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Mutations in several genes encoding components of the RNA polymerase II elongation machinery render S. cerevisiae cells sensitive to the drug 6-azauracil (6AU), an inhibitor of IMP dehydrogenase and orotidylate decarboxylase. It is thought that a reduction in nucleotide levels following drug treatment causes transcriptional elongation to be more dependent on a fully functional RNA polymerase. To gain insight into the basis of the 6AU-sensitive phenotype and discern its specificity, we screened almost 3000 deletion mutants for growth in the presence of drug; 42 (1.5%) were reproducibly sensitive to the drug. The sensitive mutants included several missing known transcription elongation factors, but the majority were in genes involved in other cellular processes. Not all of the 6AU-sensitive strains displayed cross-sensitivity to mycophenolic acid (MPA), another drug that inhibits IMP dehydrogenase and has been employed as a screening agent for elongation mutants, showing that these two drugs are mechanistically distinct. Several of the mutants were tested for the ability to induce transcription of IMP dehydrogenase-encoding genes, in response to 6-AU and MPA treatment. As expected, mutants defective in transcriptional elongation factors were unable to fully induce IMPDH expression. However, most of the 6AU-sensitive strains had normal levels of IMPDH expression. Thus, although 6AU-sensitivity often results from defects in the elongation machinery, mutations that compromise processes other than transcription and induction of IMPDH also lead to sensitivity to this drug.
S. cerevisiae strains bearing mutations in genes encoding RNA polymerase II subunits and its accessory elongation factors are often sensitive to the drugs 6-azauracil (6AU) and mycophenolic acid (MPA). Indeed, sensitivity to 6AU was the first phenotype identified for cells with defective RNA polymerase II elongation factors, and it has been widely considered to be diagnostic for proteins involved in transcriptional elongation (Hubert et al., 1983; Hampsey, 1997). The basis for this sensitivity appears to be the drug’s inhibition of IMP dehydrogenase, leading to a reduction in ribonucleotide levels and a concomitant increased dependence upon fully functional transcriptional elongation factors for efficient transcription (Archambault et al., 1992; reviewed in Wind and Reines, 2000). The gene encoding IMP dehydrogenase, IMD2, is transcriptionally induced in response to nucleotide depletion by these inhibitors and this response is dependent upon an optimally functioning elongation machinery (Shaw and Reines, 2000; Shaw et al., 2001; Escobar-Henriques and Daignan-Fornier, 2001).
The availability of a nearly complete set of gene deletion mutants of yeast (Giaever et al., 2002) enables large-scale screening of null mutants for a variety of phenotypes. Screening the yeast ‘disruptome’ has proved to be a high-throughput functional genomics tool to identify genes involved in Ty element function (Griffith et al., 2003) and responsiveness to the drugs wortmannin, rapamycin, and MPA (Chan et al., 2000; Desmoucelles et al., 2002; Zewail et al., 2003), as well as other phenotypes. Although mutations in genes encoding components of the transcriptional elongation machinery render cells sensitive to 6AU, it is clear that there is more than one mechanism to achieve this sensitivity, e.g. by mutation of a regulator of pyrimidine biosynthesis (Hubert et al., 1983; Shaw and Reines, 2000). Using the deletion collection, we set out to determine the spectrum of genes whose deletion alters growth rates in the presence of 6AU. This study complements a similar recent analysis on MPA, a biochemically distinct inhibitor of IMP dehydrogenase.
The homozygous diploid yeast gene knockout (YKO) mutants were transformed to Ura+ with pBM2636 (1.2 kb HindIII–EcoRI fragment of the HXT1 promoter fused to lacZ on the URA3-containing multicopy plasmid YEp357R; Ozcan and Johnston, 1995). Transformants were replicated to YM agar plates lacking uracil [1.7 g/l Yeast Nitrogen Base (Difco), 5 g/l ammonium sulphate, 20 g/l dextrose, 1.0 g/l drop-out mix without uracil (US Biological)] and containing either 200 μg/ml 6AU or 100 μg/ml MPA. Mutants that appeared more or less sensitive than wild-type were re-tested by plating several dilutions of the cells on the same plates. The most sensitive strains (scored ‘3’) showed little or no growth after 3 days on medium containing 6AU or 2 days for MPA; moderately sensitive strains (scored ‘2’) exhibited limited growth after 3 and 2 days for 6AU and MPA, respectively; we are not confident of results scored ‘1’.
For Northern analysis, strains were grown in YPD to an optical density at 600 nm of ≈0.5. MPA (15 μg/ml w/v; Sigma, St. Louis, MO) or 6AU (100 μg/ml w/v; Sigma, St. Louis, MO) were added and cells were collected and frozen after 2 h at 30 °C. Total RNA was isolated from thawed cell pellets by phenol extraction and quantitated by measuring absorbance at 260 nm. RNA (15 μg) was resolved on a 1% formaldehyde/agarose gel and blotted onto Zeta-probe GT nylon membrane (Bio-Rad; Hercules CA). Filters were baked at 80 °C for 2 h, pre-hybridized for a minimum of 3 h at 42 °C in 5× SSC (1 × SSC = 0.15 M NaCl, 0.015 sodium citrate), 5× Denhardt’s solution, 50% v/v formamide, 1% w/v SDS, and 100 μg/ml salmon sperm DNA. Filters were hybridized under the same conditions with ≈108cpm [32P]-labelled DNA probe for 15–18 h. Filters were washed twice at 22 °C in 2% SSC/0.1% SDS, for 5 min each, twice in 0.2% SSC/0.1% SDS for 5 min each, twice in 0.2% SSC/0.1% SDS at 42 °C for 20 min each, exposed to XOMAT film. IMD2 DNA probe (which cross-hybridizes to IMD3; Hyle et al., 2003) was prepared using the polymerase chain reaction with a wild-type yeast genomic DNA template (S288C; Research Genetics, Huntsville, AL) and primers complementary to the gene; 5′-GTGGTATGTTGGCCGGTACTACCG-3′ and 5′-TCAGTTATGTAAACGCTTTTCGTA-3′. Probes were labelled to a specific activity of approximately 107 –108 cpm/μg with Klenow DNA polymerase (Promega Life Sciences, Madison WI), random hexamer primers (Gibco BRL, Rockville MD), and [α-32P]dATP (Amersham Pharmacia Biotech, Piscataway, NJ).
We screened 2804 mutants from the YKO Collection (Giaever et al., 2002) for their sensitivity to 6AU. This screen required that YKO strains be transformed to Ura+, since uracil in the medium competes with drug and abrogates its effect (list of mutants screened available at http://www.genetics.wustl.edu/6AZAURACIL). About 200 candidates from the first screen were re-tested several times for growth on agar plates containing the drug, yielding 44 mutants that were reproducibly more sensitive or resistant than wild-type cells (Table 1, Figure 1). Each of these mutants was also tested for its ability to induce expression of IMPDH in response to 6AU and MPA (Figure 2). An example of a time course of induction for five sensitive strains compared to an isogenic wild-type control is shown in Figure 3.
As expected, deletion of several genes, including DST1, POP2 (also called CAF1 ) and RTF1, which have been implicated in transcriptional elongation (Archambault et al., 1992; Lennon et al., 1998; Denis et al., 2001; Costa and Arndt, 2000; Squazzo et al., 2002), were identified in the screen (Table 1, lines 1–7). The observed sensitivity of strains deleted for genes known to be involved in transcriptional elongation validates this screen. As in a previously reported screen of the YKO collection for MPA-sensitive strains, we found that the most sensitive mutants are those missing genes involved in transcription (Desmoucelles et al., 2002). A survey of IMPDH induction in the 6AU-sensitive strains confirmed prior work showing that induction was compromised when DST1, the gene encoding elongation factor SII (also called TFIIS) was deleted (Figure 2). Deletion of an ORF (YGL042C) considered ‘dubious’ that overlaps DST1 also conferred 6AU-sensitivity and rendered IMPDH uninducible (Table 1, line 44).
Another gene necessary for resistance to 6AU identified by this screen was SDT1 (Table 1, line 27, and Figure 1), the deletion of which is known to render cells 6AU-sensitive (Shimoaraiso et al., 2000). SDT1 was initially identified as a multi-copy suppressor of the 6AU-sensitivity of a dst1 Δ strain, and encodes a nucleotidase that detoxifies pyrimidine nucleotide derivatives (Nakanishi and Sekimizu, 2002). It was also of interest that deletion of NOT3, which encodes subunits of the Ccr4–Not complex implicated in transcriptional elongation (Denis et al., 2001), causes resistance to both drugs (Table 1, line 8). A new 6AU-sensitive mutant identified here is gal11 Δ (also called SPT13; Table 1, line 6). Gal11p is a component of the RNA polymerase II holoenzyme that stimulates the TFIIH protein kinase and is implicated in the transition from transcriptional initiation to elongation (Kim et al., 1994; Sakurai and Fukasawa, 1998; Badi and Barberis, 2001). Other members of the SPT gene family also have a demonstrated role in elongation (Yamaguchi et al. 2001). Another new 6AU-sensitive mutant found here was thp1 Δ (Table 1, line 7). The phenotype is not unexpected, since THP1 has been linked to transcription elongation and mRNA transport (Gallardo and Aguilera, 2001; Gallardo et al., 2003; Fischer et al., 2002).
Importantly, and perhaps surprisingly, a majority of the mutants are defective in processes seemingly unrelated to transcription. These include genes involved in fatty acid metabolism (Table 1, lines 9–12), amino acid transport (lines 13–15), protein ubiquitinylation (lines 16–18), stress and cell cycle progression (lines 19–20), protein trafficking (lines 21–22), RNA processing (lines 23–25) and individual mutants defective in several diverse processes. Since most of these mutants exhibit near-normal levels of IMPDH expression in the presence of the drugs, we conclude that there are multiple mechanisms of attaining drug sensitivity, only one of which is a transcriptional elongation defect. We can imagine plausible explanations for the drug sensitivity of some of these mutants. For example, tfp3 Δ is defective in vacuole function, which might affect the strain’s ability to sequester the drugs. At high concentrations, 6AU has been reported to poison amino acid metabolism (Tamaki et al., 1989). The mutations in amino acid transport and synthesis we found (Table 1, lines 13–15, 26) may exacerbate the 6AU-induced accumulation of a toxic intermediate. For many of the strains, however, it is difficult to understand the mechanistic basis for the phenotype, e.g. how do perturbations in protein degradation or trafficking lead to drug sensitivity (lines 17, 18, 21, 22)? It remains possible that some of these genes may eventually be linked to transcriptional elongation. In any case, it seems clear that sensitivity to 6AU or MPA is not a reliable diagnostic criterion for defects in transcriptional elongation, and that caution should be exercised in interpreting the 6AU-sensitive phenotype.
This report is useful in light of the recent analysis of a comprehensive identification of genes required for MPA resistance from the YKO collection (Desmoucelles et al., 2002). We found that 1.5% of the mutants we screened were sensitive to 6AU, a value similar to the fraction of mutants sensitive to MPA (2%) (Desmoucelles et al., 2002). While much is known about the mechanism of uncompetitive inhibition of IMP dehydrogenase by MPA, including a co-crystal of enzyme and inhibitor, the metabolism and mechanism of action of 6AU is less well understood (Exinger and Lacroute, 1992; Sintchak et al., 1996; Hedstrom, 1999). Nevertheless, it has become a popular test for yeast mutants because of the extensive characterization of 6AU-sensitive strains with well-demonstrated transcriptional elongation defects. Early work suggested that the sensitivity to either MPA or 6AU could serve as a biomarker for transcriptional elongation factor mutants (Exinger and Lacroute, 1992). Indeed, both we and Desmoucelles et al. (2002) observe a large degree of cross-sensitivity to 6AU and MPA and both screens independently identified several genes encoding proteins known or likely to be involved in transcriptional elongation [RTF1, POP2 (CAF1 ), SPT3, and DST1 ]. On the other hand, both studies show that not all mutants are equally affected by 6AU and MPA, e.g. this work and that of Desmoucelles et al. (2002) show that the mot3 mutant is relatively more sensitive to 6AU than MPA. We observe a similar phenotype for sdt1 Δ, which was previously shown to be 6AU-sensitive (Nakanishi and Sekimizu, 2002). SDT1 encodes a nucleotidase which was identified as a suppressor of the 6AU-sensitivity of dst1 Δ and is thought to act by hydrolysing toxic nucleotide derivatives such as 6AU (Nakanishi and Sekimizu, 2002). Since it would not be expected to affect the metabolism of MPA, which is not a nucleotide, and the sdt1Δ strain is not impaired in IMPDH induction, it is easy to see why the strain is not MPA-sensitive and was not detected in the screen of Desmoucelles et al. (2002). This is a good example of a non-transcription-related pathway that, when mutated, confers 6AU-sensitivity.
This work was supported by NIH Grant GM46331 (to D.R.) and by funds awarded to M.J. by the James S. McDonnell Foundation. We thank our colleague Ali Shilatifard for suggestions, advice, encouragement and enthusiasm.