Nrd1 is necessary for recovery from under certain stresses.

<p>(A) Disassembly of stress-induced Nrd1 granules. Wild-type cells expressing GFP-tagged Nrd1 were grown in EMM+thiamine at 27°C. After a 20-min incubation at 42°C, the cells were allowed to recover for 60-min by incubating them at 27°C (recovery from 42°C 60 min). After a 15-min exposure to 2.0 mM arsenite at 27°C (2.0 mM arsenite 15 min), the cells were washed and allowed to recover for 30- (2.0 mM arsenite 45 min) or 240-min (recovery from arsenite 240 min). After a 30-min exposure to 5.0 mM H₂O₂ at 27°C, the cells were washed and allowed to recover for 30 min (recovery from H₂O₂ 30 min). After a 120-min exposure to 10.0 mM CdCl₂ at 27°C, the cells were washed and allowed to recover for 60 min (recovery from CdCl₂ 60 min). After a 10-min exposure to 1.0 M KCl at 27°C, the cells were washed and allowed to recover for 30 min (recovery from KCl 30 min). After a 60-min exposure to 1.0 M KCl, Nrd1-positive granules resolved (1.0 M KCl 60 min). Bar, 10 µm. Lower panel: Graphs showing the number of stress granules per cell from each strain after each condition as indicated. (B) Δ<i>nrd1</i> cells displayed transient stress sensitivity. Wild-type cells transformed with control vector or the <i>nrd1</i>⁺ genes, or the Δ<i>nrd1</i> cells transformed with control vector were grown to mid-log-phase in EMM+thiamine at 27°C. The indicated cells were then exposed to thermal stress (48°C 90 min), 2.0 mM arsenite for 120 min, 5.0 mM H₂O₂ for 30 min, 10.0 mM CdCl₂ for 180 min, and 1.0 M KCl for 180 min and were then spotted onto YES plates and incubated at 27°C. (C) CPU assay of the cells as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029683#pone-0029683-g006" target="_blank">Figure 6(B)</a>. Cells were treated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029683#pone-0029683-g006" target="_blank">Figure 6(B)</a>, and the colony forming ability of each strain after each condition as indicated was determined by counting the number of viable colonies and normalized to the number of colonies in unstressed condition for each strain. This experiment is representative of two independently performed experiments.</p

<i>Schizosaccharomyces pombe</i> strains used in this study.

<p><i>Schizosaccharomyces pombe</i> strains used in this study.</p

Nrd1 regulates stress granule assembly.

<p>(A) Effects of Nrd1 deletion on Pabp-positive granule formation after heat shock. Wild-type or the Δ<i>nrd1</i> cells expressing YFP-tagged Pabp were grown in normal medium or under the conditions indicated. Bar, 10 µm. Graphs showing the number of Pabp-positive granules per cell from each strain as a function of time. (B) Overproduction of Nrd1 induced Pabp-positive granules in the absence of stress. Cells expressing tdTomato-tagged Pabp transformed with pREP1-GFP-Nrd1 or pREP1-GFP-Nrd1^DD were grown in EMM (thiamine-free medium) for 16 h, 18 h, or 22 h to induce overproduction of GFP-Nrd1 or GFP-Nrd1^DD. Bar, 10 µm. (C) Wild-type cells transformed with control vector or containing the <i>nrd1⁺</i> gene, or the Δ<i>nrd1</i> cells transformed with control vector, were grown in EMM+thiamine at 27°C and were then spotted onto YES plates, or spotted onto YES plates with 0.3 mM arsenite, 4.0 mM H₂O₂, 0.3 mM CdCl₂, or 1.0 M KCl, and then incubated at temperatures indicated.</p

Effects of Pmk1 signaling on stress granule assembly.

<p>(A) Effects of Pmk1 deletion on SG assembly. Wild-type cells and Pmk1-deleted cells expressing Nrd1-GFP were grown in YES at 27°C (untreated). Cells were treated with heat shock (42°C for 4 min, or 20 min). Lower panel: Graphs showing the number of stress granules per cell from each strain. (B) Effects of Pmk1 hyperactivation on SG assembly. Wild-type cells expressing Nrd1-GFP were transformed with the constitutively active Pek1^DD or the control vector, and grown in EMM at 27°C (untreated). Cells underwent heat shock at 42°C for 3, or 20 min. Graphs showing the number of stress granules per cell from each strain. The inset is a magnification of the results obtained in each strain at 0 min and 3 min.</p

Nrd1 localizes to stress granules under various environmental stresses.

<p>(A) Analysis of Nrd1-GFP localization under stress. Localization of Nrd1-GFP in living cells grown at 27°C (untreated) after a shift to 42°C for 20 min (42°C 20 min) and after exposure to 2.0 mM arsenite (2.0 mM arsenite 120 min), 5.0 mM H₂O₂ (5.0 mM H₂O₂ 30 min), 10.0 mM CdCl₂ (10.0 mM CdCl₂ 120 min), or 1.0 M KCl (1.0 M KCl 10 min) for the times indicated. Wild-type (wt) cells transformed with pREP1-GFP-Nrd1 were grown in EMM (thiamine-free medium) for 18 h to induce overproduction of GFP-Nrd1 (overproduction 18 hr). Bar, 10 µm. The number in the picture indicates the SG number/cell in each experiment. Right panel: Quantitative analysis of the number of SGs/cell on each stress. Graph depicting the number of stress granules per cell formed before (untreated) and after each condition as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029683#pone-0029683-g001" target="_blank">Figure 1(A)</a> plotted against time after exposure to each stress and the <i>inset</i> is a magnification of the results obtained on KCl treatment. (B) Co-localization of Nrd1 with poly(A)-binding protein (Pabp). Merged image of fluorescence micrographs showing Pabp-GFP (green) and Nrd1-tdTomato (red) in untreated cells and after a 20-min incubation at 42°C. Bar, 10 µm. (C) Fluorescence micrographs of the wild-type cells expressing mCherry-tagged Nrd1 grown at 26°C (untreated); and these were subjected to <i>in situ</i> hybridization with a digoxigenin-labeled oligo (dT)₅₀ probe after a 40-min exposure to 2.0 mM arsenite (2.0 mM arsenite 40 min). The hybridized probe was detected by treatment with mouse anti-digoxin antibody, followed by a fluorescein-conjugated goat anti-mouse IgG antibody (FITC). Nrd1 was detected using mCherry fluorescence (mCherry-Nrd1). Nuclei are counterstained using DAPI dye (DAPI). Bar, 10 µm. (D) Cycloheximide (CHX) prevents the formation of heat-shock- and arsenite-induced Nrd1 granules. Fluorescent images of cells expressing Nrd1-GFP incubated (from left to right) at 27°C with 100 µg/ml CHX for 30 min (CHX); at 42°C for 20 min; pre-incubated with CHX for 30 min at 27°C followed by 20-min incubation at 42°C (CHX then 42°C 20 min); 20-min incubation at 42°C followed by CHX incubation (42°C 20 min then CHX); with 2.0 mM arsenite for 120 min at 27°C; pre-incubated with CHX for 30 min followed by 120-min incubation with 2.0 mM arsenite; and 120-min pre-incubation with arsenite followed by 30-min incubation at with CHX. Bar, 10 µm. Lower panel: Graph depicting the number of stress granules per cell in each condition plotted against time after exposure to each stress.</p

Cpc2 regulates arsenite-induced, but not heat shock-induced formation of Nrd1 granules.

<p>(A) Nrd1 binds to Cpc2 in a phosphorylation-dependent manner. Cells expressing endogenous Cpc2-GFP were transformed with plasmids harboring GST alone, GST-Nrd1, GST-Nrd1^DD, or GST-Nrd1^AA and were grown to mid-log-phase in normal medium (EMM) at 27°C (untreated) and were exposed to a 10-min incubation at 42°C (heat shock), or 120-min incubation to 2.0 mM arsenite at 27°C (arsenite). Cell lysates (lysate) and proteins bound to glutathione sepharose (pull-down) were analyzed by immunoblotting using anti-GFP antibodies (lysates, and pull-down), and anti-GST antibodies (pull-down). (B) Effects of Cpc2 deletion on granule formation of Nrd1 and Nrd1^DD after heat shock or arsenite stress. GFP-Nrd1 or GFP-Nrd1^DD localization in the wild-type cells (wt) or Δ<i>cpc2</i> cells under the conditions indicated. Bar, 10 µm. Lower panel: Graphs showing the number of stress granules per cell from each strain. (C) Overproduction of Nrd1 induced dot-like structures in the absence of stress in a phosphorylation- and Cpc2-dependent manner. Wild-type (wt) or Δ<i>cpc2</i> cells transformed with pREP1-GFP-Nrd1 or pREP1-GFP-Nrd1^DD were grown in EMM (thiamine-free medium) for 16 h, 18 h, or 22 h to induce overproduction of GFP-Nrd1 or GFP-Nrd1^DD. Bar, 10 µm. (D) Immunoblot of GFP-Nrd1 from each strain after each condition as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029683#pone-0029683-g004" target="_blank">Figure 4(C)</a>. Lower panel: Quantification of Nrd1 protein levels against tubulin at each condition as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029683#pone-0029683-g004" target="_blank">Figure 4(D)</a>.</p