Molecular effects of cordycepin on RNA metabolism. (A) Cellular poly(A) length distribution following cordycepin treatment. Total RNA was extracted from wild-type strains following growth in the presence of 20 μg/mL cordycepin for the indicated times. RNAs were labeled with [α-³²P]cordycepin and poly(A) polymerase, and digested with RNases A and T1. RNase-resistant poly(A) tails were resolved on denaturing 15% polyacrylamide/8.3 M urea gels and visualized by PhosphorImaging (Fuji FLA7000). (B) Northern analysis of RNA obtained from a wild-type strain treated with cordycepin as described in A. RNAs were detected with random prime labeled probes directed against the open reading frames (ORF) of the indicated genes; (left panel) the probes against CYH2 were either directed against the entire ORF or (right panel) exclusively against its intron. (Asterisks) 3′-Extended transcripts. 18S rRNA and U3 snoRNA were detected with end-labeled oligonucleotides. (C) High-resolution Northern analysis of ACT1 and ASC1 3′ ends. Total RNA from a wild-type strain treated with cordycepin as described in A was treated with RNase H in the presence of oligonucleotides complementary to the 3′ end of the ORF of the (left panel) ACT1 or (right panel) ASC1 genes, respectively. Below each gel a scheme indicates the relative location of the oligonucleotide used to target RNAse H and the distribution of observed 3′ ends (I to V for ACT1; and I to IV for ASC1).

Increased ATP levels in pap1 mutants. (A) Tenfold serial dilutions of wild-type and pap1 mutant strains were spotted on SD medium containing 20 μg/mL cordycepin that was supplemented with 20 μg/mL adenine as indicated. The yeast were grown for 3 d at 25°C and photographed. (B) Wild-type and pap1 mutants strains were grown at 25°C or shifted for 1 h to 33°C as indicated and assayed for ATP content. Represented in the graph are average values for ATP content obtained from three independent yeast cultures; the error bars display standard error.

Cordycepin-responsive mutants are linked to poly(A) metabolism. (A) Mutations causing cordycepin sensitivity genetically interact with rna14-1. The temperature-sensitive rna14-1 strain, carrying a URA3-marked plasmid containing the wild-type RNA14 gene, was used for disruption of genes causing cordycepin sensitivity. Double mutants were challenged with 5-flouroorotic acid (5-FOA) to force loss of the URA3-marked plasmid exhibiting the phenotype of the double mutation. Shown are serial dilutions of indicated single and double mutant strains on synthetic (SD) medium lacking or containing 5-FOA. Growth phenotypes were categorized as synthetic lethal (no growth), synthetic sick (reduced growth), or normal (no phenotype) when compared to the rna14-1 mutant alone. (B) Cellular poly(A) length distribution in cordycepin-responsive strains. Total RNA was obtained from indicated mutant strains and analyzed as described in the legend of Figure 2A, except that poly(A) tails were resolved on 10% sequencing gels.

Cordycepin-responsive mutant yeast strains involved in RNA synthesis and turn-over. (A–C) The indicated yeast strains were grown overnight in synthetic complete (SD) medium at the appropriate temperature. Tenfold serial dilutions were produced and spotted on media lacking or containing cordycepin and grown for 3–5 d. Unless indicated otherwise, the cordycepin concentration used was 20 μg/mL.

The pap1-1 mutation neutralizes the effects of cordycepin. Gene expression profiling was done with wild-type and pap1-1 strains following a shift for 1 h to medium lacking or containing 20 μg/mL cordycepin, respectively. All experiments were performed in triplicate. (A) Heat map of gene expression changes of cordycepin treated wild-type and nontreated and treated pap1-1 mutant strains relative to the untreated wild-type control. Rows represent genes, and columns represent average values obtained from three independent measurements for the indicated condition; only those 3920 features were represented that gave three values each for all three conditions. Clustering was done with Spearman Row correlation (substance and row) using Acuity 4.0. The color code indicates the distribution of log₂ ratios. (B) Scatterplot of gene expression changes (log₂ ratios). (Upper graph) Plots signals obtained with the pap1-1 strain in the absence (y-axis) and the presence (x-axis) of cordycepin. (Lower graph) Signals obtained with the pap1-1 strain in the absence of cordycepin (y-axis) are plotted against signals obtained with cordycepin-treated wild-type (x-axis). (C) The table displays the numbers of RNA transcripts that changed at least twofold (log₂ value ±1) relative to untreated wild type in the respective conditions. The numbers of transcripts that were derived from dubious open reading frames are indicated in percent in brackets. (D) Transcripts that were increased under the indicated conditions were subjected to Gene Ontology analysis using the SGD slim mapper (http://www.yeastgenome.org/). The number of genes in the individual compartments is presented in percent of all analyzed genes. Also indicated is the background distribution of all genes. (E) Northern blot analysis of total RNA obtained from wild-type and mutant strains following growth in the absence and presence of 20 μg/mL cordycepin. The CUT NEL025c was detected using a random primed labeled probe. (Asterisk) Indicates a transcript that accumulates due to Pap1 dependent 3′ end formation at a downstream gene (Wyers et al. 2005).

Identification of cordycepin-responsive haploid deletion mutants. (A) The growth phenotypes of strains identified by drop-test screening were analyzed on medium containing or lacking 40 μg/mL cordycepin as indicated. Strains were grown for 3 d at 30°C and photographed. (B) Pools of all viable haploid deletion mutants from the systematic deletion library were grown in complete liquid medium with or without 20 μg/mL cordycepin for 12 or 18 generations (Experiments 1 and 2, respectively) by serial dilutions. Cells were then collected, the genomic DNA was extracted, and the barcode sequence tags associated with each gene deletion were amplified by PCR and hybridized on barcode DNA arrays as described (Decourty et al. 2008). The figure plots for each experiment (Experiment 1 on the x-axis and Experiment 2 on the y-axis) the log₂ of the ratio for each deletion mutant of the barcode signal between the cultures without and with cordycepin (computed as in Decourty et al. 2008). Negative log₂ (ratios) indicate strains more sensitive to cordycepin than the average population of mutants. Mutants in PEX genes, SWR1 complex genes, and SKI 2, 3, and 8 genes are color-coded as indicated. Arrows and labels point to mutants with strong phenotypes. (C) Cordycepin-sensitive growth phenotypes of pex mutants. The indicated strains were analyzed as described in A. (D) Graph depicting the overlap of mutants identified in drop-test screening versus functional profiling (after 12 generations). Also indicated are the numbers of multi-drug-resistance genes that were identified with the individual procedures or which are shared, respectively. In brackets the number of strains are indicated that are identified in the drop-test screening that could not be analyzed by functional profiling due to slow growth of the mutants.