The Ase1/Prc1 proteins constitute a conserved microtubule-associated protein family that is implicated in central spindle formation and cytokinesis. Here we characterize a role for fission yeast Ase1. Ase1 localizes to microtubule overlapping zones and displays dynamic alterations of localization during the cell cycle. In particular, its spindle localization during metaphase is reduced substantially, followed by robust appearance at the spindle midzone in anaphase. ase1 deletions are viable but defective in nuclear and septum positioning and completion of cytokinesis, which leads to diploidization and chromosome loss. Time-lapse imaging shows that elongating spindles collapse abruptly in the middle of anaphase B. Either absence or overproduction of Ase1 results in profound defects on microtubule bundling in an opposed manner, indicating that Ase1 is a dose-dependent microtubule-bundling factor. In contrast microtubule nucleating activities are not noticeably compromised in ase1 mutants. During meiosis astral microtubules are not bundled and oscillatory nuclear movement is impaired significantly. The Aurora kinase does not correctly localize to central spindles in the absence of Ase1. Finally Ase1 acts as a regulatory component in the cytokinesis checkpoint that operates to inhibit nuclear division when the cytokinesis apparatus is perturbed. Ase1, therefore, couples anaphase completion with cytokinesis upon cell division.

The selective elimination system blocks the accumulation of meiosis-specific mRNAs during the mitotic cell cycle in fission yeast. These mRNAs harbour a region, the determinant of selective removal (DSR), which is recognized by a YTH-family RNA-binding protein, Mmi1. Mmi1 directs target transcripts to destruction in association with nuclear exosomes. Hence, the interaction between DSR and Mmi1 is crucial to discriminate mitosis from meiosis. Here, we show that Mmi1 interacts with repeats of the hexanucleotide U(U/C)AAAC that are enriched in the DSR. Disruption of this ‘DSR core motif’ in a target mRNA inhibits its elimination. Tandem repeats of the motif can function as an artificial DSR. Mmi1 binds to it in vitro . Thus, a core motif cluster is responsible for the DSR activity. Furthermore, certain variant hexanucleotide motifs can augment the function of the DSR core motif. Notably, meiRNA, which composes the nuclear Mei2 dot required to suppress Mmi1 activity during meiosis, carries numerous copies of the core/augmenting motifs on its tail and is indeed degraded by the Mmi1/exosome system, indicating its likely role as decoy bait for Mmi1.

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Summary Target of rapamycin (TOR), an evolutionarily conserved serine/threonine protein kinase, plays pivotal roles in several important cellular processes in eukaryotes. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1), which includes Tor2 as a catalytic subunit, manages the switch between cell proliferation and differentiation by sensing nutrient availability. However, little is known about the direct target of TORC1 that plays key roles in nutrient-dependent TORC1 signaling in fission yeast. Here we report that in fission yeast, three AGC kinase family members, named Psk1, Sck1 and Sck2, which exhibit high homology with human S6K1, are phosphorylated under nutrient-rich conditions and are dephosphorylated by starvation conditions. Among these, Psk1 is necessary for phosphorylation of ribosomal protein S6. Furthermore, Psk1 phosphorylation is regulated by TORC1 in nutrient-dependent and rapamycin-sensitive manners in vivo. Three conserved regulatory motifs (the activation loop, the hydrophobic and the turn motifs) in Psk1 are phosphorylated and these modifications are required for Psk1 activity. In particular, phosphorylation of the hydrophobic motif is catalyzed by TORC1 in vivo and in vitro. Ksg1, a homolog of PDK1, is also important for Psk1 phosphorylation in the activation loop and for its activity. The TORC1 components Pop3, Toc1 and Tco89, are dispensable for Psk1 regulation, but disruption of pop3⁺ causes an increase in the sensitivity of TORC1 to rapamycin. Taken together, these results provide convincing evidence that TORC1/Psk1/Rps6 constitutes a nutrient-dependent signaling pathway in fission yeast.