Tpk2 and Tpk3 affect the translational response to severe heat stress.

<p>(A) mRNA expression level was determined by q-RT-PCR on samples before and after severe heat stress. The value represents mean +/- SEM, n = 2. <i>ACT1</i> mRNA was used as a control. (B) The RNA collected from sucrose gradient fractions was pooled into monosome (M) and polysome (P) fractions. The mRNA distribution was analyzed by qRT-PCR and quantified relative to a luciferase mRNA control. Translational activity change was calculated as described in Materials and Methods. The values represent mean from two independent samples. The value represents mean +/- SEM, n = 2.</p

PKA catalytic subunits differentially affect the SGs and PBs aggregation in response to severe heat stress.

<p>(A) Wild type (WT), <i>tpk1</i>Δ, <i>tpk2</i>Δ and <i>tpk3</i>Δ expressing Pbp1-GFP, Edc3-RFP, Dcp2-RFP, Pab1-GFP or Rpg1-RFP were grown to exponential phase in YPD (30°C) and incubated at 46°C during 10 minutes. PBs and SGs aggregation were analyzed by fluorescence microscopy. The arrows show granular localization. The graph shows the amount of granules/100 cells. Bars represent the mean ± SEM, n = 3. * <i>p</i> < 0.05, 30°C <i>versus</i> 46°C; # <i>p</i> < 0.05, <i>tpk2</i>Δ 46°C or <i>tpk3</i>Δ 46°C <i>versus</i> WT 46°C; & <i>p</i> < 0.05, Edc3 <i>tpk2</i>Δ <i>versus</i> Edc3 WT 30°C; ^ <i>p</i> < 0.05, Pbp1, Rpg1, Dcp2 <i>tpk3</i>Δ <i>versus</i> Pbp1, Rpg1, Dcp2 WT 30°C (ANOVA Bonferroni post-test). <i>tpk3</i>Δ Rpg1-RFP panel results from a montage of images. (B) WT and <i>tpk3</i>Δ mutant cells expressing Tpk2-GFP and eIF4E-RFP were incubated at 46°C for 10 minutes. Tpk2-GFP and eIF4E-RFP co-localization was analyzed by confocal microscopy. (C) Tpk2-GFP enrichment in granular fractions was analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185416#pone.0185416.g002" target="_blank">Fig 2</a>. Representative blots are shown. The graph shows the ratio P/S of the abundance of each protein determined by densitometric quantification of the bands. Values are mean ± SEM, n = 2.</p

Characterization of kinase dead <i>tpk2</i> and <i>tpk3</i> granule localization evoked by severe heat stress.

<p>Cells co-expressing Tpk2-GFP, <i>tpk2</i>^<i>dead</i>-GFP, Tpk3-GFP or <i>tpk3</i>^<i>dead</i>-GFP and Dcp2-RFP were subjected to severe heat stress as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185416#pone.0185416.g002" target="_blank">Fig 2A</a>. Co-localization was determined by confocal microscopy. Arrows indicate Tpk-GFP granular localization and merge.</p

Characterization of Tpk2 and Tpk3 granules evoked during mild and severe thermal stress.

<p>(A) Cells co-expressing Tpk2-GFP or Tpk3-GFP and Rpg1-RFP or eIF4E-RFP and Tpk3-GFP and Dcp2-RFP were grown to exponential phase (30°C) and then incubated at 46°C for 10 minutes or 37°C for 30 minutes. Co-localization was determined by confocal microscopy. Arrows indicate Tpk-GFP granular localization and merge. Lower graphs show the quantitation of Tpks, Dcp2, Rpg1, eIF4E and merge granules/100 cells under each thermal stress condition. Values are mean ± SEM, n = 3. * <i>p</i> < 0.05 Tpk3-GFP <i>versus</i> Tpk3-GFP/Dcp2-RFP merge; Tpk3-GFP <i>versus</i> Tpk3-GFP/Rpg1-RFP merge; Tpk3-GFP <i>versus</i> Tpk3-GFP/eIF4E-RFP merge at 37°C (ANOVA-Tukey HSD test). (B) Effect of cycloheximide on the Tpk2 and Tpk3 assembly on heat stress evoked SGs. Cells co-expressing Tpk2-GFP or Tpk3-GFP and eIF4E-RFP were pre incubated or not with cycloheximide 100 μg/ml for 10 minutes before heat stress (CHX). Tpk2-GFP and Tpk3-GFP granule formation was analyzed as described in A. (C) Biochemical analysis of Tpk2 and Tpk3 granules evoked by heat stress. Wild type cells expressing Tpk2-GFP or Tpk3-GFP were grown to exponential phase in YPD and subsequently incubated at 30°C, 37°C for 30 minutes or 46°C for 10 minutes. When indicated, cells expressing Tpk3-GFP incubated at 30°C or 37°C 30 minutes were subsequently cross-linked by treatment with 1% (v/v) formaldehyde. Representative blots are shown. The results for the translation markers in Tpk2-GFP cells or Tpk3-GFP cells were similar. Right graph shows the ratio P/S of the abundance of each protein determined by densitometric quantification of the bands. Values are mean ± SEM, n = 2.</p

PKA catalytic subunits show a different subcellular localization upon mild and severe heat stress.

<p>(A) Subcellular localization of Bcy1-GFP, Tpk1-GFP, Tpk2-GFP or Tpk3-GFP in exponentially growing cells (30°C) and after heat stress at 37°C 30 minutes or 46°C 10 minutes visualized by fluorescence microscopy. Cell nuclei were stained with DAPI. The left graph shows the % of nuclear GFP signal. Values are mean +/- SEM, n = 3. * <i>p</i> < 0.05 Tpk1-GFP 30°C <i>versus</i> 46°C; Tpk2-GFP 30°C <i>versus</i> 37°C and 46°C; Tpk3-GFP 30°C <i>versus</i> 37°C and 46°C (ANOVA Bonferroni post-test). (B) The panels show representative images. Numbers inside each photo indicate total granules/100 cells for each of the conditions tested. The arrows show granular localization in the merge channel. Values are mean +/- SEM, n = 3. * <i>p</i> < 0.05 Tpk2-GFP 30°C <i>versus</i> 46°C; Tpk3-GFP 30°C <i>versus</i> 37°C and 46°C (ANOVA-Tukey HSD test).</p

Tpk2 and Tpk3 show an opposite role in translational arrest in response to severe heat stress.

<p>Polysomal profile analysis and immunoblots of 15–50% sucrose gradient fractions from WT (A), <i>tpk2</i>Δ and <i>tpk3</i>Δ cells grown to exponential phase in YPD (30°C) and subjected to severe heat stress (46°C for 10 minutes). Free, monosome and polysome regions are indicated over the polysome profile. The numbers represent the polysome/monosome area ratio (mean +/- SEM, n = 3). * <i>p</i> < 0.05 WT and <i>tpk2</i>Δ 30°C <i>versus</i> 46°C; # <i>p</i> < 0.05 <i>tpk3</i>Δ <i>versus</i> WT and <i>tpk2</i>Δ 46°C (ANOVA Bonferroni post-test). Quantification of translation factors in monosome fraction (M) and polysome fraction (P) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185416#pone.0185416.s001" target="_blank">S1E Fig</a>.</p

Slf1p is required for oxidative stress gene expression during oxidative stress conditions.

<p>(<b>A</b>) The fold-enrichment change is shown for those proteins identified as increasing or decreasing in the wild-type after peroxide treatment compared with the <i>slf1Δ</i> mutant. All proteins on the scatter plot were found to significantly alter in abundance (FDR<0.05) in the wild-type strain following oxidative stress (315 proteins; 249 up and 66 down). Proteins encoded by Slf1p target mRNAs are indicated as red and yellow dots. These include proteins which form part of the oxidative stress response according to MIPS (red dots) as well as proteins which are not directly involved in the oxidative stress response (yellow dots, for details see text). Proteins which form part of the oxidative stress response but are not direct Slf1p targets are shown as blue dots. The dotted line shows the trend-line that would be expected if there was no difference between the wild-type and <i>slf1Δ</i> mutant. (<b>B</b>) Diagrammatic representation of the oxidative stress response highlighting changes in the <i>slf1Δ</i> strain. Hydrogen peroxide (H₂O₂) is generated by the breakdown of superoxide (O₂^.-) catalysed by superoxide dismutases (SOD). Hydrogen peroxide can be reduced by iron (Fe2+) in the Fenton reaction to produce the highly reactive hydroxyl radical (^.OH). Various antioxidant enzymes are involved in the defence against hydrogen peroxide including peroxidases, peroxiredoxins (Prx), glutathione peroxidases (Gpx), glutathione transferases (GST), glutaredoxins (Grx), thioredoxins (Trx), glutathione reductase (Glr), thioredoxin reductase (Trr) and glutathione (GSH). mRNAs bound by Slf1p where protein induction is attenuated in the <i>slf1Δ</i> are in red (corresponding to red dots in Fig. 6A), mRNAs bound by Slf1p where the corresponding protein was not detected in the <i>slf1Δ</i> are in green and mRNAs which are not bound by Slf1p, but where protein induction is attenuated in the <i>slf1Δ</i> are in blue (corresponding to blue dots in Fig. 6A).</p

Slf1p is associated with actively translating ribosomes during oxidative stress conditions.

<p>(<b>A</b>) Polyribosomal profiles of the <i>slf1</i>Δ and wild-type strains before or after hydrogen peroxide treatments for 15 min. (<b>B</b>) Quantification of the ratio of ribosomes in monosomes (80S) to Polysomes (M:P) over a 0–1 mM range of hydrogen peroxide concentrations. The polyribosomal profiles which were used to generate this data are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004903#pgen.1004903.s006" target="_blank">S6 Fig</a>. (<b>C</b>) Ribosome-association of both Slf1p-TAP and Sro9p in fractions isolated from sucrose gradients of an Slf1p-TAP tagged strain. Cultures were treated with 0.4 mM hydrogen peroxide for 15 minutes or with EDTA as shown.</p

Comparison of Slf1p and Sro9p target mRNAs.

<p>(<b>A</b>) A scatterplot comparing the mRNA targets identified in the Slf1p (green), Sro9p (blue) or in both (red) Rip-Seq experiments. The number of ORFs identified as unique to Slf1p or Sro9p or in both are indicated. (<b>B</b>) MIPS Functional categorisation of Slf1p and Sro9p target mRNA enrichment. (<b>C</b>) Slf1p maintains steady state levels of its mRNA targets. Transcript abundance of the whole transcriptome and target mRNAs of Slf1p (Left) or Sro9p (Right) were analysed as described in the legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004903#pgen-1004903-g001" target="_blank">Fig. 1A</a>. Slf1p and Sro9p targets were filtered (FDR<0.05). The x axis of the graph has been restricted to show only those data that are in bins between 3 and -3. (<b>D</b>) Translation efficiency <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004903#pgen.1004903-Subtelny1" target="_blank">[16]</a> of mRNAs bound in each IP and the total RNA. Outliers are shown (open circles) and samples with a P<2.2 -e16 (Wilcoxon rank) are indicated (asterisk).</p

Slf1p associates with ribosomes independently of the La motif.

<p>(<b>A</b>) Western blotting of fractions isolated from polyribosome gradients are shown for strains expressing <i>SLF1</i>, ΔN+, ΔLaM and ΔM mutants. (<b>B</b>) As (A) but with RNAse I treated extracts.</p