Victor A. Tallada

Genome‐wide search of Schizosaccharomyces pombe genes causing overexpression‐mediated cell cycle defects

Genetic studies in yeasts enable an in vivo analysis of gene functions required for the cell division cycle (cdc genes) in eukaryotes. In order to characterize new functions involved in cell cycle regulation, we searched for genes causing cell division defects by overexpression in the fission yeast Schizosaccharomyces pombe. By using this dominant genetic strategy, 26 independent clones were isolated from a Sz. pombe cDNA library. The cloned cDNAs were partially sequenced and identified by computer analysis. The 26 clones isolated corresponded to 21 different genes. Among them, six were genes previously characterized in Sz. pombe, 11 were homologues to genes identified and characterized in other organisms, and four represented genes with unknown functions. In addition to known cell cycle regulators encoding inhibitory protein kinases (wee1, pka1) and DNA checkpoint proteins (Pcna, rad24), we have identified genes that are involved in a number of cellular processes. This includes protein synthesis (ribosomal proteins L7, L10, L29, L41, S6, S11, S17 and the PolyA‐Binding Protein PABP), protein degradation (UBI3), nucleolar rRNA expression (fib, imp1, dbp2), cell cytoskeleton (act1) and glycolysis (pfk1). The interference caused in the cell cycle by overexpression of these genes may elucidate novel mechanisms coupling different cellular processes with the control of the cell division. The effect caused by some of them is described in more detail. Copyright

Nucleocytoplasmic transport in the midzone membrane domain controls yeast mitotic spindle disassembly

During anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the membrane domain surrounding the mitotic spindle midzone promotes spindle midzone dissolution in fission yeast.

Hsp90 interaction with Cdc2 and Plo1 kinases contributes to actomyosin ring condensation in fission yeast

In Schizosaccharomyces pombe, cytokinesis occurs by ordered recruitment of actomyosin components at the division site, followed by lateral condensation to produce a ring-like structure early in anaphase, which eventually matures and contracts at the end of mitosis. We found that in temperature-sensitive hsp90-w1 mutant cells, encoding an Hsp90 mutant protein, ring components were recruited to form a cortical network at the division site, but this network failed to condense into a compact ring, suggesting a role for Hsp90 in this particular step. hsp90-w1 mutant shows strong genetic interaction with specific mutant alleles of the fission yeast cdc2, such as cdc2-33. Interestingly, actomyosin ring defects in hsp90-w1 cdc2-33 mutant cells resembled that of hsp90-w1 single mutant at restrictive temperature. Noteworthy, similar genetic interaction was found with a mutant allele of polo-like kinase, plo1-ts4, suggesting that Hsp90 collaborates with Cdc2 and Plo1 cell cycle kinases to condense medial ring components. In vitro analyses suggested that Cdc2 and Plo1 physically interact with Hsp90. Association of Cdc2 to Hsp90 was ATP independent, while Plo1 binds to this chaperone in an ATP-dependent manner, indicating that these two kinases interact with different Hsp90 complexes. Overall, our analyses of hsp90-w1 reveal a possible role for this chaperone in medial ring condensation in association with Cdc2 and Plo1 kinases.

Fitness Landscape of the Fission Yeast Genome

Background Non-protein-coding regions of eukaryotic genomes remain poorly understood. Diversity studies, comparative genomics and biochemical outputs of genomic sites can be indicators of functional elements, but none produce fine-scale genome-wide descriptions of all functional elements. Results Towards the generation of a comprehensive description of functional elements in the haploid Schizosaccharomyces pombe genome, we generated transposon mutagenesis libraries to a density of one insertion per 13 nucleotides of the genome. We applied a five-state hidden Markov model (HMM) to characterise insertion-depleted regions at nucleotide-level resolution. HMM-defined functional constraint was consistent with genetic diversity, comparative genomics, gene-expression data and genome annotation. Conclusions We infer that transposon insertions lead to fitness consequences in 90% of the genome, including 80% of the non-protein-coding regions, reflecting the presence of numerous non-coding elements in this compact genome that have functional roles. Display of this data in genome browsers provides fine-scale views of structure-function relationships within specific genes.

RNA metabolism is the primary target of formamide in vivo

The synthesis, processing and function of coding and non-coding RNA molecules and their interacting proteins has been the focus of a great deal of research that has boosted our understanding of key molecular pathways that underlie higher order events such as cell cycle control, development, innate immune response and the occurrence of genetic diseases. In this study, we have found that formamide preferentially weakens RNA related processes in vivo. Using a non-essential Schizosaccharomyces pombe gene deletion collection, we identify deleted loci that make cells sensitive to formamide. Sensitive deletions are significantly enriched in genes involved in RNA metabolism. Accordingly, we find that previously known temperature-sensitive splicing mutants become lethal in the presence of the drug under permissive temperature. Furthermore, in a wild type background, splicing efficiency is decreased and R-loop formation is increased in the presence of formamide. In addition, we have also isolated 35 formamide-sensitive mutants, many of which display remarkable morphology and cell cycle defects potentially unveiling new players in the regulation of these processes. We conclude that formamide preferentially targets RNA related processes in vivo, probably by relaxing RNA secondary structures and/or RNA-protein interactions, and can be used as an effective tool to characterize these processes.

Gene dosis and the timing of mitosis

In all organisms, cell size is crucial for biological function and is tightly controlled by genetic, environmental and physiological factors. Cell-size homeostasis is mediated by a finely balanced coordination between cell growth and division. At a critical point in the cell cycle, the G2-M transition, cells stop growth and enter mitosis; advancing or delaying this transition leads to smaller or larger cells, respectively. Uncoordinated cell growth and mitotic entry can lead to diseases. The fission yeast, Schizosaccharomyces pombe, has long served as a potent genetic model system to study mitotic entry and cell-size regulation. The rod-shaped S. pombe cells grow by length extension until they reach the size requirement for the next cell division; thus, longer or shorter cells at division directly point to a delayed or accelerated mitotic entry. In this way, conserved core regulators and modulators of cell-cycle transitions have been identified a while ago. More recently, a systematic study of nearly all S. pombe genes has uncovered 538 mutants showing aberrant cell-cycle progression, leading to longer or shorter cells. In order to identify rate-limiting steps for mitotic entry, Moris, Shrivastava et al. have now analyzed which of these genes act in a dose-dependent manner by using diploid cells with heterozygous deletion mutants, where one of 2 gene copies is deleted and the expression level of the corresponding gene is typically halved. They screened for so-called haploinsufficient mutants in which the single gene copy is not sufficient for normal control of mitotic entry, leading to longer or shorter cells at division. Notably, only 13 such haploinsufficient mutants were more than 10% longer or shorter than control cells. This surprisingly low number points to a remarkable cellular resilience to gene copy decreases, which suggests that the expression levels of other cell-cycle regulators are not rate limiting even when reducing expression levels by 50% or that cells can adjust the expression of these genes by compensatory mechanisms. Moreover, all 13 rate-limiting factors appear to act during the G2-M transition, suggesting that regulators functioning during the G1-S transition, another key control point for cell size which is cryptic in S. pombe, are present in excess or that the screen is not sufficiently sensitive to uncover such factors. These findings highlight the distinct and complementary insights that can be obtained from haploinsufficiency screens. The 13 rate-limiting proteins identified in this screen can be grouped into 3 functional categories: 1) regulation of G2-M transition (7 proteins), including proteins well-known to control the timing of mitotic entry via the CDK network, such as Cdc2, Cdc25, Pom1 and Wee1, which validate the screen; 2) nucleocytoplasmic transport (5 proteins), including 4 nucleoporins and an importin; and 3) nucleotide metabolism (1 protein, Dea2). These results indicate that the CDK network is regulated by multiple rate-limiting steps, each contributing to the timing of mitotic entry. The proteins functioning in nucleocytoplasmic transport could be indirectly involved in controlling G2-M progression by influencing the localization of rate-limiting regulators. However, a more direct role in cell-cycle progression for certain nuclear pore proteins is plausible, because their cell cyclerelated functions besides transport are well documented and the haploinsufficient nuclear-pore mutants showed remarkably specific phenotypes. It may be informative to assess epistasis relationships in diploid cells containing different combinations of haploinsufficient mutants. The nucleotide-metabolism protein Dea2 is the most unexpected rate-limiting factor emerging from this screen. Dea2 is an adenine deaminase involved in the adenine salvage pathway and catabolism. The heterozygous dea2 mutant actually leads to cells being 32% longer than the control, by far the strongest cell elongation phenotype emerging from the screen. This substantial delay in G2-M progression probably reflects activation of DNA damage or replication checkpoints caused by aberrant nucleotide metabolism, which suggests that Dea2 is rate-limiting for nucleotide metabolism. This idea could be tested by analyzing the phenotypes of dea2 haploinsufficient mutants upon DNA damage or replication interference and in combination with checkpoint mutants. The remarkably specific cell-cycle phenotype of dea2 haploinsufficient mutants, unlike other nucleotide-metabolism mutants, raises the intriguing possibility that Dea2 plays a key role at the nexus of nucleotide metabolism ‘health’ and cell-cycle progression.