Nrd1 regulates the stability of Cdc4 mRNA. (A) Top panel, Northern blot analysis of the Cdc4 mRNA levels in wild-type cells (wt), Nrd1-deletion cells (Δnrd1), and cells overproducing Nrd1 (Nrd1 OP). Bottom panel, quantification of Cdc4 mRNA levels by densitometry of the bands was expressed relative to that of the control mRNA (leucine) . (B) Top panel. immunoblot analysis of the Cdc4 protein in cells as indicated. The whole-cell lysates were analyzed by immunoblotting with anti-Cdc4 antibodies. Bottom panel, the Cdc4 protein levels (relative to tubulin) were obtained by densitometry. (C) The effect of Nrd1 on the stability of Cdc4 mRNA. Cdc4 mRNA levels after expression from the nmt1/cdc4 construct that included the entire coding region plus the 3′-UTR and transcription repression with thiamine. (D) Quantification of Cdc4 mRNA levels from C. Cdc4 mRNA levels at each time point were calculated as a percentage of time (0 min = 100%) after normalization to leu1 levels. Shown are the mean values (± SD) from three independent experiments, identical to those shown in C.

Mutation in the UCUU sequences in Cdc4 mRNA affects Nrd1 binding and mRNA stability. (A) Affinity capillary electrophoresis mobility shift assay, as described in Figure 3B, was performed with the wild-type Cdc4 mRNA and its mutant versions. Shown are the mean values (±SD) of migration time in each sample from three independent experiments. (B) The UCUU sequences in the Cdc4 mRNA. The UCUU elements and their mutated sequences are underlined. The nucleotide number from the start codon is indicated. (C) Cells transformed with the pREP41 vector containing the indicated genes were spotted onto EMM plates containing thiamine (repressed condition) and then incubated at 27 or 37°C. (D) Cytokinesis defects of the cdc4 M1M2 mutant cells. Cells as indicated were analyzed as described in Figure 1C. Bar, 10 μm. (E) Mutation in UCUU sequences results in the destabilization of Cdc4 mRNA. Wild-type cells or wild-type cells overproducing Nrd1 (Nrd1 OP cells) and expressing either pREP41-GFP-Cdc4 (wt cdc4⁺) or pREP41-GFP-Cdc4M1M2 (cdc4M1M2) were analyzed as described in Figure 2C and Materials and Methods. (F) Quantification of Cdc4 mRNA levels from E. The mRNA stability of Cdc4 was examined as shown in Figure 2C. Results are expressed as the mean value of the results of three independent experiments.

Nrd1 is a direct target of MAPK Pmk1. (A) Inactivation of Pmk1 specifically suppressed the temperature sensitivity of the cdc4-8 mutants. The strains were streaked onto the YPD plates and incubated as indicated. (B) Phosphorylation of Nrd1 is dependent on Pmk1 signaling in vivo. The cells as indicated were transformed with pREP1-GST-Nrd1, with either the control vector or the constitutively active pek1^DD, and analyzed for T40 or T126 phosphorylation. (C) Reactivity of phospho-specific Nrd1 antibodies with wild-type Nrd1 and the mutant Nrd1^AA proteins. Wild-type cells expressing pREP1-GST-Nrd1, or the pREP1-GST-Nrd1^AA mutant protein were analyzed by immunoblotting with anti-pNrd1 (T40), anti-pNrd1 (T126), or anti-GST antibodies. (D) Pmk1 MAPK directly phosphorylates Nrd1 in vitro. GST-Nrd1 phosphorylation activity of the recombinant GST-Pmk1 was analyzed as described in Materials and Methods.

The Pmk1 MAPK pathway modulates the level of Cdc4 mRNA. (A) Inactivation of Pmk1 stabilized Cdc4 mRNA. Northern blot analysis of the Cdc4 mRNA levels in wild-type cells (wt) and Pmk1-deletion cells (Δpmk1) (top panel). Quantification of Cdc4 mRNA levels (bottom panel). The above data were quantified by densitometry and were expressed as shown in Figure 2A. (B) The effect of Pmk1 on the stability of Cdc4 mRNA. The stability of Cdc4 mRNA was examined as shown in Figure 2, C and D. (C) Inactivation of Pmk1 ameliorated the cytokinesis defects of the cdc4-8 mutants. The indicated strains were incubated at 27°C and then stained with calcofluor as shown in Figure 1C. Bar, 10 μm. (D) The Nrd1 deletion reverted the phenotypes of the cdc4-8Δpmk1 cells. Cells as indicated were streaked onto YPD plates and incubated at the temperature as indicated. (E) Top panel, Northern blot analysis of the Cdc4 mRNA levels in the indicated cells, as described in Figure 2A. Bottom panel, immunoblot analysis of the Cdc4 protein in the indicated cells. The whole-cell lysates were analyzed by immunoblotting with anti-Cdc4 antibodies. (F) Cdc4 mRNA and protein levels were quantified as described in Figure 2, A and B.

Nrd1 was isolated as a multicopy suppressor of the cdc4 mutant cells. (A) Overexpression of nrd1⁺ suppresses the temperature sensitivity of the cdc4-8 mutants. Cells were transformed with a multicopy plasmid containing the indicated genes, streaked onto YPD plates and then incubated at the temperatures as indicated. (B) Nrd1 deletion exacerbated the temperature sensitivity of the cdc4-8 mutants. Cells as indicated were streaked onto YPD plates and incubated at the temperature as indicated. (C) Nrd1 affects the cytokinesis defects of the cdc4-8 mutants. Cells as indicated were incubated at 27°C and then stained with calcofluor to visualize the cell wall and septum. Arrows indicate cells with abnormally thickened septa (+vector) or multiseptated cells (Δnrd1), respectively. Bar, 10 μm. (D) Cells as indicated were incubated at 36°C for 8 h, and the septation index was determined microscopically with calcofluor staining. (E) Overexpression of nrd1⁺ suppresses the temperature sensitivity of the cdc4-312 mutants. Cells were transformed with a multicopy plasmid containing the indicated genes and analyzed as described in A. (F) Nrd1 deletion exacerbated the temperature sensitivity of the cdc4-312 mutants. Cells as indicated were analyzed as described in B. (G) Nrd1 affects the cytokinesis defects of the cdc4-312 mutants. Cells as indicated were incubated at 27°C and then stained with calcofluor to visualize the cell wall and septum. Bar, 10 μm.

Nrd1 binds to Cdc4 mRNA in vivo and in vitro. (A) GST-Nrd1 associates with Cdc4 mRNA in vivo. Wild-type cells expressing GST with either a control vector or Cdc4 with or without its 3′-UTR or GST-Nrd1 with either a control vector or Cdc4 with or without its 3′-UTR were analyzed for their mRNA binding as described in Materials and Methods. (B) GST-Nrd1 associates with Cdc4 mRNA in vitro. Affinity capillary electrophoresis mobility shift assay of the recombinant Nrd1 protein concentrations of Cdc4 mRNA as indicated were analyzed as described in Materials and Methods.

The RNA-binding ability of Nrd1 is phosphorylation dependent. (A) The amino acid sequence containing the two putative MAPK phosphorylation sites. The identified phosphorylation sites, T(40) and T(126), are denoted by arrows. (B) Phosphorylation of T(40) and T(126) abrogates the ability of Nrd1 to suppress the cdc4-8 mutant. Cells transformed with the pREP1 vector containing the indicated genes were spotted onto the EMM plates containing thiamine (repressed condition) and then incubated at 27 or 33°C. (C) Phosphorylation of T(40) and T(126) abrogates the ability of Nrd1 to suppress the temperature sensitivity of the pat1-114 mutants. Cells transformed with the pREP1 vector containing the indicated genes were spotted onto EMM plates containing thiamine (repressed condition) and then incubated at 27 or 34°C. (D) Wild-type cells expressing GST with a control vector, expressing GST-Nrd1 with a control vector, or expressing either GST-Nrd1, GST-Nrd1^DD, or GST-Nrd1^AA with Cdc4 were analyzed as shown in Figure 3A. (E) The amount of Cdc4 mRNA bound by either GST, GST-Nrd1, or GST-Nrd1 mutants was quantified by densitometry of the bands from D and was expressed relative to the control GST pulldown precipitates run in the same experiment.

Pmk1-mediated phosphorylation negatively regulates the ability of Nrd1 to bind and regulate Cdc4 mRNA. (A) Pmk1 phosphorylation of Nrd1 impairs the binding activity of Nrd1 to Cdc4 mRNA. The cells indicated are transformed either with the control vector or cdc4⁺ and analyzed as shown in Figure 3A. Data were quantified and expressed as shown in Figure 5D. (B) Affinity capillary electrophoresis mobility shift assay as shown in Figure 3B was performed with the Nrd1 protein phosphorylated by the activated Pmk1 MAPK.

Cell cycle–dependent regulation of Pmk1-Nrd1 signaling. (A) Cell cycle–dependent activation of Pmk1 MAPK. Top, cells from SP628 strains (cdc25-22 pmk1-GST∷KanMx6) were grown at 25°C, shifted to 37°C for 4 h, and then released from growth arrest by transferring them back to 25°C. Aliquots were obtained at different time points, and activated or total Pmk1 was detected by immunoblotting with anti-phospho-p42/44 or anti-GST antibodies, respectively. Bottom, quantification of the Pmk1 activity during the cell cycle by normalization to the loading control value. The septation index (□) is also shown, which indicates good cell cycle synchrony in the culture. (B) Cell cycle–dependent phosphorylation of Nrd1. Top, cells from strain cdc25-22 were transformed with pREP1-GFP-Nrd1, grown in EMM containing thiamine, and analyzed as shown in A. Phosphorylated or total Nrd1 was detected by immunoblotting with anti-phospho-Nrd1 (T40), anti-phospho-Nrd1 (T126), or anti-GFP antibodies. Tubulin served as a loading control. Bottom, quantification of Nrd1 phosphorylation by normalization to the loading control value. (C) Cell cycle–dependent oscillation of Cdc4 mRNA. Top, cells from the SP628 strains were grown at 25°C, shifted to 37°C for 4 h, and then released from growth arrest by transferring them back to 25°C. Aliquots were obtained at different time points, and Cdc4 mRNA was detected by Northern blot analysis as described in Figure 2A. Bottom, quantification of Pmk1 activation (▵), T40 Nrd1 phosphorylation (○), and Cdc4 mRNA levels (■).