Biology subjects

Rhind, N.

Publications and source records attributed to Rhind, N..

12 recordsLinked to original sources

bioRxiv2023

Size-Dependent Expression of the Fission Yeast Cdc13 Cyclin is Conferred by Translational Regulation

The major fission yeast cyclin, Cdc13, has been shown to increase in concentration in correlation with cell size, and has been proposed to thereby regulate cell size at division. However, the mechanism of its cell-size regulation has been unknown. Here, we show that Cdc13 is regulated post-transcriptionally. Its transcript is not expressed in a size-dependent manner, rather a size-dependent concentration of protein is expressed from a size-independent concentration of mRNA. Moreover, we show that the expression of Cdc13 is, in fact, size dependent, as opposed to simply size-correlated due to time-dependent expression. We identify a 20-amino-acid motif, which includes the APC D-box degron, as necessary and sufficient for size-dependent expression, which allowed us to construct a size-independent allele of cdc13. Using this allele, we show that size-dependent expression of Cdc13 is not required for size control in fission yeast cells.

cell biology↗

bioRxiv2022

pomBseen: An Automated Pipeline for Analysis of Fission Yeast Images

pomBseen is a image analysis pipeline for the quantitation of fission yeast micrographs containing a brightfield channel and up to two fluorescent channels. It accepts a wide range of image formats and produces a table with the number, size and total and nuclear fluorescent intensities of the cells in the image. Written in MATLAB, pomBseen is also available as a standalone application.

cell biology↗

bioRxiv2022

A high-quality reference genome for the fission yeast Schizosaccharomyces osmophilus

Fission yeasts are an ancient group of fungal species that diverged from each other from tens to hundreds of million years ago. Among them is the preeminent model organism Schizosaccharomyces pombe, which has significantly contributed to our understandings of molecular mechanisms underlying fundamental cellular processes. The availability of the genomes of S. pombe and three other fission yeast species S. japonicus, S. octosporus, and S. cryophilus has enabled cross-species comparisons that provide insights into the evolution of genes, pathways, and genomes. Here, we performed genome sequencing on the type strain of the recently identified fission yeast species S. osmophilus and obtained a complete mitochondrial genome and a nuclear genome assembly with gaps only at rRNA gene arrays. A total of 5098 protein-coding nuclear genes were annotated and orthologs for more than 95% of them were identified. Genome-based phylogenetic analysis showed that S. osmophilus is most closely related to S. octosporus and these two species diverged around 16 million years ago. To demonstrate the utility of this S. osmophilus reference genome, we conducted cross-species comparative analyses of centromeres, telomeres, transposons, the mating-type region, Cbp1 family proteins, and mitochondrial genomes. These analyses revealed conservation of repeat arrangements and sequence motifs in centromere cores, identified telomeric sequences composed of two types of repeats, delineated relationships among Tf1/sushi group retrotransposons, characterized the evolutionary origins and trajectories of Cbp1 family domesticated transposases, and discovered signs of interspecific transfer of two types of mitochondrial selfish elements.

genomics↗

bioRxiv2022

Molecular origins of genome instability following a single chromosome mis-segregation event

Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures were upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors.

cell biology↗

bioRxiv2020

Competition for MCM Loading at Origins Establishes Replication Timing Patterns

Loading of the MCM replicative helicase onto origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The number of MCM loaded on origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide characteristics of MCM loading and their direct effect on replication timing remain unclear. In order to probe MCM loading dynamics and its effect on replication timing, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their initiation during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels not limiting for MCM loading. Our results support a model in which the loading activity of origins, controlled by their ability to recruit ORC and compete for MCM, determines the number of helicases loaded, which in turn affects replication timing.

cell biology↗

bioRxiv2020

Genome-Wide Mapping of Human DNA Replication by Optical Replication Mapping Supports a Stochastic Model of Eukaryotic Replication Timing

DNA replication is regulated by the location and timing of replication initiation. Therefore, much effort has been invested in identifying and analyzing the sites of human replication initiation. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation site utilization in metazoans has made mapping the location and timing of replication initiation in human cells difficult. A potential solution to the problem of human replication mapping is single-molecule analysis. However, current approaches do not provide the throughput required for genome-wide experiments. To address this challenge, we have developed Optical Replication Mapping (ORM), a high-throughput single-molecule approach to map newly replicated DNA, and used it to map early initiation events in human cells. The single-molecule nature of our data, and a total of more than 2000-fold coverage of the human genome on 27 million fibers averaging ~300 kb in length, allow us to identify initiation sites and their firing probability with high confidence. In particular, for the first time, we are able to measure genome-wide the absolute efficiency of human replication initiation. We find that the distribution of human replication initiation is consistent with inefficient, stochastic initiation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. In particular, we find sites of human replication initiation are not confined to well-defined replication origins but are instead distributed across broad initiation zones consisting of many initiation sites. Furthermore, we find no correlation of initiation events between neighboring initiation zones. Although most early initiation events occur in early-replicating regions of the genome, a significant number occur in late-replicating regions. The fact that initiation sites in typically late-replicating regions have some probability of firing in early S phase suggests that the major difference between initiation events in early and late replicating regions is their intrinsic probability of firing, as opposed to a qualitative difference in their firing-time distributions. Moreover, modeling of replication kinetics demonstrates that measuring the efficiency of initiation-zone firing in early S phase suffices to predict the average firing time of such initiation zones throughout S phase, further suggesting that the differences between the firing times of early and late initiation zones are quantitative, rather than qualitative. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

genomics↗

bioRxiv2018

Fission Yeast Cells Grow Approximately Exponentially

How the rate of cell growth is influenced by cell size is a fundamental question of cell biology. The simple model that cell growth is proportional to cell size, based on the proposition that larger cells have proportionally greater synthetic capacity than smaller cells, leads to the predication that the rate of cell growth increases exponentially with cell size. However, other modes of cell growth, including bilinear growth, have been reported. The distinction between exponential and bilinear growth has been explored in particular detail in the fission yeast Schizosaccharomyces pombe. We have revisited the mode of fission yeast cell growth using high-resolution time-lapse microscopy and find, as previously reported, that these two growth models are difficult to distinguish both because of the similarity in shapes between exponential and bilinear curves over the two-fold change in length of a normal cell cycle and because of the substantial biological and experimental noise inherent to these experiments. Therefore, we contrived to have cells grow more than two fold, by holding them in G2 for up to eight hours. Over this extended growth period, in which cells grow up to 5.5-fold, the two growth models diverge to the point that we can confidently exclude bilinear growth as a general model for fission yeast growth. Although the growth we observe is clearly more complicated than predicted by simple exponential growth, we find that exponential growth is a robust approximation of fission yeast growth, both during an unperturbed cell cycle and during extended periods of growth.

cell biology↗

bioRxiv2017

Transcriptome-wide analysis of the functional intronome using spliceosome profiling

Full understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although RNA-Seq provides much of this information, the low abundance of many spliced transcripts (often due to their rapid cytoplasmic decay) limits the ability of RNA-Seq alone to reveal the full repertoire of spliced species. Here we present \"spliceosome profiling\", a strategy based on deep sequencing of RNAs co-purifying with late stage spliceosomes. Spliceosome profiling allows for unambiguous mapping of intron ends to single nucleotide resolution and branchpoint identification at unprecedented depths. Our data reveal hundreds of new introns in S. pombe and numerous others that were previously misannotated. By providing a means to directly interrogate sites of spliceosome assembly and catalysis genome-wide, spliceosome profiling promises to transform our understanding of RNA processing in the nucleus much like ribosome profiling has transformed our understanding mRNA translation in the cytoplasm.

genomics↗

bioRxiv2017

Genome-Wide Identification of Early-Firing Human Replication Origins by Optical Replication Mapping

The timing of DNA replication is largely regulated by the location and timing of replication origin firing. Therefore, much effort has been invested in identifying and analyzing human replication origins. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual origins in metazoans has made mapping the location and timing of replication initiation in human cells difficult. We have mapped early-firing origins in HeLa cells using Optical Replication Mapping, a high-throughput single-molecule approach based on Bionano Genomics genomic mapping technology. The single-molecule nature and 290-fold coverage of our dataset allowed us to identify origins that fire with as little as 1% efficiency. We find sites of human replication initiation in early S phase are not confined to well-defined efficient replication origins, but are instead distributed across broad initiation zones consisting of many inefficient origins. These early-firing initiation zones co-localize with initiation zones inferred from Okazaki-fragment-mapping analysis and are enriched in ORC1 binding sites. Although most early-firing origins fire in early-replication regions of the genome, a significant number fire in late-replicating regions, suggesting that the major difference between origins in early and late replicating regions is their probability of firing in early S-phase, as opposed to qualitative differences in their firing-time distributions. This observation is consistent with stochastic models of origin timing regulation, which explain the regulation of replication timing in yeast.

genomics↗

bioRxiv2017

The Fission Yeast S-Phase Cyclin Cig2 Can Drive Mitosis

Commitment to mitosis is regulated by cyclin-dependent kinase (CDK) activity. In the fission yeast Schizosaccharomyces pombe, the major B-type cyclin, Cdc13, is necessary and sufficient to drive mitotic entry. Furthermore, Cdc13 is also sufficient to drive S phase, demonstrating that a single cyclin can regulate alternating rounds of replication and mitosis and providing the foundation of the quantitative model of CDK function. It has been assumed that Cig2, a B-type cyclin expressed only during S-phase and incapable of driving mitosis in wild-type cells, was specialized for S-phase regulation. Here, we show that Cig2 is capable of driving mitosis. Cig2/CDK activity drives mitotic catastrophe--lethal mitosis in inviably small cells--in cells that lack CDK inhibition by tyrosine-phosphorylation. Moreover, Cig2/CDK can drive mitosis in the absence of Cdc13/CDK activity and constitutive expression of Cig2 can rescue loss of Cdc13 activity. These results demonstrate that in fission yeast, not only can the presumptive M-phase cyclin drive S phase, but the presumptive S-phase cyclin can drive M phase, further supporting the quantitative model of CDK function. Furthermore, these results provide an explanation, previously proposed on the basis of computational analyses, for the surprising observation that cells expressing a single-chain Cdc13-Cdc2 CDK do not require Y15 phosphorylation for viability. Their viability is due to the fact that in such cells, which lack Cig2/CDK complexes, Cdc13/CDK activity is unable to drive mitotic catastrophe.

cell biology↗

bioRxiv2017

Replication Fork Slowing And Stalling are Distinct, Checkpoint-Independent Consequences of Replicating Damaged DNA

In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents--MMS, 4NQO and bleomycin--that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.\n\nAuthor SummaryFaithful duplication of the genome is essential for genetic stability of organisms and species. To ensure faithful duplication, cells must be able to replicate damaged DNA. To do so, they employ checkpoints that regulate replication in response to DNA damage. However, the mechanisms by which checkpoints regulate DNA replication forks, the macromolecular machines that contain the helicases and polymerases required to unwind and copy the parental DNA, is unknown. We have used DNA combing, a single-molecule technique that allows us to monitor the progression of individual replication forks, to characterize the response of fission yeast replication forks to DNA damage that blocks the replicative polymerases. We find that forks pass most lesions with only a brief pause and that this lesion bypass is checkpoint independent. However, at a low frequency, forks stall at lesions, and that the checkpoint is required to prevent these stalls from accumulating single-stranded DNA. Our results suggest that the major role of the checkpoint is not to regulate the interaction of replication forks with DNA damage, per se, but to mitigate the consequences of fork stalling when forks are unable to successfully navigate DNA damage on their own.

cell biology↗

bioRxiv2016

An Estradiol-Inducible Promoter Enables Fast, Graduated Control of Gene Expression in Fission Yeast

The fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on {beta}-estradiol-regulated function of the human estrogen receptor, for use in S. pombe. We show that this promoter system, termed Z3EV, turns on quickly, can reach a maximal induction of 20 fold, and exhibits a linear dose response over its entire induction range, with few off target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by {beta}-estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast.

cell biology↗