<i>URA3</i> and <i>APE2</i> mRNA expression levels are not significantly altered in heterozygous <i>ESS1</i> mutants and controls.

<p>qRT-PCR showing a quantitative measurement of <i>URA3</i> and <i>APE2</i> mRNA expression in the indicated strains. (<b>A</b>, <b>C</b>) Cells were grown at 30°C in complete synthetic medium (CSM), CSM minus uracil, or CSM minus histidine and leucine as appropriate for each strain. (<b>B</b>, <b>D</b>) Cells were grown at 37°C in serum-containing medium (10% FBS in YPD). Strains are as described in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-g003" target="_blank">Figure 3</a>.</p

RNA-sequencing indicates transcription readthrough at SnoRNA loci.

<p>Results are visualized using Integrated Genome Viewer. The total number of RNA-sequencing reads (y-axis) and their position along the chromosome (x-axis) is indicated in grey. Forward oriented reads are indicated in red; reverse oriented reads are indicated in blue. Not all reads (blue, red) are visible. Results for the CaEss1 <i>ts</i>-mutant strain are in the upper panels, and results for the isogenic control are in the lower panels (<b>A</b>–<b>C</b>). Solid bars indicate the previously annotated gene positions, while the dotted (grey) boxes indicate the positions implied based on actual transcript data from RNA-sequencing of the the wild-type strain (<i>ESS1</i>/<i>ESS1</i>) (not shown) and the control strain (lower panels). A putative CUT is indicated in panel (B). Approximate positions of primers used for strand-specific cDNA synthesis are shown (black), as are the positions of the primer sets (green) used for qRT-PCR in (D). The positions of likely readthrough transcription are identified by red dashed arrows. (<b>D</b>) Results of qRT-PCR to detect readthrough transcripts for different snoRNA genes (x-axis), expressed as a fold-change (y-axis) of <i>ts</i>-mutant (CaDS-C) over isogenic control (CaDS-FC), normalized to <i>ACT1</i>. For RNA-sequencing, cell growth conditions are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-t002" target="_blank">Table 2</a> (shift to 42°C, then serum induction at 42°C), for qRT-PCR, samples were serum-induced at 37°C.</p

Changes in gene expression based on RNA-sequencing results.

<p>The log₂ fold changes are based on results from the Cuffdiff program in the Galaxy server. Cuffdiff was used to select significant gene expression changes depending on whether the <i>p</i> value was greater than the allowed false discovery rate after Benjamini-Hochberg correction for multiple-testing. The highest and lowest log₂ fold changes of the genes considered to be significantly differentially expressed in each group are documented it in the table.</p

Primers used in this study.

<p>Lower case sequences added to obtain appropriate length. Bold indicates a mutation or restriction site.</p

Structure-function analysis of CaEss1.

<p>(<b>A</b>) X-ray crystallographic structure of the human homolog, Pin1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Ranganathan1" target="_blank">[40]</a> and CaEss1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Li1" target="_blank">[50]</a>. Substitutions made in the structured linker α-helix of CaEss1 to construct the helix mutant (<i>hm</i>) strains are indicated. (<b>B</b>) Complementation of CaEss1 linker mutants in <i>S. cerevisiae</i>. Plasmids encoding the indicated mutant proteins (independent clone isolates of linker-swapped plasmid, pDS426(sw) and helix substitution plasmid, pDS426(pm)) were separately transformed into a <i>ts</i>-mutant strain of <i>S. cerevisiae</i> (<i>Scess1^H164R</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Wu1" target="_blank">[42]</a>. Plasmids were constructed using a pRS426 backbone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Sikorski1" target="_blank">[88]</a>. Serial dilution assays (1∶5) shows the growth of these independent transformants (a, b). Growth at 37°C, the restrictive temperature for the <i>S. cerevisiae ess1</i> mutant, indicates complementing activity. The CaEss1 helix substitution mutant complements but the linker swap mutant does not. pGDCaEss1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Devasahayam1" target="_blank">[52]</a> (pRS426 backbone) was used as a positive control and pRS426 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Sikorski1" target="_blank">[88]</a> was used as an empty vector control. (<b>C</b>) Western analysis of whole cell extracts of <i>S. cerevisiae</i> expressing the indicated CaEss1 proteins. A total of 15 µg of protein was used per lane and the blot was probed using anti-CaEss1 polyclonal antibody at a 1∶500 dilution. For the mutant strains, two independent clone isolates of pDS413(sw) (lanes 3 and 4) and pDS413(pm) (lanes 5 and 6) were transformed into <i>S. cerevisae</i> and analyzed. Plasmids were constructed using a pRS413 backbone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Sikorski1" target="_blank">[88]</a>. The <i>S. cerevisiae</i> strain used (CBW22; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Wilcox1" target="_blank">[44]</a> does not express endogenous Ess1 protein but is viable due to a suppressor mutation (<i>ess1</i>Δ<i>srb10</i>Δ). The linker-swapped protein appears to be absent, or present at a very low level compared to the vector control, while the helix mutant protein is easily detected. pGD-CaESS1 (pRS413 backbone) encoding the wild-type protein (Devasahayam and Hanes, unpublished) was used as a positive control, and pRS413 was used as the empty vector control.</p

<i>CaESS1</i> is expressed at reduced levels in heterozygous mutants as expected.

<p>(<b>A</b>) Quantitative reverse transcription PCR (qRT-PCR) shows <i>CaESS1</i> mRNA expression levels in the indicated strains. (<b>B</b>) Western blot analysis showing the expression of CaEss1 protein in the indicated strains. A total of 3 µg of protein was used per lane. The blot was probed using anti-CaEss1 polyclonal antibody at a 1:500 dilution. CaEss1 is ∼19 kD. Strains correspond to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-g002" target="_blank">2:</a> (<i>ESS1</i>/<i>ESS1</i>) <i>URA3</i> at <i>CaESS1</i> is R6; (<i>ess1</i>Δ/<i>ESS1</i>) <i>URA3</i> at <i>CaESS1</i> is CaGD1; (<i>ESS1</i>/<i>ESS1</i>) <i>HIS1</i>/<i>LEU2</i> is CaDS-B5; (<i>ess1</i>Δ/<i>ESS1</i>) <i>HIS1</i>/<i>LEU2</i> is CaDS-B5.5.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Schematic representation of the strains used in this study.

<p>Strains <b>A</b>–<b>H</b> are made in the CAI4(*) background using <i>URA3</i> as a selectable marker. Strains <b>I</b>–<b>O</b> are made in SN87 background with <i>HIS1</i> and <i>LEU2</i> selectable markers. The <i>CaESS1</i> coding region and promoter region (pCaEss1) are shown in green; the <i>APE2</i> gene is shown in purple. <i>APE2</i> gene has two exons and the wavy line represents the intronic region. <i>CaESS1</i> and <i>APE2</i> are 210 bp apart in the genome (grey). The <i>URA3</i> gene (blue) with the two flanking <i>hisG</i> direct repeats from <i>S. typhimurium</i> (yellow) is part of the construct used in the <i>URA</i>-Blaster method to target genes and later recycle <i>URA3</i>. <i>RPS1</i> is shown in brown, <i>LEU2</i> in red, <i>HIS1</i> in pink and (p) indicates promoter sequence. The sequence upstream of <i>APE2</i> that is repeated is shown in a gray-white gradient. The CAI4(*) indicates that this strain appears to have acquired a mutation that affects filamentation as per this study. Figure is not drawn to scale.</p

Filamentation of strains used in the study.

<p>Filamentation was tested on solid medium at 37°C. (<b>Upper panel</b>) Serum medium (4% FBS in 2% agar), (<b>Lower panel</b>) Spider Medium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Liu1" target="_blank">[80]</a>. 2 µl of the 0.25 OD₆₀₀ of fresh overnight cultures were spotted and grown for 4 days at 37°C before documentation. Small letters (A–O) within each panel refer to the constructs shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-g001" target="_blank">Figure 1</a>.</p

<i>CaESS1</i> is essential for the growth of <i>C. albicans</i>.

<p>(<b>A</b>) Western analysis of <i>C. albicans</i> whole cell extracts to detect expression of wild-type CaEss1 (TAA) and a larger protein product encoded by the <i>Caess1^TGC</i> “readthrough” allele (TGC). The level of read-through protein is reduced significantly at 60 min and is nearly absent after 2 hrs at non-permissive temperature (42°C). A total of 7.5 µg of protein was used per lane. The blot was probed using anti-CaEss1 polyclonal antibody at a 1∶500 dilution. (<b>B</b>) Serial dilution (1∶3) of cells of the indicated genotype grown on solid medium (YPD) at different temperatures. The readthrough strain, <i>Caess1</i>Δ/<i>Caess1^TGC</i> shows a clear temperature-sensitive phenotype at 42°C, but no growth defect at 37°C. (<b>C</b>) Filamentation on the indicated solid medium (4 days) (upper two rows), and germ tube formation in liquid Spider medium (2 hrs) (lower row). Upper two rows are reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-g002" target="_blank"><b>Figure 2</b></a> for comparison. In (B) and (C), CaDS-B5 (<i>CaESS1</i>/<i>CaESS1</i>) is used as the wild-type, and is an isogenic control for both the TAA and TGC strains.</p