RNA-Binding Protein Rnc1 Regulates Cell Length at Division and Acute Stress Response in Fission Yeast through Negative Feedback Modulation of the Stress-Activated Mitogen-Activated Protein Kinase Pathway

(A) mRNA levels of the indicated genes were measured by qPCR from total RNA extracted from cell samples corresponding to S. pombe wild-type and rnc1Δ strains growing exponentially in YES medium. Results are shown as relative fold expression (mean ± SD) from three biological repeats. *, P < 0.05; ns, not significant, as calculated by unpaired Student’s t test. RR, response regulator; TF, transcription factor. (B) (Upper panel) Total extracts from growing cultures of wild-type and rnc1Δ strains or those expressing Mcs4-GFP, Wak1-13myc, Wis1-13myc, Sty1-HA, Pyp1-13myc, Pyp2-13myc, and Ptc1-13myc genomic fusions were resolved by SDS-PAGE, and the levels of the respective proteins were detected by incubation with anti-Atf1, anti-HA, anti-GFP, and anti-c-myc antibodies. Anti-Cdc2 was used as a loading control. (Lower panel) Quantification of Western blot experiments. *, P < 0.05; ns, not significant, as calculated by unpaired Student’s t test. (C) S. pombe wild-type, pyp1Δ, rnc1Δ, and pyp1Δ rnc1Δ cells expressing a genomic Sty1-HA6his fusion were grown in YES medium to mid-log phase. Activated and total Sty1 were detected with anti-phospho-p38 and anti-HA antibodies, respectively. Total levels of Atf1 and Pyp2-13myc fusion were determined as described for panel B.

(A) mRNA levels of the indicated genes were measured by qPCR from total RNA extracted from cell samples corresponding to S. pombe wild-type and rnc1Δ strains growing exponentially in YES medium and treated with 0.6 M KCl for the indicated times. Results are shown as relative fold expression (mean ± SD) from three biological repeats. *, P < 0.05; ns, not significant, for the comparisons of rnc1Δ cells with the corresponding incubation times of wild-type cells as calculated by unpaired Student’s t test. (B) S. pombe wild-type and rnc1Δ cells expressing a genomic Sty1-HA6his fusion were grown in YES medium to mid-log phase and treated with 0.6 M KCl for the indicated times. Activated and total Sty1 were detected with anti-phospho-p38 and anti-HA antibodies, respectively. Relative units as mean ± SD (biological triplicates) for Sty1 phosphorylation (anti-phospho-p38 blot) were determined with respect to the internal control (anti-HA blot). *, P < 0.05, as calculated by unpaired Student’s t test. (C and D) S. pombe wild-type and rnc1Δ cells expressing either genomic Wak1-13myc or Wis1-13myc fusions were grown in YES medium to mid-log phase and treated with 0.6 M KCl for the indicated times, and total levels of Wak1-13myc and Wis1-13myc were detected by incubation with anti-c-myc antibodies. Anti-Cdc2 was used as a loading control. *, P < 0.05, as calculated by unpaired Student’s t test. (E) Total extracts from growing cultures of wild-type and rnc1Δ strains or those expressing Pyp1-13myc or Pyp2-13myc genomic fusions, treated with 0.6 M KCl for the indicated times, were resolved by SDS-PAGE, and the levels of the respective proteins were detected by incubation with anti-Atf1 and anti-c-myc antibodies. Anti-Cdc2 was used as a loading control. *, P < 0.05, as calculated by unpaired Student’s t test. (F) S. pombe wild-type and rnc1Δ cells expressing a genomic Pmk1-HA6his fusion were grown in YES medium to mid-log phase and treated with 0.6 M KCl for the indicated times. Activated and total Pmk1 were detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. **, P < 0.005; ns, not significant, as calculated by unpaired Student’s t test.

(A) Secondary structure of Rnc1. KH domains appear colored in light blue. Putative S/T MAPK-phosphosites are shown. Prediction of intrinsically disordered regions (light green boxes) with IUpred2 (https://iupred2a.elte.hu/) is shown below. (B) Coimmunoprecipitation of Rnc1-3HA and Sty-GFP genomic fusions from yeast extracts obtained from vegetatively growing cultures of the indicated genotypes. Results from a representative experiment are shown. IP, immunoprecipitation; WB, Western blot. (C) Bacterially purified GST-Rnc1, GST-Rnc1(T50A), or GST-Rnc1(S/T56A) fusions were incubated with ATP-γ-S and GST-Wis1DD (constitutively active MAPKK) and GST-Sty1-(T97A) (analog-sensitive MAP kinase), in the presence or absence of a specific kinase inhibitor (3-Br-PP1). Rnc1 thiophosphorylation was detected with anti-thioP-ester antibody. Total Wis1, Sty1, and Rnc1 levels in the reaction mixture were determined after incubation with anti-GST antibody. Results from a representative experiment are shown. (D) S. pombe cells expressing Rnc1-3HA or Rnc1(S/T6A)-3HA genomic fusions were grown in YES medium with 7% glucose, recovered by filtration, resuspended in the same medium lacking glucose, and osmotically equilibrated with 3% glycerol for the indicated times. Total and phosphorylated Rnc1 levels were determined by immunoblotting TCA-precipitated protein extracts with anti-HA antibody. Anti-Cdc2 was used as a loading control. Results from a representative experiment are shown. P-species, Rnc1-phosphorylated species. (E) Extracts from S. pombe growing cells starved for glucose for 60 min and expressing a genomic Rnc1-3HA fusion were treated with lambda phosphatase in the presence/absence of specific phosphatase inhibitor. Total and phosphorylated Rnc1 levels were determined by immunoblotting with anti-HA antibody. Anti-Cdc2 was used as a loading control. Results from a representative experiment are shown. (F) cdc10-129 (G₁-phase arrest), cdc25-22 (G₂-phase arrest), and nda3-km311 (M-phase arrest) mutants expressing a genomic Rnc1-3HA fusion were incubated at either 36.5°C for 3.5 h (cdc10-129 and cdc25-22 backgrounds) or 18°C for 7 h (nda3-km311 background). Total and phosphorylated Rnc1 levels were determined by immunoblotting with anti-HA antibody. Anti-Cdc2 was used as a loading control. Results from a representative experiment are shown. (G) Cells from cdc25-22 and cdc25-22 pmk1Δ strains expressing a genomic Rnc1-3HA fusion were grown to an A₆₀₀ of 0.3 at 25°C, shifted to 37°C for 3.5 h, and then released from the growth arrest by transfer back to 25°C. Aliquots were taken at the indicated time intervals, and Rnc1, Cdc2 phosphorylation at Y15, or total Cdc2 was detected by immunoblotting with anti-HA, anti-Cdc2 pY15, and anti-Cdk1/Cdc2 (PSTAIR) antibodies, respectively. Numbers at bottom show the corresponding percentages of binucleated and septated cells. Results from representative experiments are shown. (H) Wild-type, pmk1Δ, sty1Δ, and sty1Δ pmk1Δ strains expressing an Rnc1-3HA genomic fusion were grown in YES medium, resuspended in the same medium lacking glucose, and osmotically equilibrated with 3% glycerol (top panel), incubated at 40°C (middle panel), or treated with 0.5 mM sodium arsenite (bottom panel) for the indicated times. Total and phosphorylated Rnc1 levels were determined by immunoblotting of TCA-precipitated protein extracts with anti-HA antibody. Anti-Cdc2 was used as a loading control. Results from representative experiments are shown.

(A) GST, GST-Rnc1, GST-Rnc1(mKH), and GST-Rnc1(S/T6A) fusions purified from S. pombe cultures were separately incubated with total RNA, and after extensive washes, the RNA-binding ability of Rnc1 with respect to the indicated transcripts was measured by RT-qPCR and normalized with leu⁺ mRNA. (B) mRNA levels of the indicated genes were measured by qPCR from total RNA extracted from cell samples corresponding to S. pombe cells growing exponentially in YES medium and expressing either Rnc1-3HA (wild type), Rnc1(mKH)-3HA, or Rnc1(S/T6A)-3HA genomic fusions. Results are shown as relative fold expression (mean ± SD) from three biological repeats. *, P < 0.05; ns, not significant, as calculated by unpaired Student’s t test. (C) (Left) Total extracts from growing cultures of strains coexpressing either Rnc1-3HA (wild type), Rnc1(mKH)-3HA, or Rnc1(S/T6A)-3HA with Wak1-13myc, Wis1-13myc, Pyp1-13myc, or Pyp2-13myc genomic fusions were resolved by SDS-PAGE, and the levels of the indicated proteins were detected by incubation with anti-Atf1 and anti-c-myc antibodies. Anti-Cdc2 was used as a loading control. (Right) Quantification of Western blot experiments. *, P < 0.05; ns, not significant, as calculated by unpaired Student’s t test. (D) Rnc1-3HA (wild type), Rnc1(mKH)-3HA, and Rnc1(S/T6A)-3HA cells expressing genomic Sty1-HA6his fusions were grown in YES medium to mid-log phase. Activated and total Sty1 were detected with anti-phospho-p38 and anti-HA antibodies, respectively. Relative units as mean ± SD (biological triplicates) for Sty1 phosphorylation (anti-phospho-p38 blot) were determined with respect to the internal control (anti-HA blot). *, P < 0.05, as calculated by unpaired Student’s t test. (E) Cell lengths at division of S. pombe Rnc1-3HA (wild type), Rnc1(mKH)-3HA, and Rnc1(S/T6A)-3HA cells growing exponentially in YES medium are presented as scatter plots showing the average values ± SD (number of independent biological replicates = 3; number of cells ≥ 200/strain). Significant differences were assessed by Tukey’s test following one-way ANOVA for the comparisons with respective values of wild-type cells. *, P < 0.05. Cell morphology of each strain was analyzed by fluorescence microscopy after staining with calcofluor white. Bar, 10 μm.