0.004 2 10.02 sce00450:Selenoamino acid metabolism 5 6.76 2.31E-04 0.007 3 9.92 RNA polymerase II transcription factor activity 23 31.08 1.14E-15 1.44E-13 3 9.92 regulation of transcription from RNA polymerase II promoter 25 33.78 1.14E-13 4.68E-11 3 9.92 positive regulation of transcription 18 24.32 2.35E-10 9.68E-08 3 9.92 positive regulation of biosynthetic process 19 25.68 2.56E-10 1.05E-07 3 9.92 positive regulation of cellular biosynthetic process 19 25.68 2.56E-10 1.05E-07 3 9.92 positive regulation of gene expression 18 24.32 2.58E-10 1.06E-07 3 9.92 positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process 18 24.32 5.77E-10 2.37E-07 3 9.92 positive regulation of nitrogen compound metabolic process 18 24.32 5.77E-10 2.37E-07 3 9.92 positive regulation of transcription, DNA-dependent 16 21.62 6.05E-10 2.49E-07 3 9.92 positive regulation of transcription from RNA polymerase II promoter 14 18.92 9.26E-10 3.80E-07 3 9.92 positive regulation of RNA metabolic process 16 21.62 1.02E-09 4.18E-07 3 9.92 positive regulation of macromolecule biosynthetic process 18 24.32 1.57E-09 6.46E-07 3 9.92 positive regulation of macromolecule metabolic process 18 24.32 6.20E-09 2.55E-06 4 5.59 DNA-binding region:Basic motif 10 13.51 2.10E-12 3.63E-10 4 5.59 IPR004827:Basic-leucine zipper (bZIP) transcription factor 7 9.46 7.65E-08 8.11E-06 4 5.59 domain:Leucine-zipper 7 9.46 4.19E-07 7.25E-05 4 5.59 SM00338:BRLZ 7 9.46 2.04E-06 2.85E-05 5 5.29 IPR001138:Fungal transcriptional regulatory protein, N-terminal 9 12.16 1.08E-06 1.15E-04 5 5.29 DNA-binding region:Zn(2)-C6 fungal-type 8 10.81 2.06E-06 3.57E-04 5 5.29 SM00066:GAL4 9 12.16 6.12E-05 8.57E-04 6 4.63 metal-binding 25 33.78 8.70E-08 8.79E-06 6 4.63 zinc 21 28.38 1.03E-07 1.04E-05 6 4.63 transition metal ion binding 27 36.49 1.93E-05 0.003 6 4.63 zinc ion binding 22 29.73 4.08E-05 0.005 6 4.63 zinc-finger 12 16.22 1.53E-04 0.015 6 4.63 metal ion binding 28 37.84 3.39E-04 0.043 7 4.35 response to inorganic substance 9 12.16 2.56E-07 1.05E-04 7 4.35 response to cadmium ion 5 6.76 5.24E-06 0.002 8 3.85 response to oxidative stress 13 17.57 1.55E-09 6.37E-07 8 3.85 antioxidant activity 8 10.81 2.92E-07 3.80E-05 8 3.85 peroxidase activity 7 9.46 3.29E-07 4.28E-05 8 3.85 oxidoreductase activity, acting on peroxide as acceptor 7 9.46 3.29E-07 4.28E-05 8 3.85 peroxidase 5 6.76 4.70E-06 4.75E-04 8 3.85 oxidoreductase 12 16.22 1.20E-04 0.012 8 3.85 glutathione peroxidase activity 4 5.41 1.62E-04 0.021 9 3.58 Transcription 8 10.81 1.25E-05 7.51E-05 9 3.58 IPR013088:Zinc finger, NHR/GATA-type 4 5.41 3.43E-05 0.004 9 3.58 zinc finger region:GATA-type 4 5.41 1.11E-04 0.019 9 3.58 IPR000679:Zinc finger, GATA-type 4 5.41 3.89E-04 0.04 9 3.58 SM00401:ZnF_GATA 4 5.41 0.002 0.029 10 3.31 IPR007087:Zinc finger, C2H2-type 7 9.46 8.95E-05 0.009 10 3.31 IPR015880:Zinc finger, C2H2-like 7 9.46 1.12E-04 0.012 10 3.31 zinc-finger 12 16.22 1.53E-04 0.015 10 3.31 IPR013087:Zinc finger, C2H2-type/integrase, DNA-binding 5 6.76 4.35E-04 0.045 10 3.31 SM00355:ZnF_C2H2 7 9.46 0.002 0.031 11 2.96 sulfate assimilation 5 6.76 8.63E-06 0.004 13 2.41 response to reactive oxygen species 5 6.76 9.04E-05 0.036 13 2.41 Inorganic ion transport and metabolism 4 5.41 0.008 0.045 18 1.72 response to oxidative stress 13 17.57 1.55E-09 6.37E-07 21 1.48 sce00450:Selenoamino acid metabolism 5 6.76 2.31E-04 0.007



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