Biology subjects

Rallis, C.

Publications and source records attributed to Rallis, C..

13 recordsLinked to original sources

bioRxiv2026

Pharmacological stress exposes hidden allelic background effects in genetic interaction screen normalisation

Synthetic Genetic Array (SGA) analysis comprises the high-throughput crossing of a query deletion strain against a genome-wide deletion library to score fitness interactions in thousands of double mutants. SGAs have produced comprehensive genetic interaction maps in yeasts and have emerged as a leading platform for pharmacogenomics: mapping genetic modifiers of drug response, identifying synthetic lethal targets and illuminating mechanisms of drug action and resistance. We have previously demonstrated that in fission yeast, the ade6 mutant is functionally neutral relative to the parental library and can serve as a standard negative control for SGA screens. Here, while we confirm our previous observation, we show that this neutrality fails under pharmacological stress. Using Torin1, an ATP-competitive TOR kinase inhibitor, we demonstrate that the ade6 SGA fitness profile diverges from that of the parental library in a dose-dependent and genomically widespread manner. At 2 M Torin1 only 12.2% of scored genes exceed a 1.5-fold fitness difference between backgrounds; at 3 M this proportion rises to 43.2% -a 3.5-fold increase driven by qualitative reorganisation of the genetic interaction landscape rather than simple scaling of pre-existing differences. Gene ontology analysis of divergent genes implicates autophagy, iron starvation responses, central carbon metabolism, and vesicle trafficking, consistent with TOR-regulated nutrient adaptation being differentially affected by the ade6-M210/M216 point mutations in the library versus the ade6 null in the SGA control. Our results have implications in fission yeast and beyond and we propose solutions towards reliable retrieval of genetic interactions.

genetics↗

bioRxiv2026

Bro1-Mediated Trafficking Couples TOR Signalling to Cellular Metabolism and Longevity

Adaptation to nutrient availability requires coordination between growth control, metabolism, and intracellular trafficking. In eukaryotes, inhibition of Target of Rapamycin (TOR) signalling robustly promotes stress resistance and longevity, yet how reduced growth signalling is coupled to organelle dynamics and proteome remodelling remains unclear. Here, we identify the conserved ESCRT-associated protein Bro1 as a central integrator of TOR signalling, vacuolar trafficking, and metabolic adaptation. Using fission yeast, we show that Bro1 is required for normal lifespan and for the global proteomic reprogramming that accompanies TOR inhibition. In Bro1 mutant cells, repression of ribosome biogenesis is uncoupled from activation of catabolic, vacuolar, and metabolic pathways, resulting in an altered metabolic state characterised by elevated lipid metabolism and increased abundance of nutrient transporters. Mechanistically, Bro1 promotes TOR-dependent cargo deubiquitination, vacuolar trafficking, and turnover of plasma membrane hexose transporters and enables appropriate nuclear relocalisation of the transcriptional repressor Scr1. In the absence of Bro1, nutrient transporters persist at the cell surface despite TOR inhibition, conferring resistance to TOR inhibitors while impairing stress responses and reducing lifespan. Together, our findings establish Bro1 as a key coordinator linking ESCRT-mediated endosomal-vacuolar trafficking to TOR-dependent metabolic control. By coupling growth suppression to enhanced recycling and cellular maintenance, Bro1 enables the transition from growth to longevity-promoting states, revealing a mechanism connecting intracellular trafficking, metabolism, and ageing.

genetics↗

bioRxiv2025

Compromised ESCRT signalling is sufficient for resistance to the Target of Rapamycin Complex inhibitor Torin1 in fission yeast

BackgroundFission yeast cells defective in Golgi-endosomal sorting display high resistance to Torin1, a pan-Target of Rapamycin (TOR) inhibitor. TOR complexes regulate the ESCRT system to integrate nutrient availability with cell division. TOR activity is frequently deregulated in cancer, making it an attractive therapeutic target. Deregulated ESCRT activity has also been associated with cancer, but its role in mediating drug resistance is not fully understood. Herein, we have investigated the role of the ESCRT system in regulating sensitivity to Torin1. MethodsGrowth assays were used to monitor the growth of yeast cells. The effect of Torin1 on protein expression was monitored by immunoblotting. Fluorescence microscopy was used to investigate the action of Torin1 on protein localization. ResultsThe ESCRT system mediates Torin1-induced degradation of amino acid and glucose transporters. The expression of these transporters at the plasma membrane is not abolished in ESCRT mutants. Mutants unable to effectively ubiquitylate these transporters are also resistant to Torin1. Impaired ESCRT-mediated protein degradation is associated with strong resistance to Torin1. ConclusionsMutations in genes encoding ESCRT components have been reported in cancer. We present evidence that compromised ESCRT signalling is sufficient for resistance to Torin1. Cells defective in ESCRT signalling or ubiquitin homeostasis are highly resistant to Torin1. Our studies demonstrate that defective ESCRT-mediated proteolysis can suppress sensitivity to Torin1.

cell biology↗

bioRxiv2025

A Time-Resolved High-Throughput Screening of Fission Yeast Deletion Mutants for Oxidative Stress Resistance

Cells typically balance growth with stress responses - growing rapidly in low stress conditions and halting growth to defend against stress or to repair stress-induced damage. While numerous genome-wide screens have identified mutants resistant to oxidative stress, these have largely relied on static, end-point measurements. Here, we take a dynamic, time-resolved approach to uncover how fission yeast, Schizosaccharomyces pombe, adapts to oxidative stress over time. We have tracked the growth of 3,420 deletion mutants across nine time points spanning four days on both nutrient-rich solid media and that containing oxidative stress induced by hydrogen peroxide. This kinetic strategy revealed not just resistant or sensitive mutants. It allowed clustering of growth patterns across time and uncovered mutants that are capable of transiently uncoupling growth from stress response. Hydrogen peroxide induced a dose-dependent delay in colony expansion in most deletion strains, yet 15 mutants consistently maintained robust growth. These belong to different functional categories, highlighting diverse potential mechanisms ranging from altered DNA damage checkpoints to metabolic rewiring and growth regulation. By capturing dynamic trajectories rather than static outcomes, this study exposes hidden layers of growth under oxidative stress and identifies new genetic determinants of cellular resilience in fission yeast.

cell biology↗

bioRxiv2025

Evolutionary rewiring of host metabolism and interferon signalling by SARS-CoV-2 variants

SARS-CoV-2 variants differ in transmissibility and immune evasion, but their effects on host-cell metabolism and signalling remain less defined. Using integrated transcriptomic, phosphoproteomic, and amino acid profiling in primary nasal epithelial cells, we compared early and late host responses to pre-Omicron variants (Alpha, Beta), Delta, and Omicron subvariants (BA.1, BA.5). Pre-Omicron strains broadly suppressed antiviral interferon-stimulated gene expression and reprogrammed metabolism by reducing mitochondrial oxidative phosphorylation and {beta}-oxidation. Delta infection was associated with extensive transcriptional and metabolic remodelling, characterised by activation of stress- and growth-related kinases and selective retention of biosynthetic amino acids, consistent with a host response to stress and viral modulation of interferon-associated signalling. In contrast, Omicron infection elicited a more restrained response dominated by cytokine and survival pathways, with limited metabolic activation and interferon suppression. Together, these findings suggest SARS-CoV-2 has progressively evolved toward a strategy that maintains efficient upper-airway replication while minimising epithelial stress and inflammation.

systems biology↗

bioRxiv2024

Rapalink-1 reveals novel mTOR-dependent genes and the role of agmatinases in cellular growth and chronological lifespan

The mechanistic Target of Rapamycin, mTOR, is a conserved pro-ageing pathway with characterised inhibitors such as rapamycin, rapalogues and torins. Bi-steric third-generation inhibitors, such as rapalink-1 have been developed, however, their effects on organismal gene expression and lifespan have not been characterised. Here, we demonstrate that rapalink-1 affects fission yeast spatial and temporal growth and prolongs chronological lifespan with a distinct TORC1 selectivity profile. Endosome and vesicle-mediated transport and homeostasis processes related to autophagy and Pik3, the orthologue of human PI3K, render cells resistant to rapalink-1. Our study reveals mTOR-regulated genes with unknown roles in ageing including all fission yeast agmatinases, the enzymes that convert agmatine to putrescine and urea. Through genome-wide screens, we identify sensitive and resistant mutants to agmatine and putrescine. Genetic interactome assays for the agmatinase agm1 and further cell and molecular analyses, demonstrate that impairing the agmatinergic branch of arginine catabolism results in mTOR activity levels that are beneficial for growth but detrimental for chronological ageing. Our study reveals the anti-ageing action of agmatinases within a metabolic feedback circuit regulating mTOR activity levels and ageing and with possible implications to other systems, including human cells.

cell biology↗

bioRxiv2022

The AMPK-TORC1 signalling axis regulates caffeine-mediated DNA damage checkpoint override and cell cycle effects in fission yeast

Caffeine is a widely consumed neuroactive substance. It can modulate cell cycle progression, override DNA damage checkpoint signalling and increase chronological lifespan (CLS) in various model systems. Early studies suggested that caffeine inhibits the phosphatidylinositol 3-kinase-related kinase (PIKK) Rad3 to override DNA damage-induced cell cycle arrest in fission yeast. We have previously suggested that caffeine modulates cell cycle progression and lifespan by inhibiting the Target of Rapamycin Complex 1 (TORC1). Nevertheless, whether this inhibition is direct or not, has remained elusive. TORC1 controls metabolism and mitosis timing by integrating nutrients and environmental stress response (ESR) signalling. Nutritional or other stresses activate the Sty1-Ssp1-Ssp2 (AMP-activated protein kinase complex, AMPK) pathway, which inhibits TORC1 and accelerates mitosis through Sck2 inhibition. Additionally, activation of the ESR pathway can extend lifespan in fission yeast. Here, we demonstrate that caffeine activates Ssp1, Ssp2 and the AMPK{beta} regulatory subunit Amk2 to advance mitosis. Ssp2 is phosphorylated in an Ssp1-dependent manner following exposure to caffeine. Furthermore, Ssp1 and Amk2, are required for resistance to caffeine under conditions of prolonged genotoxic stress. The effects of caffeine on DNA damage sensitivity are uncoupled from mitosis in AMPK pathway mutants. We propose that caffeine interacts synergistically with other genotoxic agents to increase DNA damage sensitivity. Our findings suggest that caffeine accelerates mitotic division and is beneficial for CLS through AMPK. Direct pharmacological targeting of AMPK may serve towards health span and lifespan benefits beyond yeasts, given the highly conserved nature of this key regulatory cellular energy sensor.

cell biology↗

bioRxiv2020

Amino acids whose intracellular levels change most during aging alter chronological lifespan of fission yeast

Amino acid deprivation or supplementation can affect cellular and organismal lifespan, but we know little about the role of concentration changes in free, intracellular amino acids during aging. Here, we determine free amino-acid levels during chronological aging of non-dividing fission yeast cells. We compare wild-type with long-lived mutant cells that lack the Pka1 protein of the protein kinase A signalling pathway. In wild-type cells, total amino-acid levels decrease during aging, but much less so in pka1 mutants. Two amino acids strongly change as a function of age: glutamine decreases, especially in wild-type cells, while aspartate increases, especially in pka1 mutants. Supplementation of glutamine is sufficient to extend the chronological lifespan of wild-type but not of pka1{Delta} cells. Supplementation of aspartate, on the other hand, shortens the lifespan of pka1{Delta} but not of wild-type cells. Our results raise the possibility that certain amino acids are biomarkers of aging, and their concentrations during aging can promote or limit cellular lifespan.

physiology↗

bioRxiv2020

Caffeine stabilises fission yeast Wee1 in a Rad24-dependent manner but attenuates its expression in response to DNA damage identifying a putative role for TORC1 in mediating its effects on cell cycle progression

The widely consumed neuroactive compound caffeine has generated much interest due to its ability to override the DNA damage and replication checkpoints. Previously Rad3 and its homologues was thought to be the target of caffeines inhibitory activity. Later findings indicate that the Target of Rapamycin Complex 1 (TORC1) is the preferred target of caffeine. Effective Cdc2 inhibition requires both the activation of the Wee1 kinase and inhibition of the Cdc25 phosphatase. The TORC1, DNA damage, and environmental stress response pathways all converge on Cdc25 and Wee1. We previously demonstrated that caffeine overrides DNA damage checkpoints by modulating Cdc25 stability. The effect of caffeine on cell cycle progression resembles that of TORC1 inhibition. Furthermore, caffeine activates the Sty1 regulated environmental stress response. Caffeine may thus modulate multiple signalling pathways that regulate Cdc25 and Wee1 levels, localisation and activity. Here we show that the activity of caffeine stabilises both Cdc25 and Wee1. The stabilising effect of caffeine and genotoxic agents on Wee1 was dependent on the Rad24 chaperone. Interestingly, caffeine inhibited the accumulation of Wee1 in response to DNA damage. Caffeine therefore modulates cell cycle progression contextually through increased Cdc25 activity and Wee1 repression following DNA damage via TORC1 inhibition.

cell biology↗

bioRxiv2019

The GATA transcription factor Gaf1 represses tRNA genes, inhibits growth, and extends chronological lifespan downstream of fission yeast TORC1

Target of Rapamycin Complex 1 (TORC1) signaling promotes growth and ageing. Inhibition of TORC1 leads to a down-regulation of factors that stimulate protein translation, including RNA polymerase III, which in turn contributes to longevity. TORC1-mediated post-transcriptional regulation of protein translation has been well studied, while analogous transcriptional regulation is less well understood. Here we screened fission yeast deletion mutants for resistance to Torin1, which inhibits TORC1 and cell growth. Mutants lacking the GATA transcription factor Gaf1 (gaf1{Delta}) grew normally even in high doses of Torin1. The gaf1{Delta} mutants shortened the chronological lifespan of non-dividing cells and diminished the lifespan extension triggered by Torin1 treatment. Expression profiling and genome-wide binding experiments showed that, after TORC1 inhibition, Gaf1 directly up-regulated genes for small-molecule metabolic pathways and indirectly repressed genes for protein translation. Surprisingly, Gaf1 bound to, and down-regulated the tRNA genes, so also functions as a transcription factor for genes transcribed by RNA polymerase III. We conclude that Gaf1 controls the transcription of both coding and tRNA genes to inhibit translation and growth downstream of TORC1.

genetics↗

bioRxiv2018

An essential role for dNTP homeostasis following CDK-induced replication stress

Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here we show that CDK-induced replication stress is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, Mus81-dependent DNA damage, and to genome instability. Cells respond to this replication stress by increasing dNTP supply through Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a dNTP supply and demand model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

molecular biology↗

bioRxiv2018

Uncovering natural longevity alleles from intercrossed pools of aging fission yeast cells

Quantitative traits often show large variation caused by multiple genetic factors. One such trait is the chronological lifespan of non-dividing yeast cells, serving as a model for cellular aging. Screens for genetic factors involved in ageing typically assay mutants of protein-coding genes. To identify natural genetic variants contributing to cellular aging, we exploited two strains of the fission yeast, Schizosaccharomyces pombe, that differ in chronological lifespan. We generated segregant pools from these strains and subjected them to advanced intercrossing over multiple generations to break up linkage groups. We chronologically aged the intercrossed segregant pool, followed by genome sequencing at different times to detect genetic variants that became reproducibly enriched as a function of age. A region on Chromosome II showed strong positive selection during ageing. Based on expected functions, two candidate variants from this region in the long-lived strain were most promising to be causal: small insertions and deletions in the 5-untranslated regions of ppk31 and SPBC409.08. Ppk31 is an orthologue of Rim15, a conserved kinase controlling cell proliferation in response to nutrients, while SPBC409.08 is a predicted spermine transmembrane transporter. Both Rim15 and the spermine-precursor, spermidine, are implicated in ageing as they are involved in autophagy-dependent lifespan extension. Single and double allele replacement suggests that both variants, alone or combined, have subtle effects on cellular longevity. Furthermore, deletion mutants of both ppk31 and SPBC409.08 rescued growth defects caused by spermidine. We propose that Ppk31 and SPBC409.08 may function together to modulate lifespan, thus linking Rim15/Ppk31 with spermidine metabolism.

genetics↗

bioRxiv2017

Long non-coding RNA repertoire and regulation by nuclear exosome, cytoplasmic exonuclease and RNAi in fission yeast

Transcriptomes feature pervasive, but poorly defined long non-coding RNAs (lncRNAs). We identify 5775 novel lncRNAs in Schizosaccharomyces pombe, nearly 4-times the previously annotated lncRNAs. Most lncRNAs become derepressed under genetic and physiological perturbations, especially during late meiosis. These lncRNAs are targeted by three RNA-processing pathways: the nuclear exosome, cytoplasmic exonuclease and RNAi, with substantial coordination and redundancy among pathways. We classify lncRNAs into cryptic unstable transcripts (CUTs), Xrn1-sensitive unstable transcripts (XUTs), and Dicer-sensitive unstable transcripts (DUTs). XUTs and DUTs are enriched for antisense lncRNAs, while CUTs are often bidirectional and actively translated. The cytoplasmic exonuclease and RNAi repress thousands of meiotically induced RNAs. Antisense lncRNA and sense mRNA expression often negatively correlate in the physiological, but not the genetic conditions. Intergenic and bidirectional lncRNAs emerge from nucleosome-depleted regions, upstream of positioned nucleosomes. This broad survey of the S. pombe lncRNA repertoire and characteristics provides a rich resource for functional analyses.

genomics↗