Rrd1 Regulates Elongation of RNA Polymerase II Figure 5. rrd1D mutants exhibit phenotypes consistent with a defect in transcriptional elongation. (A) Transcription elongation assay measuring the b-galactosidase activity of the lacZ gene containing the artificial arrest site expressed from a plasmid transformed into the indicated strains. The dst1D mutant strain was co-transformed with a plasmid expressing RRD1 (pRRD1). The measurements are expressed as Miller units and are displayed as the average of at least 5 independent assays with the corresponding error bars. (B) Spot test analysis of WT, rrd1D and dst1D mutant strains on agar containing selective media lacking uracil (URA) and containing 6-azauracil (6AU) with the indicated concentration. (C) Linear correlations of the mean enrichment of Ser5 phosphorylated form of RNAPII (Ser5-P) at all promoters and Ser2 phosphorylated form of RNAPII (Ser2- P) of all ORFs, between WT and rrd1D mutant without (2 RAP) and with (+ RAP) rapamycin treatment. (D) Mapping analysis of Ser5-P and Ser2-P forms of RNAPII for up and downregulated genes in response to rapamycin. WT cells (green line) and rrd1D mutant cells (red line) under normal growth conditions (full line) and rapamycin treated conditions (dotted line). doi:10.1371/journal.pone.0023159.g005 environmental stresses. Previously work has shown that rrd1D mutants exhibit multiple phenotypes including resistance to caffeine, but sensitivity towards vanadate, 4-NQO and calcium [13,37]. Both vanadate and 4-NQO are known to cause oxidative Figure 6. rrd1D mutants display hypersensitivity to agents causing oxidative stress. (A) Survival curve of WT (open circle) and rrd1D mutant strains (closed circle) upon H2O2 treatment. H2O2 concentrations are indicated below; result shown is an average of three independent experiments and error bars indicate the standard deviation. (B) Spot test analysis of WT and rrd1D mutant strain on YPD agar plates, containing 1 mM sodium arsenite (NaAs), where indicated. (C) LacZ reporter expression analysis from the ACR3 promoter-lacZ fusion plasmid, expressed in Miller units. WT (open circle) and rrd1D mutant (closed circle) show the lacZ expression over time in response to 1 mM NaAs treatment (n = 5). doi:10.1371/journal.pone.0023159.g006 stress [37,38]. To test whether Rrd1 is required for resistance to other oxidizing agents, we challenged cells with hydrogen peroxide (H2O2) and sodium arsenite (NaAs) and found that the rrd1D mutant was indeed sensitive to these agents (Fig. 6A and B). To determine whether the sensitivity was the result of a defect in gene regulation, we introduced a known arsenite-response reporter that bears the promoter of the ACR3 gene fused to lacZ [39]. ACR3 encodes a plasma membrane efflux pump that is upregulated via the Yap8 transcriptional activator in response to arsenite [39]. While there was a strong induction of the ACR3-lacZ reporter in the wild-type, it was hardly induced in the rrd1D mutant (Fig. 6C). This data suggests that the transcriptional response to oxidative stress is also affected in the rrd1D mutant. To explore this further, we monitored expression of 10 stress responsive genes using the GeXP multiplex PCR system in response to rapamycin, H2O2, NaAs and heat shock (see Materials and Methods). We chose genes that are known to be upregulated (PRX1, ARR3, HSP12, HXK1 and TSL1) or downregulated (RPL3, RPL32, and RPS2) in response to environmental stresses as well as control genes which are not significantly altered under these conditions (ACT1, GAL1) [35]. The Kanamycin resistance marker was added to the samples and used to normalize the data (see materials and methods). First, the untreated and rapamycin- treated expression data was compared to the RNAPII median enrichment on these ORFs (see Suppl. Fig. S3). This analysis revealed that for both conditions RNAPII occupancy correlated with mRNA expression for all of the genes except the ribosomal genes (see Suppl. Fig. S3). This can be explained by the mRNA half life of the downregulated genes: Although RNAPII is depleted from these genes, mRNA is still present. We next compared gene expression in wild-type and rrd1D yeast for each condition (Fig. 7). Genes that are known to be induced were indeed upregulated in wild-type, but this was inhibited in the rrd1D mutant (Fig. 7A). Genes that are known to be downregulated upon stress were unaffected by RRD1 deletion (Fig. 7B). It might be possible that for these genes other regulatory mechanisms are active such as the above mentioned mRNA half life (see discussion). This is consistent with the observation that RNAPII was strongly depleted from these genes in response to rapamycin, but mRNA levels remained unchanged (Suppl. Fig. S3). Finally, the control genes ACT1 and GAL1 remained similar between wild-type and rrd1D throughout the various conditions (Fig. 7C). Taken together, the expression analysis and the multiple phenotypes of rrd1D mutants towards environmental stresses suggest that the role of Rrd1 is not limited to rapamycin, but instead that this peptidyl-prolyl isomerase plays a general role in transcriptional stress responses. Discussion We have previously shown that Rrd1 is associated to chromatin and interacts with RNAPII [22]. Consistently with this, we now demonstrate that Rrd1 directly co-localizes with RNAPII on actively transcribed genes independently of the massive transcrip- tional changes that are induced by rapamycin. Interestingly, our PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23159