Figure 1.

Ndc80 degradation is temporally regulated during meiosis. (A) LUTI-based regulation of Ndc80 protein synthesis in budding yeast meiosis. In meiotic prophase, the Ime1–Ume6 transcription factor complex induces a long undecoded transcript isoform of NDC80 called NDC80^LUTI, which cannot produce Ndc80 protein due to the upstream open reading frames in its 5′ extension. NDC80^LUTI represses transcription of a protein-coding isoform of NDC80, NDC80^ORF. Through this combined act of transcriptional and translational repression, NDC80^LUTI inhibits Ndc80 protein synthesis. In the meiotic divisions, NDC80^ORF is induced by a second meiotic transcription factor, Ndt80. URS1 (upstream regulatory sequence 1), a DNA-binding motif for Ume6. MSE (mid-sporulation element), a DNA-binding motif for Ndt80. (B) The lexO-LUTI system induces NDC80^LUTI expression upon β-estradiol addition, thus conditionally inhibiting NDC80^ORF expression and Ndc80 protein synthesis. (Top) Regulatory elements of the NDC80 gene. (Bottom) The lexO-LUTI system. mse, a mutant MSE site defective in Ndt80 binding. (C) Ndc80 turnover in early or late meiotic prophase. The strain carrying the lexO-LUTI (UB14883) was transferred to the sporulation medium (SPO) at 0 h to induce meiosis, and β-estradiol was added at either 1.5 or at 4 h after meiosis induction. The strain was halted in meiotic prophase using an ndt80Δ block. Here and throughout, Ndc80 levels were determined by anti-V5 immunoblot. Hxk2, loading control. Unless specified, the numbers below the immunoblots were calculated by first normalizing Ndc80 levels to Hxk2 levels in each lane, and then dividing the ratio to the 0-h time point. All the experiments in this study were performed at least twice, and one representative biological replicate is shown. (D) Induction levels of NDC80^LUTI mRNA for the experiment in C, measured by reverse transcription followed by quantitative PCR (RT-qPCR). For all RT-qPCR experiments, NDC80^LUTI signals were normalized to that of PFY1. (a.u.) Arbitrary unit. The mean from three independent experiments, along with the standard error of the mean, is displayed. The P-values were calculated by a two-tailed Student's t-test. (E) Ndc80 turnover in late meiotic prophase or in metaphase I arrest. The ndt80Δ (UB19616) and the ndt80Δ cdc20-mn (UB19618) strains were cultured in SPO for 4 h before β-estradiol addition. Both strains were halted in meiotic prophase with an ndt80Δ block. The cdc20 meiotic null mutant (cdc20-mn, UB19678) was cultured in SPO for 5 h before β-estradiol addition and subsequently halted in metaphase I for 3 h.