Polyadenylation in eukaryotes is conventionally associated with increased nuclear export, translation, and stability of mRNAs. In contrast, recent studies suggest that the Trf4 and Trf5 proteins, members of a widespread family of noncanonical poly(A) polymerases, share an essential function in Saccharomyces cerevisiae that involves polyadenylation of nuclear RNAs as part of a pathway of exosome-mediated RNA turnover. Substrates for this pathway include aberrantly modified tRNAs and precursors of snoRNAs and rRNAs. Here we show that Cid14 is a Trf4/5 functional homolog in the distantly related fission yeast Schizosaccharomyces pombe. Unlike trf4 trf5 double mutants, cells lacking Cid14 are viable, though they suffer an increased frequency of chromosome missegregation. The Cid14 protein is constitutively nucleolar and is required for normal nucleolar structure. A minor population of polyadenylated rRNAs was identified. These RNAs accumulated in an exosome mutant, and their presence was largely dependent on Cid14, in line with a role for Cid14 in rRNA degradation. Surprisingly, both fully processed 25S rRNA and rRNA processing intermediates appear to be channeled into this pathway. Our data suggest that additional substrates may include the mRNAs of genes involved in meiotic regulation. Polyadenylation-assisted nuclear RNA turnover is therefore likely to be a common eukaryotic mechanism affecting diverse biological processes.In eukaryotic cells, most genes are transcribed into precursor messenger RNAs that have to undergo several processing events before maturation, including addition of a cap structure to the 5Ј terminus, removal of introns by splicing, and 3Ј cleavage and polyadenylation. A key player in the polyadenylation process is the nuclear poly(A) polymerase (PAP

Schizosaccharomyces pombe Rqh1 protein is a member of the RecQ DNA helicase family. Members of this protein family are mutated in several human genome instability syndromes, including Bloom, Werner and Rothmund-Thomson syndromes. RecQ helicases participate in recombination repair of stalled replication forks or DNA breaks, but the precise mechanisms that lead to the development of cancer in these diseases have remained obscure. Here, we reveal a function for Rqh1 in chromosome segregation even in the absence of exogenous insult to the DNA. We show that cells lacking Rqh1 are delayed in anaphase progression, and show lagging chromosomal DNA, which is particularly apparent in the rDNA locus. This mitotic delay is dependent on the spindle checkpoint, as deletion of mad2 abolishes the delay as well as the accumulation of Cut2 in rqh1Δ cells. Furthermore, relieving replication fork arrest in the rDNA repeat by deletion of reb1+ partially suppresses rqh1Δ phenotypes. These data are consistent with the function of the Top3-RecQ complex in maintenance of the rDNA structure by processing aberrant chromosome structures arising from DNA replication. The chromosome segregation defects seen in the absence of functional RecQ helicases may contribute to the pathogenesis of human RecQ helicase disorders.

In Schizosaccharomyces pombe, topoisomerase III is encoded by a single gene, top3+, which is essential for cell viability and proper chromosome segregation. Deletion of rqh1+, which encodes the sole RecQ family helicase in S. pombe, suppresses the lethality caused by loss of top3. Here, we provide evidence suggesting that the lethality in top3 mutants is due to accumulation of aberrant DNA structures that arise during S phase, as judged by pulsed-field gel electrophoresis. Using a top3 shut-off strain, we show here that depletion of Top3 activates the DNA damage checkpoint associated with phosphorylation of the checkpoint kinase Chk1. Despite activation of this checkpoint, top3 cells exit the arrest but fail to undergo faithful chromosome segregation. However, these mitotic defects are secondary to chromosomal abnormalities that lead to the lethality, because advance into mitosis did not adversely affect cell survival. Furthermore, top3 function is required for maintenance of nucleolar structure, possibly due to its ability to prevent recombination at the rDNA loci. Our data are consistent with the notion that Top3 has a key function in homologous recombinational repair during S phase that is essential for ensuring subsequent fidelity of chromosome segregation.

Faithful chromosome segregation is fundamentally important for the maintenance of genome integrity and ploidy. By isolating conditional mutants defective in chromosome segregation in the fission yeast Schizosaccharomyces pombe, we identified a role for the essential gene pfs2 in chromosome dynamics. In the absence offunctionalPfs2,chromosomalattachmenttothemitoticspindlewasdefective,withconsequentchromosomemissegregation. Under these circumstances, multiple intracellular foci of spindle checkpoint proteins Bub1 and Mad2 were seen, and deletion of bub1 exacerbated the mitotic defects and the loss of cell viability that resulted from the loss of pfs2 function. Progression from G 1 into S phase following release from nitrogen starvation also required pfs2؉ function. The product of the orthologous Saccharomyces cerevisiae gene PFS2 is a component of a multiprotein complex required for 3-end cleavage and polyadenylation of pre-mRNAs and, in keeping with the conservation of this essential function, an S. pombe pfs2 mutant was defective in mRNA 3-end processing. Mutations in pfs2 were suppressed by overexpression of the putative mRNA 3-end cleavage factor Cft1. These data suggest unexpected links between mRNA 3-end processing and chromosome replication and segregation.Following DNA replication in eukaryotic cells, accurate mitotic chromosome segregation requires the bivalent attachment of the replicated chromosomes to the spindle via sister kinetochores, followed by anaphase movement of the chromosomes to opposite spindle poles. Defects in this process result either in catastrophic failure of mitosis and cell death or in aneuploidy. Spindle checkpoint mechanisms provide protection against these eventualities by imposing a delay over the onset of anaphase until all chromosomes have established symmetrical, bivalent attachments to tense spindle microtubules (14). The molecular basis of this checkpoint is incompletely understood, but many of the proteins involved have been identified and characterized in yeast models and shown to perform analogous checkpoint functions in diverse species, including metazoans and plants. Such proteins include Bub1, Bub3, Mps1, Mad1, Mad2, and Mad3, which are thought to form a heterooligomeric complex at unattached kinetochores (2). During progression through normal, unperturbed mitosis, such complexes would be expected to be short-lived due to the highly dynamic interactions between spindle microtubules and kinetochores. Once all kinetochores have achieved bivalent attachment, the reduction in Mad-and Bub-dependent signaling allows activation of the multisubunit ubiquitin ligase known as the anaphase-promoting complex/cyclosome and subsequent progression into anaphase.Normal chromosome behavior requires the assembly of specialized protein-DNA complexes at telomeres, centromeres, and origins of replication as well as the establishment and maintenance of cohesion between the sister chromatids following DNA replication. The integrity of these chromosome-associated complexes is potentially thre...

The class V myosins are actin-based motors that move a variety of cellular cargoes [1]. In budding yeast, their activity includes the relocation of a portion of the vacuole from the mother cell to the bud [2, 3]. Fission yeast cells contain numerous (approximately 80) small vacuoles. When S. pombe cells are placed in water, vacuoles fuse in response to osmotic stress [4]. Fission yeast possess two type V myosin genes, myo51(+) and myo52(+) [5]. In a myo51Delta strain, vacuoles were distributed throughout the cell, and mean vacuole diameter was identical to that seen in wild-type cells. When myo51Delta and wild-type cells were placed in water, vacuoles enlarged by fusion. In myo52Delta cells, by contrast, vacuoles were smaller and mostly clustered around the nucleus, and fusion in water was largely inhibited. When cells containing GFP-Myo52 were placed in water, Myo52 was seen to redistribute from the cell poles to the surface of the fusing vacuoles. Vacuole fusion in fission yeast was inhibited by the microtubule drug thiabendazole (TBZ) but not by the actin inhibitor latrunculin B. This is the first demonstration of the involvement of a type V myosin, possibly via an interaction with microtubules, in homotypic membrane fusion.