interacts with RNAPII and directly isomerizes in vivo and in vitro it’s CTD in response to rapamycin [22]. Many transcription elongation factors are known to interact with and modify the CTD, including Pin1, Fcp1 and the Bur kinases and the CTD is thought to be a recruitment platform for elongation, RNA processing and termination factors [28,29,30,31]. (ii) Here we have shown that overexpression of RRD1 suppresses the elongation defect of DST1 (TFIIS) deletion in an artificial elongation assay (ARTAR). Dst1 is crucial to transcribe this ARTAR as it is required to restart paused RNAPII [25,36]. Although we currently cannot explain how RRD1 increased dosage rescues the dst1D deficiency, we presume that this might be via its function during elongation. (iii) Rrd1D mutants displayed hypersensitivity against the agent 6-AU, a phenotype which is common for elongation factors although it was not as sensitive as dst1D mutants, possibly because Rrd1 affects elongation at only a subset of genes whereas Dst1 acts globally [36,40,46,47,48,49]. Finally, (iv) rrd1D mutants displayed an altered phosphorylation pattern for Ser5-P and Ser2-P on most genes (Fig. 5D and Suppl. Fig. S2). Phosphorylation of RNAPII changes throughout elongation and this pattern is altered in the rrd1D mutant. First of all, the phosphorylation pattern appears to be similar under normal growth conditions correlating with RNAPII levels. However, one observes a distinctive pattern of Ser5-P and Ser2- P in both up and downregulated genes in response to rapamycin. In the rrd1D mutant Ser5-P and Ser2-P are strongly enriched in the 39 region of the genes consistently throughout all up and down regulated genes. So, how can the same phenomenon account for the failure to up and down regulate gene expression? This may be explained by the fact that for upregulated genes Ser5-P and Ser2-P is much higher in the WT throughout the ORF but not at the 39 end of the gene compared to the rrd1D mutant. In this case, rrd1D fail to up regulate CTD phosphorylation and thus RNAPII levels do not increase. For down regulated genes rrd1D mutants retain the altered phosphorylation patterns at the 39 end of the genes thereby prohibiting an adequate downregulation of RNAPII. Thus, Rrd1 would be required to modulate the phosphorylation of RNAPII so that they remain flexible for up and down regulation. If Rrd1 affects the phosphorylation status of RNAPII one would expect to see changes in the global phosphorylation status of RNAPII, for example analyzed by Western blot on total cell extract. However, we previously monitored the total phosphory- lation status of RNAPII in response to rapamycin in the rrd1D mutant and could not find any significant differences [22]. This apparent discrepancy can be explained by the fact that the phosphorylation changes are very local (at the 39 end of genes) and that for example, for the upregulated genes there is less phosphorylation in the rrd1D mutant in the ORF but then retains a higher level of phosphorylation in the 39-end of downregulated genes. These subtle changes are therefore unlikely to be visible using the immunoblot approaches. We propose a model in which Rrd1 regulates elongation by modulating the level of Ser5-P and Ser2-P via isomerisation of the CTD of RNAPII. The isomerization of the CTD of RNAPII would allow the efficient up and downregulation of RNAPII on stress regulated genes. Our model has some precedent, as another peptidyl-prolyl isomerase, Ess1, has been shown to regulate Ser5-P of RNAPII at the end of snRNAs genes, thereby promoting transcription termination via the Nrd1 pathway [24]. In addition, over expression of Pin1 (the human homolog of Ess1) results in hyper phosphorylation of RNAPII and its release from the chromatin [27]. It is known that RNAPII occupancy is regulated during transcription elongation, for example, it was previously reported that RNAPII was enriched on ribosomal genes but Rrd1 Regulates Elongation of RNA Polymerase II associated with a slow transcriptional rate [50]. Interestingly, when these cells were transferred from glucose to galactose containing medium, the level of RNAPII decreased on these ribosomal genes and their transcriptional rate increased. Simultaneously, RNAPII was recruited to other genes including those involved in mitochondrial function [50]. Similar to a switch from glucose to galactose, rapamycin induces a transcriptional response which requires some genes to be turned off and others to be induced. Rrd1 might promote this transcriptional reorganization by allowing Ser5-P and Ser2-P changes thereby fine-tuning the elongation efficiency. Based on our model, we predicted that Rrd1 might play a similar role in other stress response situations, notably the environmental stress responses that induce a similar pattern of gene expression as rapamycin [35,51]. Indeed, rrd1D mutants are sensitive to agents that cause oxidative stress, which is known to induce a drastic transcriptional response (Fig. 6) [35,51]. Although these phenotypes may at first glance seem opposite of the one observed for rapamycin, they are actually consistent with our model of Rrd1 function: In both cases, the response to the stress condition is inhibited in rrd1D cells. This leads to resistance to rapamycin (because the environment remains nutrient-rich despite the presence of the drug), but sensitivity to oxidative stress (because the cells fail to arrest and adapt to high ROS conditions). In accordance with this, we show that Rrd1 is required to adequately induce gene expression on a subset of stress responsive genes upon various stress conditions (Fig. 7). Surprisingly, ribosomal genes were not strongly downregulated in wild-type cells as predicted from the ChIP-chip data. Since mRNA levels were measured at 30 min, long mRNA half-lives could obscure the drop in transcription that was apparent in the ChIP-chip data. Rrd1 was required for induction of stress-induced genes, however, consistent with our model. Interestingly, rrd1D cells showed stronger defects for some stresses than others. For example, HSP12 induction was dependent on Rrd1 after rapamycin and H2O2, but not heat shock and to a lesser extend with NaAs (Fig. 7). This might be due to effects specific to each condition, as not all of the genes we tested were induced in the same manner by each condition. For example, the NaAs exporter ACR3 was only expressed in response to NaAs, and this was dependent on Rrd1 despite the fact that expression was specific to one condition (Fig. 7). Taken together, we have shown that Rrd1 regulates the transcriptional stress response via two mechanisms, through regulation of PIC assembly, and more drastically through regulation of RNAPII elongation. The latter mechanism is likely to be via CTD isomerisation and alteration of the phosphorylation status of RNAPII, thereby regulating the elongation of RNAPII. This is a general stress response mechanism since rrd1D mutants display multiple phenotypes and are unable to adequately regulate gene expression in response to environmental changes. As such, we propose that Rrd1 is a novel transcription elongation factor required to modulate the expression in response to environmental stresses. This study goes along with many others as it further supports that elongation is also subject to transcriptional regulation. Finally, as Rrd1 is conserved throughout evolution, future work should examine whether this function is also remaining in higher eukaryotes as this could be a potential target for the TOR signalling pathway and cancer treatment. Materials and Methods Strains, cell growth and crosslinking conditions All strains used in this study were from the BY4741 background (Mat a, his3-1, leu2-0, met15-0, ura3-0). The RRD1 and SPT15 genes PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e23159