The fission yeast stress-responsive MAPK pathway promotes meiosis via the phosphorylation of Pol II CTD in response to environmental and feedback cues

Abstract

The RRM-type RNA-binding protein Mei2 is a master regulator of meiosis in fission yeast, in which it stabilizes meiosis-specific mRNAs by blocking their destruction. Artificial activation of Mei2 can provoke the entire meiotic process, and it is suspected that Mei2 may do more than the stabilization of meiosis-specific mRNAs. In our current study using a new screening system, we show that Mei2 genetically interacts with subunits of CTDK-I, which phosphorylates serine-2 residues on the C-terminal domain of RNA polymerase II (Pol II CTD). Phosphorylation of CTD Ser-2 is essential to enable the robust transcription of ste11, which encodes an HMG-type transcription factor that regulates the expression of mei2 and other genes necessary for sexual development. CTD Ser-2 phosphorylation increases under nitrogen starvation, and the stress-responsive MAP kinase pathway, mediated by Wis1 MAPKK and Sty1 MAPK, is critical for this stress response. Sty1 phosphorylates Lsk1, the catalytic subunit of CTDK-I. Furthermore, a feedback loop stemming from activated Mei2 to Win1 and Wis4 MAPKKKs operates in this pathway and eventually enhances CTD Ser-2 phosphorylation and ste11 transcription. Hence, in addition to starting meiosis, Mei2 functions to reinforce the commitment to it, once cells have entered this process. This study also demonstrates clearly that the stress-responsive MAP kinase pathway can modulates gene expression through phosphorylation of Pol II CTD.

Figure 1. Phenotypes of the mutants defective in each CTDK-I subunit.

(A) Sensitivity to Latrunculin A. Growth of haploid strains JY450 (wild-type), JT659 (lsg1Δ), JT660 (lsk1Δ) and JT661 (lsc1Δ) was examined on YE plates with or without addition of 0.5 µM Latrunculin A. Ten-fold serial dilutions of each strain were spotted and incubated at 30°C for four days. (B) Suppression of mei2-L-SATA by lsg1Δ, lsk1Δ, or lsc1Δ. Ten-fold serial dilutions of haploid strains JV312 (mei2-L-SATA), JT662 (mei2-L-SATA lsg1Δ), JT663 (mei2-L-SATA lsk1Δ), and JT664 (mei2-L-SATA lsc1Δ) were spotted onto SD plates and incubated either at 32°C or 25°C for four days. (C) Reduced mating and sporulation frequencies in the CTDK-I deletion mutants. Cells of the homothallic (h ⁹⁰) haploid strains JY450 (wild-type), JT659 (lsg1Δ), JT660 (lsk1Δ), and JT661 (lsc1Δ) were examined microscopically for their conjugation frequency after incubation on SSA plates at 30°C for three days (left panel). Cells of heterozygous diploid (h ⁺/h ⁻) strains JY362 (wild-type), JT665 (lsg1Δ/lsg1Δ), JT666 (lsk1Δ/lsk1Δ), and JT667 (lsc1Δ/lsc1Δ) were examined microscopically for their sporulation frequency after incubation on SSA plates at 30°C for two days (right panel). (D) DNA content of the diploid strains JY362, JT665, JT666, and JT667 exposed to nitrogen starvation. Cells were cultured in liquid MM medium to mid-log phase and then shifted to MM-N medium. Aliquots were taken at the indicated intervals and the cellular DNA content was determined by FACS analysis.

Figure 2. The phosphorylation of Ser-2 residues on Pol II CTD is required for ste11 expression.

(A) Nitrogen starvation induces the phosphorylation of Ser-2 residues on Pol II CTD in wild-type (JY450) but not in lsg1Δ (JT659) cells. Cells of the two strains were subjected to nitrogen starvation for the indicated periods and analyzed by immunoblotting with antibodies against Ser-2 phosphorylated CTD, Ser-5 phosphorylated CTD, or unphosphorylated CTD. α-tubulin is shown as a loading control. (B) Comparison of the mating and sporulation frequencies among wild-type (JY450), rpb1-12×CTD (JT668), and rpb1-12×S2ACTD (JT669) strains. Cells were incubated on SSA plates at 30°C for three days, and the frequencies were determined microscopically. (C) Expression of ste11 in cells examined in (B). Cells were grown to mid-log phase and shifted to nitrogen-free medium. They were then harvested right before and at 4 hours after this shift, and subjected to northern blot analysis. rRNAs stained with ethidium bromide are shown as a loading control. Expression of rbp1, which was not affected by nitrogen starvation, is also shown for comparison. The rbp1 transcripts in JT668 and JT669 were larger than the authentic transcript due to a vector sequence inserted during the strain construction . (D) Effects of ste11 overexpression on mating and sporulation in the rpb1-12×CTD and rpb1-12×S2ACTD strains. JY450, JT668, and JT669 cells harboring either pREP41-ste11 or pREP41 were examined for their mating and sporulation frequencies after incubation on SSA plates at 30°C for three days.

Figure 3. The stress-responsive MAP kinase Sty1 is essential for the phosphorylation of CTD Ser-2 residues.

(A) CTD Ser-2 phosphorylation was examined in JY450 (wild-type), JT674 (sty1Δ), JX303 (atf1Δ), JZ496 (ste11Δ), and JZ127 (mei2Δ) cells. The cultures were subjected to nitrogen starvation, sampled at the indicated intervals, and analyzed by immunoblotting with antibodies specific for Ser-2 phosphorylated CTD. α-tubulin was detected as a loading control. (B) JY450 and JT660 (lsk1Δ) cells were transformed with either pREP81 or pREP81-wis1*, the latter of which expresses a constitutively active form of MAPKK Wis1. Each transformant was cultured in liquid MM without thiamine for 12 to 18 hours to derepress the weakened nmt1 promoter on pREP81 (nmt1-81). Cells were harvested at the indicated times and analyzed by immunoblotting as in (A). (C) Phosphorylation of GST-Lsg1, GST-Lsk1 and GST-Lsc1 by Sty1 was examined in vitro (right panel, ³²P). Substrates were stained with Coomassie brilliant blue to indicate their quantities (left panel, CBB). (D) GST-Lsk1-N (1–270), GST-Lsk1-C (271–593), and GST as a control were analyzed for phosphorylation (³²P) and quantities (CBB) as in (C). A schematic illustration of the structure of Lsk1 is also shown.

Figure 4. The activation of Mei2 leads to elevated CTD Ser-2 phosphorylation.

(A) Cells of the JX382 fission yeast strain, which carries a mei2 ORF driven by the attenuated nmt1 promoter (nmt1-41) on the chromosome, and of the JX383 strain, which carries the mei2-SATA ORF but is otherwise identical to JX382, were cultured in liquid MM with no supplementation of thiamine. The nmt1-41 promoter was therefore derepressed under these growth conditions. Cells were sampled at the indicated times and the phosphorylation of Ser-2 residues within the CTD repeats was examined by immunoblotting. These samples were also examined for the expression of Mei2 protein by western blot. α-tubulin is shown as a loading control. (B) The Pnmt1-41-mei2-SATA allele in JX383 was combined with either lsk1Δ (JT675), sty1Δ (JT676), wis4Δ (JT677), win1Δ (JT678), wis4Δ win1Δ (JT679), mcs4Δ (JT680), or ste11Δ (JT681). Cells of each strain were cultured in liquid MM with no thiamine addition for 16 and 18 hours, and harvested. Lysates were prepared and then analyzed by immunoblotting with anti-phospho-Ser-2 CTD. The production of Mei2 protein was also evaluated by immunoblotting with α-tubulin detected as a loading control. (C) Cells of the JX382 and JX383 strains were cultured and processed as described in (A). Immunoblotting was performed using antibodies specific for the phosphorylated form of Sty1 MAPK. The Mei2 protein and a loading control α-tubulin were also immunoblotted.

Figure 5. Activated Mei2 enhances expression of ste11 via CTDK-I.

(A) Northern blot analysis of ste11 expression in JY741 (wild-type), JV312 (mei2-L-SATA) and JT663 (mei2-L-SATA lsk1Δ). Cells were grown to the mid-log phase in MM at 32°C, shifted to 25°C, and sampled at the indicated intervals. Total RNA (10 µg) from each sample was resolved by gel electrophoresis and subjected to northern blot analysis to detect transcripts of ste11 and rpb1. rRNAs stained with ethidium bromide are shown as a loading control. (B) Northern blot analysis of ste11 expression in JY333 (wild-type), JZ409 (pat1-114), JT915 (pat1-114 lsk1Δ) and JW92 (pat1-114 mei2Δ). Cells were grown to the mid-log phase in MM at 25°C, shifted to 34°C, sampled at the indicated intervals, and analyzed as in (A). (C) Northern blot analysis of ste11 and mei2 expression in heterozygous diploid (h ⁺/h ⁻) strains JY362 (wild-type) and JT908 (mei2-FA/mei2-FA). Cells were grown to the mid-log phase at 30°C, shifted to nitrogen-free medium, and sampled at the indicated intervals. Total RNA (10 µg) from each sample was resolved by gel electrophoresis and subjected to northern blot analysis to detect ste11 and mei2 transcripts. The level of their expression was normalized by the amount of rRNAs stained with ethidium bromide, and is displayed in a graph for quantitative comparison (lower panel). (D) A diagram of the regulatory pathway leading to the activation of CTDK-I and expression of ste11, which incorporates a feedback loop from Mei2 to MAPKKK Wis4 and Win1.