scholarly journals Inactivation of the Pre-mRNA Cleavage and Polyadenylation Factor Pfs2 in Fission Yeast Causes Lethal Cell Cycle Defects

2005 ◽  
Vol 25 (6) ◽  
pp. 2288-2296 ◽  
Author(s):  
Shao-Win Wang ◽  
Kazuhide Asakawa ◽  
Thein Z. Win ◽  
Takashi Toda ◽  
Chris J. Norbury
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Nitrogen Starvation ◽  
Essential Gene ◽  
Genome Integrity ◽  
Checkpoint Proteins ◽  
Cleavage And Polyadenylation ◽  
Essential Function ◽  
Polyadenylation Factor

ABSTRACT 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.

scholarly journals Intermediates in the assembly of mitotic checkpoint complexes and their role in the regulation of the anaphase-promoting complex

2016 ◽  
Vol 113 (4) ◽  
pp. 966-971 ◽  
Author(s):  
Sharon Kaisari ◽  
Danielle Sitry-Shevah ◽  
Shirly Miniowitz-Shemtov ◽  
Avram Hershko
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Proteins ◽  
Sister Chromatids ◽  
Unknown Species ◽  
Mitotic Checkpoint Complex

The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. Kinetochores that are not attached properly to the mitotic spindle produce an inhibitory signal that prevents progression into anaphase. The checkpoint system acts on the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, which targets for degradation inhibitors of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which assembles when the checkpoint is activated. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20. The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some subcomplexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. We used purified subcomplexes of mitotic checkpoint proteins to examine these problems. Our results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. We also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. A previously unknown species of MCC, which consists of Mad2, BubR1, and two molecules of Cdc20, contributes to the inhibition of APC/C by the mitotic checkpoint system.

scholarly journals A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Pooja Sharma ◽  
Robert Mahen ◽  
Maxim Rossmann ◽  
Jamie E. Stokes ◽  
Bryn Hardwick ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Substrate Recognition ◽  
Genome Integrity ◽  
Mitotic Progression ◽  
New Approach ◽  
Molecule Inhibitor ◽  
Evolutionarily Conserved ◽  
Essential Function ◽  
Carboxyl Terminal

Abstract The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.

scholarly journals Nnf1p, Dsn1p, Mtw1p, and Nsl1p: a New Group of Proteins Important for Chromosome Segregation in Saccharomyces cerevisiae

2002 ◽  
Vol 1 (2) ◽  
pp. 229-240 ◽  
Author(s):  
Ghia M. Euskirchen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Envelope ◽  
Chromosome Segregation ◽  
Indirect Immunofluorescence ◽  
Mitotic Spindle ◽  
Reverse Genetics ◽  
Essential Gene ◽  
Genetic Interactions ◽  
The Novel ◽  
Spindle Poles

ABSTRACT Previously, antibodies were raised against a nuclear envelope-enriched fraction of yeast, and the essential gene NNF1 was cloned by reverse genetics. Here it is shown that the conditional nnf1-17 mutant has decreased stability of a minichromosome in addition to mitotic spindle defects. I have identified the novel essential genes DSN1, DSN3, and NSL1 through genetic interactions with nnf1-17. Dsn3p was found to be equivalent to the kinetochore protein Mtw1p. By indirect immunofluorescence, all four proteins, Nnf1p, Mtw1p, Dsn1p, and Nsl1p, colocalize and are found in the region of the spindle poles. Based on the colocalization of these four proteins, the minichromosome instability and the spindle defects seen in nnf1 mutants, I propose that Nnf1p is part of a new group of proteins necessary for chromosome segregation.

scholarly journals Histone Deacetylase Homologs Regulate Epigenetic Inheritance of Transcriptional Silencing and Chromosome Segregation in Fission Yeast

Genetics ◽  
1998 ◽  
Vol 150 (2) ◽  
pp. 563-576 ◽  
Author(s):  
Shiv I S Grewal ◽  
Michael J Bonaduce ◽  
Amar J S Klar
Keyword(s):  
Fission Yeast ◽  
Histone Deacetylase ◽  
Chromosome Segregation ◽  
Histone Deacetylases ◽  
Essential Gene ◽  
Trichostatin A ◽  
Position Effect ◽  
Epigenetic Inheritance ◽  
Histone Deacetylation ◽  
Mat Locus

Abstract Position-effect control at the silent mat2-mat3 interval and at centromeres and telomeres in fission yeast is suggested to be mediated through the assembly of heterochromatin-like structures. Therefore, trans-acting genes that affect silencing may encode either chromatin proteins, factors that modify them, or factors that affect chromatin assembly. Here, we report the identification of an essential gene, clr6 (c ryptic loci r egulator), which encodes a putative histone deacetylase that when mutated affects epigenetically maintained repression at the mat2-mat3 region and at centromeres and reduces the fidelity of chromosome segregation. Furthermore, we show that the Clr3 protein, when mutated, alleviates recombination block at mat region as well as silencing at donor loci and at centromeres and telomeres, also shares strong homology to known histone deacetylases. Genetic analyses indicate that silencing might be regulated by at least two overlapping histone deacetylase activities. We also found that transient inhibition of histone deacetylase activity by trichostatin A results in the increased missegregation of chromosomes in subsequent generations and, remarkably, alters the imprint at the mat locus, causing the heritable conversion of the repressed epigenetic state to the expressed state. This work supports the model that the level of histone deacetylation has a role in the assembly of repressive heterochromatin and provides insight into the mechanism of epigenetic inheritance.

scholarly journals Fission Yeast dim1+ Encodes a Functionally Conserved Polypeptide Essential for Mitosis

1997 ◽  
Vol 137 (6) ◽  
pp. 1337-1354 ◽  
Author(s):  
Lynne D. Berry ◽  
Kathleen L. Gould
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Nuclear Division ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Sensitive Mutant ◽  
Evolutionarily Conserved ◽  
Mutant Cells

In a screen for second site mutations capable of reducing the restrictive temperature of the fission yeast mutant cdc2-D217N, we have isolated a novel temperature-sensitive mutant, dim1-35. When shifted to restrictive temperature, dim1-35 mutant cells arrest before entry into mitosis or proceed through mitosis in the absence of nuclear division, demonstrating an uncoupling of proper DNA segregation from other cell cycle events. Deletion of dim1 from the Schizosaccharomyces pombe genome produces a lethal G2 arrest phenotype. Lethality is rescued by overexpression of the mouse dim1 homolog, mdim1. Likewise, deletion of the Saccharomyces cerevisiae dim1 homolog, CDH1, is lethal. Both mdim1 and dim1+ are capable of rescuing lethality in the cdh1::HIS3 mutant. Although dim1-35 displays no striking genetic interactions with various other G2/M or mitotic mutants, dim1-35 cells incubated at restrictive temperature arrest with low histone H1 kinase activity. Morevoer, dim1-35 displays sensitivity to the microtubule destabilizing drug, thiabendazole (TBZ). We conclude that Dim1p plays a fundamental, evolutionarily conserved role as a protein essential for entry into mitosis as well as for chromosome segregation during mitosis. Based on TBZ sensitivity and failed chromosome segregation in dim1-35, we further speculate that Dim1p may play a role in mitotic spindle formation and/or function.

scholarly journals Fission yeast mal2+ is required for chromosome segregation.

1996 ◽  
Vol 16 (11) ◽  
pp. 6169-6177 ◽  
Author(s):  
U Fleig ◽  
M Sen-Gupta ◽  
J H Hegemann
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Null Allele ◽  
Fluorescent Protein ◽  
Essential Gene ◽  
Permissive Temperature ◽  
Chromosome Movement ◽  
Green Fluorescent ◽  
Yeast Genes ◽  
Novel Protein

By a screen designed to isolate new fission yeast genes required for chromosome segregation, we have identified mal2+. The conditionally lethal mal2-1 allele gives rise to increased loss of a nonessential minichromosome at the permissive temperature and leads to severe missegregation of the chromosomes at the nonpermissive temperature. Cloning by complementation and subsequent sequence analysis revealed that mal2 is a novel protein with a mass of 34 kDa. Cells containing a mal2 null allele were inviable, indicating that mal2+ is an essential gene. Fusion of mal2 protein to the green fluorescent protein (GFP) showed that mal2 was predominantly localized in the nucleus. Sensitivity to microtubule-destabilizing drugs and strong genetic interactions with alpha1-tubulin suggest an interaction of the mal2 protein with the microtubule system. Spindle formation and elongation were not detectably affected in the mal2-1 mutant as determined by indirect immunofluorescence. However, anomalous chromosome movement on the spindle leading to aberrant distribution of the chromosomal material was observed.

scholarly journals Fission Yeast Bub1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis

1998 ◽  
Vol 143 (7) ◽  
pp. 1775-1787 ◽  
Author(s):  
Pascal Bernard ◽  
Kevin Hardwick ◽  
Jean-Paul Javerzat
Keyword(s):  
Fission Yeast ◽  
Genomic Instability ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Checkpoint ◽  
Chromosome Loss ◽  
Checkpoint Response ◽  
Chromosome Missegregation ◽  
Centromere Function

The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1+ is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Δbub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1+ function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase–anaphase transition.

scholarly journals Structure-function analysis of fission yeast cleavage and polyadenylation factor (CPF) subunit Ppn1 and its interactions with Dis2 and Swd22

PLoS Genetics ◽  
2021 ◽  
Vol 17 (3) ◽  
pp. e1009452
Author(s):  
Bradley Benjamin ◽  
Ana M. Sanchez ◽  
Angad Garg ◽  
Beate Schwer ◽  
Stewart Shuman
Keyword(s):  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Function Analysis ◽  
Transcriptional Profiling ◽  
Missense Mutations ◽  
Rna Seq ◽  
Phosphate Homeostasis ◽  
Protein Coding ◽  
Cleavage And Polyadenylation ◽  
Polyadenylation Factor

Fission yeast Cleavage and Polyadenylation Factor (CPF), a 13-subunit complex, executes the cotranscriptional 3’ processing of RNA polymerase II (Pol2) transcripts that precedes transcription termination. The three-subunit DPS sub-complex of CPF, consisting of a PP1-type phosphoprotein phosphatase Dis2, a WD-repeat protein Swd22, and a putative phosphatase regulatory factor Ppn1, associates with the CPF core to form the holo-CPF assembly. Here we probed the functional, physical, and genetic interactions of DPS by focusing on the Ppn1 subunit, which mediates association of DPS with the core. Transcriptional profiling by RNA-seq defined limited but highly concordant sets of protein-coding genes that were dysregulated in ppn1Δ, swd22Δ and dis2Δ cells, which included the DPSΔ down-regulated phosphate homeostasis genes pho1 and pho84 that are controlled by lncRNA-mediated transcriptional interference. Essential and inessential modules of the 710-aa Ppn1 protein were defined by testing the effects of Ppn1 truncations in multiple genetic backgrounds in which Ppn1 is required for growth. An N-terminal 172-aa disordered region was dispensable and its deletion alleviated hypomorphic phenotypes caused by deleting C-terminal aa 640–710. A TFIIS-like domain (aa 173–330) was not required for viability but was important for Ppn1 activity in phosphate homeostasis. Distinct sites within Ppn1 for binding to Dis2 (spanning Ppn1 aa 506 to 532) and Swd22 (from Ppn1 aa 533 to 578) were demarcated by yeast two-hybrid assays. Dis2 interaction-defective missense mutants of full-length Ppn1 (that retained Swd22 interaction) were employed to show that binding to Dis2 (or its paralog Sds21) was necessary for Ppn1 biological activity. Ppn1 function was severely compromised by missense mutations that selectively affected its binding to Swd22.

scholarly journals Fission Yeast cdc31p Is a Component of the Half-bridge and Controls SPB Duplication

2003 ◽  
Vol 14 (7) ◽  
pp. 2793-2808 ◽  
Author(s):  
Anne Paoletti ◽  
Nicole Bordes ◽  
Raphaël Haddad ◽  
Cindi L. Schwartz ◽  
Fred Chang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Mitotic Spindle ◽  
Calcium Binding ◽  
Essential Gene ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Conditional Mutant ◽  
Mutant Cells ◽  
Identification And Characterization

The fission yeast spindle pole body (SPB) is a nucleus-associated organelle that duplicates once each cell cycle during interphase. Duplicated SPBs serve as the poles of an intranuclear mitotic spindle after their insertion into the nuclear envelope in mitosis (Ding et al., Mol. Biol. Cell 8, 1461–1479). Here, we report the identification and characterization of Schizosaccharomyces pombe cdc31p, a member of the conserved calcium-binding centrin/CDC31 family. Immunofluorescence and immunoelectron microscopy show that cdc31p is a SPB component localized at the half-bridge structure of the SPB. cdc31 is an essential gene and Δcdc31 cells and cdc31 conditional mutant cells arrest in mitosis with a monopolar mitotic spindle organized from a single SPB. EM analysis demonstrates that mutant cdc31 cells fail to duplicate the SPB. In addition, cdc31p exhibits genetic interactions with the SPB component sad1p and is required for sad1p localization. Finally, cdc31 mutant can undergo single or multiple rounds of septation before the exit from mitosis, suggesting that cdc31p activity or SPB duplication may be required for the proper coordination between the exit from mitosis and the initiation of septation.

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