The spindle checkpoint is the prime cell-cycle control mechanism that ensures sister chromatids are bioriented before anaphase takes place. Aurora B kinase, the catalytic subunit of the chromosome passenger complex, both destabilizes kinetochore attachments that do not generate tension and simultaneously maintains the spindle checkpoint signal. However, it is unclear how the checkpoint is silenced following chromosome biorientation. We demonstrate that association of type 1 phosphatase (PP1(Dis2)) with both the N terminus of Spc7 and the nonmotor domains of the Klp5-Klp6 (kinesin-8) complex is necessary to counteract Aurora B kinase to efficiently silence the spindle checkpoint. The role of Klp5 and Klp6 in checkpoint silencing is specific to this class of kinesin and independent of their motor activities. These data demonstrate that at least two distinct pools of PP1, one kinetochore associated and the other motor associated, are needed to silence the spindle checkpoint.

SummaryThe spindle checkpoint is a surveillance system acting in mitosis to delay anaphase onset until all chromosomes are properly attached to the mitotic spindle [1, 2]. When the checkpoint is activated, the Mad2 and Mad3 proteins directly bind and inhibit Cdc20, which is an essential activator of an E3 ubiquitin ligase known as the anaphase-promoting complex (APC) [3]. When the checkpoint is satisfied, Cdc20-APC is activated and polyubiquitinates securin and cyclin, leading to the dissolution of sister chromatid cohesion and mitotic progression. Several protein kinases play critical roles in spindle checkpoint signaling, but the mechanism (or mechanisms) by which they inhibit mitotic progression remains unclear [4]. Furthermore, it is not known whether their activity needs to be reversed by protein phosphatases before anaphase onset can occur. Here we employ fission yeast to show that Aurora (Ark1) kinase activity is directly required to maintain spindle checkpoint arrest, even in the presence of many unattached kinetochores. Upon Ark1 inhibition, checkpoint complexes are disassembled and cyclin B is rapidly degraded. Importantly, checkpoint silencing and cyclin B degradation require the kinetochore-localized isoform of protein phosphatase 1 (PP1Dis2). We propose that PP1Dis2-mediated dephosphorylation of checkpoint components forms a novel spindle checkpoint silencing mechanism.

Fission yeast has two members of the Shugoshin family, Sgo1 and Sgo2. Although Sgo1 has clearly been established as a protector of centromere cohesion in meiosis I, the roles of Sgo2 remain elusive. Here we show that Sgo2 is required to ensure proper chromosome biorientation upon recovery from a prolonged spindle checkpoint arrest. Consistent with this, Sgo2 is essential for maintaining the Passenger proteins on centromeres upon checkpoint activation. Interestingly, lack of Sgo2 has a more penetrant effect on the localization of Survivin than on the two other Passenger proteins INCENP and Aurora B, and the Survivin-INCENP complex but not the INCENP-Aurora B complex is destabilized in the absence of Sgo2. Finally we show that the conserved C-terminus of Sgo2 is crucial to maintain Sgo2 and Passenger proteins localization on centromeres upon prolonged checkpoint activation. Taken together, our results demonstrate that Sgo2 is important for chromosome biorientation and that it controls docking of the Passenger proteins on chromosomes in early mitotic cells. INTRODUCTIONTo ensure the accuracy of chromosome segregation in mitosis, duplicated sister-chromatids must attach their kinetochores to microtubules emanating from opposite poles, a process referred to as chromosome biorientation. A single mal-orientated chromosome can be recognized by the spindle checkpoint that will block anaphase onset by inhibiting the activity of the anaphase-promoting complex (APC/C). Once all chromosomes are properly biorientated on the metaphase spindle, the spindle checkpoint is silenced and cells proceed through anaphase (for review, see Pinsky and Biggins, 2005).One of the best characterized roles of the kinase Aurora B is to correct defective kinetochore-microtubule attachment before anaphase onset and therefore ensure proper chromosome biorientation (Tanaka et al., 2002;Ditchfield et al., 2003;Hauf et al., 2003;Tanaka, 2005;Pinsky et al., 2006). Aurora B is also crucial for the recruitment of some spindle checkpoint components to kinetochores (Ditchfield et al., 2003;Vigneron et al., 2004). Thus Aurora B regulates both the physical connections of chromosomes onto the spindle and mitotic progression. When Aurora B activity is compromised, chromosomes mis-segregate massively, leading to aneuploidy (reviewed in Giet et al., 2005). Defective kinetochore-microtubules attachments accumulate in these cells and chromosomes never reach a proper metaphase plate (Hauf et al., 2003).Aurora B is one of the Chromosomal Passenger proteins, first identified in vertebrates as proteins sharing a complex and highly regulated localization pattern in mitosis (Earnshaw and Bernat, 1991). In particular they transfer abruptly from the inner-centromeres to the spindle midzone at the metaphase to anaphase transition. Each Chromosomal Passenger protein, Survivin, Borealin, TD60, INCENP, and Aurora B has long been recognized as major regulators of mitosis (see Vagnarelli and Earnshaw, 2004 for review). In fission yeast, homologues of only three of the Chromoso...

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions.

Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a "wait anaphase" signal in cases of incomplete or improper kinetochoremicrotubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochoremicrotubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation.

SummaryHistone variant H2A.Z has a conserved role in genome stability, although it remains unclear how this is mediated. Here we demonstrate in fission yeast that the Swr1 ATPase inserts H2A.Z (Pht1) into chromatin and Kat5 acetyltransferase (Mst1) acetylates it. Deletion or unacetylatable mutation of Pht1 leads to genome instability, primarily caused by chromosome entanglement/breakage at anaphase. This leads to the loss of telomere-proximal markers, though telomere protection and repeat length are unaffected by the absence of Pht1. Strikingly the chromosome entanglement in pht1Δ anaphase cells can be rescued by forcing chromosome condensation prior to anaphase onset. We show that the condensin complex, required for the maintenance of anaphase chromosome condensation, prematurely dissociates from chromatin in the absence of Pht1. This and other findings suggest an important role for H2A.Z in the architecture of anaphase chromosomes.

Recognition of self-pollen during the self-incompatibility response in Brassica oleracea is mediated by the binding of a secreted peptide (the S locus cysteine-rich protein) to the S locus receptor kinase (SRK), a member of the plant receptor kinase (PRK) superfamily. Here, we describe the characterization of three proteins that interact with the cytosolic kinase domain of SRK. A B. oleracea homolog of Arabidopsis kinase-associated protein phosphatase was shown to interact with and dephosphorylate SRK and was itself phosphorylated by SRK. Yeast (Saccharomyces cerevisiae) two-hybrid screens identified two additional interactors, calmodulin and a sorting nexin, both of which have been implicated in receptor kinase down-regulation in animals. A calmodulin-binding site was identified in sub-domain VIa of the SRK kinase domain. The binding site is conserved and functional in several other members of the PRK family. The sorting nexin also interacted with diverse members of the PRK family, suggesting that all three of the interacting proteins described here may play a general role in signal transduction by this family of proteins.Plant genomes encode large numbers of receptors with Ser/Thr protein kinase activity. These plant receptor kinases (PRKs) are related to, but phylogenetically distinct from, the receptor Tyr kinase (RTK) and receptor Ser/Thr kinase (RSK) families in animals, suggesting that the three families have evolved independently from a common ancestor (Shiu and Bleecker, 2001). The independent evolution of these gene families is reflected in the fact that, to date, none of the proteins that have been shown to interact with PRKs are closely homologous to RTK-and RSKinteracting proteins. However, despite the many differences between plant and animal receptor kinase systems, there are indications that they function in a similar manner with, for example, unrelated interacting proteins carrying out analogous roles in the two kingdoms (Cock et al., 2002).One of the best characterized PRKs is the S locus receptor kinase (SRK). SRK is located in the plasma membrane of the stigmatic papillar cells and is the female component of the self-incompatibility (SI) response in Brassica spp. (Stein et al., 1991; Delorme et al., 1995;Takasaki et al., 2000). The SI response allows the recognition and rejection of self-pollen grains on the stigma surface and, hence, promotes outcrossing (Cock, 2000). There is now strong evidence that SRK mediates self-pollen recognition by binding to a small, secreted peptide, S locus Cys rich (SCR), located in the pollen coat (Schopfer et al., 1999;Takayama et al., 2000Takayama et al., , 2001Shiba et al., 2001;Kachroo et al., 2001).SRK is phosphorylated after an incompatible pollination (Cabrillac et al., 2001), and in vitro experiments indicate that activation of SRK may involve autophosphorylation between SRK molecules associated in the membrane (Giranton et al., 2000). This suggests that SRK is activated by a mechanism similar to that described for animal receptor kinases (Heldin, 1995...

Functional links connecting gene transcription and condensin-mediated chromosome condensation have been established in species ranging from prokaryotes to vertebrates. However, the exact nature of these links remains misunderstood. Here we show in fission yeast that the 3′ end RNA processing factor Swd2.2, a component of the Cleavage and Polyadenylation Factor (CPF), is a negative regulator of condensin-mediated chromosome condensation. Lack of Swd2.2 does not affect the assembly of the CPF but reduces its association with chromatin. This causes only limited, context-dependent effects on gene expression and transcription termination. However, CPF-associated Swd2.2 is required for the association of Protein Phosphatase 1 PP1Dis2 with chromatin, through an interaction with Ppn1, a protein that we identify as the fission yeast homologue of vertebrate PNUTS. We demonstrate that Swd2.2, Ppn1 and PP1Dis2 form an independent module within the CPF, which provides an essential function in the absence of the CPF-associated Ssu72 phosphatase. We show that Ppn1 and Ssu72, like Swd2.2, are also negative regulators of condensin-mediated chromosome condensation. We conclude that Swd2.2 opposes condensin-mediated chromosome condensation by facilitating the function of the two CPF-associated phosphatases PP1 and Ssu72.