Role of the RNA-Binding Protein Nrd1 in Stress Granule Formation and Its Implication in the Stress Response in Fission Yeast

We have previously identified the RNA recognition motif (RRM)-type RNA-binding protein Nrd1 as an important regulator of the posttranscriptional expression of myosin in fission yeast. Pmk1 MAPK-dependent phosphorylation negatively regulates the RNA-binding activity of Nrd1. Here, we report the role of Nrd1 in stress-induced RNA granules. Nrd1 can localize to poly(A)-binding protein (Pabp)-positive RNA granules in response to various stress stimuli, including heat shock, arsenite treatment, and oxidative stress. Interestingly, compared with the unphosphorylatable Nrd1, Nrd1DD (phosphorylation-mimic version of Nrd1) translocates more quickly from the cytoplasm to the stress granules in response to various stimuli; this suggests that the phosphorylation of Nrd1 by MAPK enhances its localization to stress-induced cytoplasmic granules. Nrd1 binds to Cpc2 (fission yeast RACK) in a phosphorylation-dependent manner and deletion of Cpc2 affects the formation of Nrd1-positive granules upon arsenite treatment. Moreover, the depletion of Nrd1 leads to a delay in Pabp-positive RNA granule formation, and overexpression of Nrd1 results in an increased size and number of Pabp-positive granules. Interestingly, Nrd1 deletion induced resistance to sustained stresses and enhanced sensitivity to transient stresses. In conclusion, our results indicate that Nrd1 plays a role in stress-induced granule formation, which affects stress resistance in fission yeast

Survey on chest CT findings in COVID-19 patients in Okinawa, Japan: differences between the delta and omicron variants

Abstract To investigate the frequency of pneumonia and chest computed tomography (CT) findings in patients with coronavirus disease 2019 (COVID-19) during the fifth Delta variant-predominant and sixth Omicron variant-predominant waves of the COVID-19 pandemic in Okinawa, Japan. A survey on chest CT examinations for patients with COVID-19 was conducted byhospitals with board-certified radiologists who provided treatment for COVID-19 pneumonia in Okinawa Prefecture. Data from 11 facilities were investigated. Indications for chest CT; number of COVID-19 patients undergoing chest CT; number of patients with late-onset pneumonia, tracheal intubation, and number of deaths; and COVID-19 Reporting and Data System classifications of initial chest CT scans were compared by the chi-squared test between the two pandemic waves (Delta vs. Omicron variants). A total of 1944 CT scans were performed during the fifth wave, and 1178 were performed during the sixth wave. CT implementation rates, which were the number of patients with COVID-19 undergoing CT examinations divided by the total number of COVID-19 cases in Okinawa Prefecture during the waves, were 7.1% for the fifth wave and 2.1% for the sixth wave. The rates of tracheal intubation and mortality were higher in the fifth wave. Differences between the distributions of the CO-RADS classifications were statistically significant for the fifth and sixth waves (p < 0.0001). In the fifth wave, CO-RADS 5 (typical of COVID-19) was most common (65%); in the sixth wave, CO-RADS 1 (no findings of pneumonia) was most common (50%). The finding of “typical for other infection but not COVID-19” was more frequent in the sixth than in the fifth wave (13.6% vs. 1.9%, respectively). The frequencies of pneumonia and typical CT findings were higher in the fifth Delta variant-predominant wave, and nontypical CT findings were more frequent in the sixth Omicron variant-predominant wave of the COVID-19 pandemic in Okinawa, Japan

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

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

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

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

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