Sequence-specific targeting of Caenorhabditis elegans C-Ala to the D-loop of tRNAAla.

Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Alac) robustly binds both ligands. How Ce-C-Alac targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Alac are responsible for DNA and tRNA binding, respectively. Ce-C-Alac specifically recognized the conserved invariant base G18 in the D-loop of tRNAAla through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala robustly bound both tRNAAla and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNAAla.

Protein S-palmitoylation regulates different stages of meiosis in Schizosaccharomyces pombe.

Posttranslational protein S-palmitoylation regulates the localization and function of its target proteins involved in diverse cellular processes including meiosis. In this study, we demonstrate that S-palmitoylation mediated by Erf2-Erf4 and Akr1 palmitoylacyltransferases is required at multiple meiotic stages in the fission yeast Schizosaccharomyces pombe We find that S-palmitoylation by Erf2-Erf4 is required for Ras1 localization at the cell periphery to enrich at the cell conjugation site for mating pheromone response. In the absence of Erf2 or Erf4, mutant cells are sterile. A role of Akr1 S-palmitoylating the nuclear fusion protein Tht1 to function in karyogamy is identified. We demonstrate that S-palmitoylation stabilizes and localizes Tht1 to ER, interacting with Sey1 ER fusion GTPase for proper meiotic nuclear fusion. In akr1, tht1, or sey1 mutant, meiotic cells, haploid nuclei are unfused with subsequent chromosome segregation defects. Erf2-Erf4 has an additional substrate of the spore coat protein Isp3. In the absence of Erf2, Isp3 is mislocalized from the spore coat. Together, these results highlight the versatility of the cellular processes in which protein S-palmitoylation participates.

The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription.

Pin1 is a peptidyl-prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II (Pol II) through interaction with the C-terminal domain (CTD) of Rpb1, the largest subunit of Pol II. We demonstrated that this function is important for cellular response to oxidative stress in the fission yeast Schizosaccharomyces pombe. In response to oxidative stress, the Atf1 transcription factor targets Sty1, the mitogen-activated protein kinase (MAPK), to specific stress-responsive promoters. Anchored Sty1 recruits Pol II through direct association with Rpb1-CTD and phosphorylates the reiterated heptad sequence at Serine 5. Pin1 binds phosphorylated CTD to promote dissociation of Sty1 from it, and directly recruits Ssu72 phosphatase to facilitate dephosphorylation of CTD for transcription elongation. In the absence of Pin1, the association of Sty1-Atf1 with Rpb1 persists on stress-responsive promoters failed to generate transcripts of the corresponding genes effectively. The identified characteristic features of the fission yeast Pin1 are conserved in humans. We demonstrated that elevated Pin1 level in cancer cells might help to sustain survival under oxidative stress generated from their altered metabolic pathways. Together, these results suggest a conserved function of Pin1 in cellular response to oxidative stress among eukaryotic cells that might have clinical implication.

Promiscuous Binding of Microprotein Mozart1 to γ-Tubulin Complex Mediates Specific Subcellular Targeting to Control Microtubule Array Formation.

How γ-tubulin ring complex (γ-TuRC), a master template for microtubule nucleation, is spatially and temporally regulated for the assembly of new microtubule arrays remains unclear. Here, we report that an evolutionarily conserved microprotein, Mozart1 (Mzt1), regulates subcellular targeting and microtubule formation activity of γ-TuRC at different cell cycle stages. Crystal structures of protein complexes demonstrate that Mzt1 promiscuously interacts with the N-terminal domains of multiple γ-tubulin complex protein subunits in γ-TuRC via an intercalative binding mode. Genetic- and microscopy-based analyses show that promiscuous binding of Mzt1 in γ-TuRC controls specific subcellular localization of γ-TuRC to modulate microtubule nucleation and stabilization in fission yeast. Moreover, we find Mzt1-independent targeting of γ-TuRC to be crucial for mitotic spindle assembly, demonstrating the cell-cycle-dependent regulation and function of γ-TuRC. Our findings reveal a microprotein-mediated regulatory mechanism underlying microtubule cytoskeleton formation, whereby Mzt1 binding promiscuity confers localization specificity on the multi-protein complex γ-TuRC.

Fission Yeast Puf2, a Pumilio and FBF Family RNA-Binding Protein, Links Stress Granules to Processing Bodies.

Stress granules (SGs) are cytoplasmic aggregates formed upon stress when untranslated messenger ribonucleoproteins accumulate in the cells. In a green fluorescent protein library screening of the fission yeast SG proteins, Puf2 of the PUF family of RNA-binding proteins was identified that is required for SG formation after deprivation of glucose. Accordingly, the puf2 mutant is defective in recovery from glucose starvation with a much longer lag to reenter the cell cycle. In keeping with these results, Puf2 contains several low-complexity and intrinsically disordered protein regions with a tendency to form aggregates and, when overexpressed, it represses translation to induce aggregation of poly(A) binding protein Pabp, the signature constituent of SGs. Intriguingly, overexpression of Puf2 also enhances the structure of processing bodies (PBs), another type of cytoplasmic RNA granule, a complex of factors involved in mRNA degradation. In this study, we demonstrate a function of the fission yeast PB in SG formation and show Puf2 may provide a link between these two structures.

Fission Yeast Asc1 Stabilizes the Interaction between Eukaryotic Initiation Factor 3a and Rps0A/uS2 for Protein Synthesis.

Aminoacyl-tRNA synthetase cofactors play important roles in coordinating aminoacylation and translation. In this study, we describe an additional function of the fission yeast aminoacyl-tRNA synthetase cofactor 1 (Asc1) in translation. We found that Asc1 directly binds and stabilizes the interaction between small ribosomal protein Rps0A/uS2 and eukaryotic initiation factor 3a (eIF3a). In the absence of Asc1, the interaction between eIF3a and Rps0A/uS2 was compromised. The interaction between Rps0A/uS2 and eIF3a mediated the 40S ribosomal subunit binding of eIF3 in 43S preinitiation complex formation to stimulate translation initiation. Keeping with this idea, in an asc1 mutant, the association of mRNA with the 40S ribosomal subunit was defective and protein synthesis was compromised. To show that Asc1 is directly involved in translation, we demonstrate that the addition of recombinant Asc1 is able to rescue the translation defect of the asc1 mutant in a cell-free system. Furthermore, this function of Asc1 is likely to be evolutionarily conserved, as a similar interaction with eIF3a and Rps0A/uS2 could be identified in the budding yeast Saccharomyces cerevisiae and human aminoacyl-tRNA synthetase cofactors. Together, these results identify a function of aminoacyl-tRNA synthetase cofactors in translation preinitiation complex formation, which adds significantly to the expanded functions associated with aminoacyl-tRNA synthetases and their cofactors.

Involvement of fission yeast Pdc2 in RNA degradation and P-body function.

In this study we identified Pdc2, the fission yeast ortholog of human Pat1b protein, which forms a complex with Lsm1-7 and plays a role in coupling deadenylation and decapping. The involvement of Pdc2 in RNA degradation and P-body function was also determined. We found that Pdc2 interacts with Dcp2 and is required for decapping in vivo. Although not absolutely essential for P-body assembly, overexpression of Pdc2 enhanced P-body formation even in the absence of Pdc1, the fission yeast functional homolog of human Edc4 protein, indicating that Pdc2 also plays a role in P-body formation. Intriguingly, in the absence of Pdc2, Lsm1 was found to accumulate in the nucleus, suggesting that Pdc2 shuttling between nucleus and cytoplasm plays a role in decreasing the nuclear concentration of Lsm1 to increase Lsm1 in the cytoplasm. Furthermore, unlike other components of P-bodies, the deadenylase Ccr4 did not accumulate in P-bodies in cells growing under favorable conditions and was only recruited to P-bodies after deprivation of glucose in a Pdc2-Lsm1-dependent manner, indicating a function of Pdc2 in cellular response to environmental stress. In supporting this idea, pdc2 mutants are defective in recovery from glucose starvation with a much longer time to re-enter the cell cycle. In keeping with the notion that Pat1 is a nucleocytoplasmic protein, functioning also in the nucleus, we found that Pdc2 physically and genetically interacts with the nuclear 5'-3' exonuclease Dhp1. A function of Pdc2-Lsm1, in concert with Dhp1, regulating RNA by promoting its decapping/destruction in the nucleus was suggested.

Modulating the Structure and Function of an Aminoacyl-tRNA Synthetase Cofactor by Biotinylation.

Arc1p is a yeast-specific tRNA-binding protein that forms a ternary complex with glutamyl-tRNA synthetase (GluRSc) and methionyl-tRNA synthetase (MetRS) in the cytoplasm to regulate their catalytic activities and subcellular distributions. Despite Arc1p not being involved in any known biotin-dependent reaction, it is a natural target of biotin modification. Results presented herein show that biotin modification had no obvious effect on the growth-supporting activity, subcellular distribution, tRNA binding, or interactions of Arc1p with GluRSc and MetRS. Nevertheless, biotinylation of Arc1p was temperature dependent; raising the growth temperature from 30 to 37 °C drastically reduced its biotinylation level. As a result, Arc1p purified from a yeast culture that had been grown overnight at 37 °C was essentially biotin free. Non-biotinylated Arc1p was more heat stable, more flexible in structure, and more effective than its biotinylated counterpart in promoting glutamylation activity of the otherwise inactive GluRSc at 37 °C in vitro Our study suggests that the structure and function of Arc1p can be modulated via biotinylation in response to temperature changes.

Doubly Spliced RNA of Hepatitis B Virus Suppresses Viral Transcription via TATA-Binding Protein and Induces Stress Granule Assembly.

UNLABELLED: The risk of liver cancer in patients infected with the hepatitis B virus (HBV) and their clinical response to interferon alpha therapy vary based on the HBV genotype. The mechanisms underlying these differences in HBV pathogenesis remain unclear. In HepG2 cells transfected with a mutant HBV(G2335A) expression plasmid that does not transcribe the 2.2-kb doubly spliced RNA (2.2DS-RNA) expressed by wild-type HBV genotype A, the level of HBV pregenomic RNA (pgRNA) was higher than that in cells transfected with an HBV genotype A expression plasmid. By using cotransfection with HBV genotype D and 2.2DS-RNA expression plasmids, we found that a reduction of pgRNA was observed in the cells even in the presence of small amounts of the 2.2DS-RNA plasmid. Moreover, ectopic expression of 2.2DS-RNA in the HBV-producing cell line 1.3ES2 reduced the expression of pgRNA. Further analysis showed that exogenously transcribed 2.2DS-RNA inhibited a reconstituted transcription in vitro. In Huh7 cells ectopically expressing 2.2DS-RNA, RNA immunoprecipitation revealed that 2.2DS-RNA interacted with the TATA-binding protein (TBP) and that nucleotides 432 to 832 of 2.2DS-RNA were required for efficient TBP binding. Immunofluorescence experiments showed that 2.2DS-RNA colocalized with cytoplasmic TBP and the stress granule components, G3BP and poly(A)-binding protein 1 (PABP1), in Huh7 cells. In conclusion, our study reveals that 2.2DS-RNA acts as a repressor of HBV transcription through an interaction with TBP that induces stress granule formation. The expression of 2.2DS-RNA may be one of the viral factors involved in viral replication, which may underlie differences in clinical outcomes of liver disease and responses to interferon alpha therapy between patients infected with different HBV genotypes. IMPORTANCE: Patients infected with certain genotypes of HBV have a lower risk of hepatocellular carcinoma and exhibit a more favorable response to antiviral therapy than patients infected with other HBV genotypes. Using cultured human hepatoma cells as a model of HBV infection, we found that the expression of 2.2DS-RNA caused a decrease in HBV replication. In cultured cells, the ectopic expression of 2.2DS-RNA obviously reduced the intracellular levels of HBV mRNAs. Our analysis of the 2.2DS-RNA-mediated suppression of viral RNA expression showed that 2.2DS-RNA inhibited transcription via binding to the TATA-binding protein and stress granule proteins. Our findings suggest that the 2.2DS-RNA acts as a suppressive noncoding RNA that modulates HBV replication, which may in turn influence the development of chronic hepatitis B.

Pdc1 functions in the assembly of P bodies in Schizosaccharomyces pombe.

P bodies are cytoplasmic RNA granules containing the Dcp1-Dcp2 decapping enzymes where mRNA decay can occur. Here, we describe the characterization of P bodies in the fission yeast Schizosaccharomyces pombe. Most information on the property and function of P bodies stems from studies in the distantly related budding yeast Saccharomyces cerevisiae, and Edc3 was identified as a scaffold protein required for P-body assembly. However, we found that, unlike in S. cerevisiae, fission yeast Edc3 was dispensable for P-body formation. Pdc1, a novel partner of the fission yeast decapping enzyme, with a limited similarity to plant Edc4/Varicose that is required for the assembly of P bodies, was identified (tandem affinity purification-matrix-assisted laser desorption ionization tandem mass spectrometry [TAP-MALDI MS/MS]). Pdc1 interacts with Dcp2 through its C terminus and contains a coiled-coil region for self-interaction to mediate P-body formation. In line with the model that Pdc1 cross-bridges different proteins, additional interactions can be demonstrated with components such as Edc3 and Ste13. Although Pdc1 is not required for the interaction between Dcp1 and Dcp2, our data suggest that Pdc1 acts as a functional homologue of Edc4, a third component of the decapping enzymes that is thought to be absent from fungi. Together, these results highlight the diverse P-body protein compositions between different species and might help to provide insight into their evolutionary paths.

Analysis of stress granule assembly in Schizosaccharomyces pombe.

Stress granules (SGs) are cytoplasmic aggregates of RNA and proteins in eukaryotic cells that are rapidly induced in response to environmental stress, but are not seen in cells growing under favorable conditions. SGs have been primarily studied in mammalian cells. The existence of SGs in the fission yeast and the distantly related budding yeast was demonstrated only recently. In both species, they contain many orthologs of the proteins seen in mammalian SGs. In this study, we have characterized these proteins and determined their involvement in the assembly of fission yeast SGs, in particular, the homolog of human G3BP proteins. G3BP interacts with the deubiquitinating protease USP10 and plays an important role in the assembly of SGs. We have also identified Ubp3, an ortholog of USP10, as an interaction partner of the fission yeast G3BP-like protein Nxt3 and required for its stability. Under thermal stress, like their human orthologs, both Nxt3 and Ubp3 rapidly relocalize to cytoplasmic foci that contain the SG marker poly(A)-binding protein Pabp. However, in contrast to G3BP1 and USP10, neither deletion nor overexpression of nxt3(+) or ubp3(+) affected the assembly of fission yeast SGs as judged by the relocalization of Pabp. Similar results were observed in mutants defective in orthologs of SG components that are known to affect SG assembly in human and in budding yeast, such as ataxia-2 and TIA-like proteins. Together, our data indicate that despite similar protein compositions, the underlying molecular mechanisms for the assembly of SGs could be distinct between species.

Vgl1, a multi-KH domain protein, is a novel component of the fission yeast stress granules required for cell survival under thermal stress.

Multiple KH-domain proteins, collectively known as vigilins, are evolutionarily highly conserved proteins that are present in eukaryotic organisms from yeast to metazoa. Proposed roles for vigilins include chromosome segregation, messenger RNA (mRNA) metabolism, translation and tRNA transport. As a step toward understanding its biological function, we have identified the fission yeast vigilin, designated Vgl1, and have investigated its role in cellular response to environmental stress. Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe. Instead, Vgl1 is required for cell survival under thermal stress, and vgl1Δ mutants lose their viability more rapidly than wild-type cells when incubated at high temperature. As for Scp160 in S. cerevisiae, Vgl1 bound polysomes accumulated at endoplasmic reticulum (ER) but in a microtubule-independent manner. Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3. Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.

Schizosaccharomyces pombe possesses two paralogous valyl-tRNA synthetase genes of mitochondrial origin.

Previous studies showed that VAS1 of Saccharomyces cerevisiae encodes both cytosolic and mitochondrial forms of valyl-tRNA synthetase (ValRS) through alternative initiation of translation. We show herein that except for Schizosaccharomyces pombe, all yeast species studied contained a single ValRS gene encoding both forms, and all of the mature protein forms deduced from those genes possessed an N-terminal appended domain (Ad) that was absent from their bacterial relatives. In contrast, S. pombe contained two distinct nuclear ValRS genes, one encoding the mitochondrial form and the other its cytosolic counterpart. Although the cytosolic form closely resembles other yeast ValRS sequences (approximately 60% identity), the mitochondrial form exhibits significant divergence from others (approximately 35% identity). Both genes are active and essential for the survival of the yeast. Most conspicuously, the mitochondrial form lacks the characteristic Ad. A phylogenetic analysis further suggested that both forms of S. pombe ValRS are of mitochondrial origin, and the mitochondrial form is ancestral to the cytoplasmic form.

GINS inactivation phenotypes reveal two pathways for chromatin association of replicative alpha and epsilon DNA polymerases in fission yeast.

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of beta-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase epsilon, and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase alpha is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase epsilon chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase alpha.

Global role for polyadenylation-assisted nuclear RNA degradation in posttranscriptional gene silencing.

Fission yeast Cid14, a component of the TRAMP (Cid14/Trf4-Air1-Mtr4 polyadenylation) complex, polyadenylates nuclear RNA and stimulates degradation by the exosome for RNA quality control. Here, we analyze patterns of global gene expression in cells lacking the Cid14 or the Dis3/Rpr44 subunit of the nuclear exosome. We found that transcripts from many genes induced during meiosis, including key regulators, accumulated in the absence of Cid14 or Dis3. Moreover, our data suggest that additional substrates include transcripts involved in heterochromatin assembly. Mutant cells lacking Cid14 and/or Dis3 accumulate transcripts corresponding to naturally silenced repeat elements within heterochromatic domains, reflecting defects in centromeric gene silencing and derepression of subtelomeric gene expression. We also uncover roles for Cid14 and Dis3 in maintaining the genomic integrity of ribosomal DNA. Our data indicate that polyadenylation-assisted nuclear RNA turnover functions in eliminating a variety of RNA targets to control diverse processes, such as heterochromatic gene silencing, meiotic differentiation, and maintenance of genomic integrity.

Fission yeast Cut8 is required for the repair of DNA double-strand breaks, ribosomal DNA maintenance, and cell survival in the absence of Rqh1 helicase.

Schizosaccharomyces pombe Rqh1 is a member of the RecQ DNA helicase family. Members of this protein family are mutated in cancer predisposition diseases, causing Bloom's, Werner, and Rothmund-Thomson syndromes. Rqh1 forms a complex with topoisomerase III and is proposed to process or disrupt aberrant recombination structures that arise during S phase to allow proper chromosome segregation during mitosis. Intriguingly, in the absence of Rqh1, processing of these structures appears to be dependent on Rad3 (human ATR) in a manner that is distinct from its role in checkpoint control. Here, we show that rad3 rqh1 mutants are normally committed to a lethal pathway of DNA repair requiring homologous recombination, but blocking this pathway by Rhp51 inactivation restores viability. Remarkably, viability is also restored by overexpression of Cut8, a nuclear envelope protein involved in tethering and proper function of the proteasome. In keeping with a recently described function of the proteasome in the repair of DNA double-strand breaks, we found that Cut8 is also required for DNA double-strand break repair and is essential for proper chromosome segregation in the absence of Rqh1, suggesting that these proteins might function in a common pathway in homologous recombination repair to ensure accurate nuclear division in S. pombe.

Fission yeast Cid12 has dual functions in chromosome segregation and checkpoint control.

Fission yeast Cid12 is a member of the Cid1 family of specialized poly(A) polymerases. Like cells lacking cid1, cid12Delta mutants were shown to have checkpoint defects when DNA replication was inhibited. Here, we show that Cid12 is also required for faithful chromosome segregation and that mutation of amino acid residues predicted to be essential for poly(A) polymerase activity resulted in loss of Cid12 function in vivo. Cells lacking Cid12 had an increased chromosome segregation failure rate due to precocious loss of sister chromatid cohesion at the centromere but not along the chromosome arms. In keeping with a recently described function for Cid12 in RNA interference (RNAi)-mediated heterochromatin assembly, this was accompanied by an accumulation of polyadenylated transcripts corresponding to naturally silenced repeat elements within heterochromatic domains, with consequent defects in centromeric gene silencing. These cells also suffered increased meiotic defects, and their viability was dependent on the spindle checkpoint protein Bub1. To account for the effects of Cid12 on various aspects of DNA metabolism, including chromosome segregation and the checkpoint control, we suggest that Cid12 has dual functions in RNAi silencing and regulating mRNA stability.

Requirement of fission yeast Cid14 in polyadenylation of rRNAs.

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.

A role for the fission yeast Rqh1 helicase in chromosome segregation.

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 rqh1delta cells. Furthermore, relieving replication fork arrest in the rDNA repeat by deletion of reb1+ partially suppresses rqh1delta 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.

Inactivation of the pre-mRNA cleavage and polyadenylation factor Pfs2 in fission yeast causes lethal cell cycle defects.

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 of functional Pfs2, chromosomal attachment to the mitotic spindle was defective, with consequent chromosome missegregation. 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 G1 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.