Biology subjects

Rodriguez-Lopez, M.

Publications and source records attributed to Rodriguez-Lopez, M..

14 recordsLinked to original sources

bioRxiv2026

Gene function and expression profiling in yeast spores, killifish diapause embryos, and their post-dormant offspring cells

Dormancy is a reversible cellular state characterised by suspended proliferation and increased stress resilience, enabling long-term viability under adverse conditions. Although dormant cells are critical for the life cycle of diverse organisms, from microbes to humans, they are understudied compared to proliferating cells. We present a comparative investigation of dormant cells in two divergent species: spores of fission yeast and diapause embryos of turquoise killifish. A genome-wide screen for genes affecting the lifespan and heat-shock resilience of spores uncovered a trade-off between longevity and heat resistance, and considerable differences in the genetic basis for lifespan between spores and chronologically aging yeast cells. RNA-seq and mass-spectrometry analyses revealed substantial transcriptomic and proteomic changes in spores and diapause embryos, with ribosomal proteins induced as transcripts but repressed as proteins. Transcriptomic regulation of biological processes, but less so of specific genes, is broadly conserved across yeast spores, killifish diapause, and human dormant cancer cells, including the induction of autophagy- and translation-related processes and the repression of cell cycle-related processes. Spores and diapause embryos modulate their transcriptomes and proteomes in response to heat stress and prolonged time. These RNA and protein expression changes are uncoupled and differ from aging-related expression signatures in yeast cells and adult fish. Cells derived from older or stressed spores retain phenotypic differences for several cell divisions, reflected in altered expression signatures, lifespan and stress resilience. Similarly, diapause duration and heat exposure are associated with long-term expression signatures in post-diapause embryos before hatching. This study highlights core biological processes and principles that are remarkably conserved in distinct types of dormant cells.

genetics↗

bioRxiv2025

Microglial activation and alpha-synuclein oligomers drive the early inflammatory phase of Parkinson's disease

Parkinsons disease (PD) is characterised by insoluble -synuclein (Syn) aggregates in Lewy bodies (LBs) within the substantia nigra, with cortical pathology appearing as the disease progresses. Late-stage LB deposition, cellular stress, and neuronal loss obscure disease-driving events, we therefore performed multi-regional transcriptomic and aggregate profiling in early-midstage PD brains (Braak 3-4), where cortical regions are pathologically unaffected. We report neuroimmune activation as an early PD feature, characterised by the expansion of a high-SNCA-expressing microglial state. This robust immune signature occurs prior to LB formation, but is associated with oligomeric Syn within cortical microglia. In hiPSC-derived microglia, both endogenous Syn oligomerisation, and exogenous oligomer uptake, trigger transcriptional reprogramming, characterised by interferon-driven inflammation, antigen presentation, and mitochondrial suppression, closely mirroring the early PD brain. These findings describe mechanisms by which Syn oligomerisation potently initiates early neuroinflammation, highlighting a critical interplay between proteinopathy and immune activation at the earliest stages of disease.

neuroscience↗

bioRxiv2023

Broad functional profiling of fission yeast proteins using phenomics and machine learning

Many proteins remain poorly characterized even in well-studied organisms, presenting a bottleneck for research. We applied phenomics and machine-learning approaches with Schizosaccharomyces pombe for broad cues on protein functions. We assayed colony-growth phenotypes to measure the fitness of deletion mutants for 3509 non-essential genes in 131 conditions with different nutrients, drugs, and stresses. These analyses exposed phenotypes for 3492 mutants, including 124 mutants of priority unstudied proteins conserved in humans, providing varied functional clues. For example, over 900 proteins were newly implicated in the resistance to oxidative stress. Phenotype-correlation networks suggested roles for poorly characterized proteins through guilt by association with known proteins. For complementary functional insights, we predicted Gene Ontology (GO) terms using machine learning methods exploiting protein-network and protein-homology data (NET-FF). We obtained 56,594 high-scoring GO predictions, of which 22,060 also featured high information content. Our phenotype-correlation data and NET-FF predictions showed a strong concordance with existing PomBase GO annotations and protein networks, with integrated analyses revealing 1,675 novel GO predictions for 783 genes, including 47 predictions for 23 priority unstudied proteins. Experimental validation identified new proteins involved in cellular ageing, showing that these predictions and phenomics data provide a rich resource to uncover new protein functions.

genetics↗

bioRxiv2022

Large-scale changes of molecular network states explain complex traits

The complexity of many cellular and organismal traits results from poorly understood mechanisms integrating genetic and environmental factors via molecular networks. Here, we show when and how genetic perturbations lead to molecular changes that are confined to small parts of a network versus when they lead to large-scale adaptations of global network states. Integrating multi-omics profiling of genetically heterogeneous budding and fission yeast strains with an array of cellular traits identified a central state transition of the yeast molecular network that is related to PKA and TOR (PT) signaling. Genetic variants affecting this PT state globally shifted the molecular network along a single-dimensional axis, thereby modulating processes including energy- and amino acid metabolism, transcription, translation, cell cycle control and cellular stress response. We propose that genetic effects can propagate through large parts of molecular networks because of the functional requirement to centrally coordinate the activity of fundamental cellular processes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/519111v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@18d3323org.highwire.dtl.DTLVardef@1185111org.highwire.dtl.DTLVardef@17238c7org.highwire.dtl.DTLVardef@1edc6e6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Genetic variants directly or indirectly affecting the activity of PKA and/or TOR signaling cause global changes of transcriptomic and proteomic network states by modulating the activity of diverse cellular functions and network modules. Using marker genes acting downstream of PKA and TOR signaling we are able to quantify the activity status of combined PKA and TOR signaling (PT Score). This PT Score correlates with major transcriptomic and proteomic changes in response to genetic variability. Those large-scale molecular adaptations correlate with and explain phenotypic consequences for multiple cellular traits. Variants affecting the stoichiometry of proteins within a specific module have regional effects that remain confined to smaller parts of the molecular network. Variants affecting only one or very few proteins change molecular networks only locally. The global reorganization of network states caused by variants of the first type result in consequences for many cellular traits (i.e. pleiotropic effects), such as growth on different carbon sources, stress response, energy metabolism and replicative lifespan. (Created with BioRender.com) C_FIG

systems biology↗

bioRxiv2021

Functional profiling of long intergenic non-coding RNAs in fission yeast

Eukaryotic genomes express numerous long intergenic non-coding RNAs (lincRNAs) that do not overlap any coding genes. Some lincRNAs function in various aspects of gene regulation, but it is not clear in general to what extent lincRNAs contribute to the information flow from genotype to phenotype. To explore this question, we systematically analyzed cellular roles of lincRNAs in Schizosaccharomyces pombe. Using seamless CRISPR/Cas9-based genome editing, we deleted 141 lincRNA genes to broadly phenotype these mutants, together with 238 diverse coding-gene mutants for functional context. We applied high-throughput colony-based assays to determine mutant growth and viability in benign conditions and in response to 145 different nutrient, drug and stress conditions. These analyses uncovered phenotypes for 47.5% of the lincRNAs and 96% of the protein-coding genes. For 110 lincRNA mutants, we also performed high-throughput microscopy and flow-cytometry assays, linking 37% of these lincRNAs with cell-size and/or cell-cycle control. With all assays combined, we detected phenotypes for 84 (59.6%) of all lincRNA deletion mutants tested. For complementary functional inference, we analyzed colony growth of strains ectopically overexpressing 113 lincRNA genes under 47 different conditions. Of these overexpression strains, 102 (90.3%) showed altered growth under certain conditions. Clustering analyses provided further functional clues and relationships for some of the lincRNAs. These rich phenomics datasets associate lincRNA mutants with hundreds of phenotypes, indicating that most of the lincRNAs analyzed exert cellular functions in specific environmental or physiological contexts. This study provides groundwork to further dissect the roles of these lincRNAs in the relevant conditions.

genetics↗

bioRxiv2021

Barcode Sequencing and a High-throughput Assay for Chronological Lifespan Uncover Ageing-associated Genes in Fission Yeast

Ageing-related processes are largely conserved, with simple organisms remaining the main platform to discover and dissect new ageing-associated genes. Yeasts provide potent model systems to study cellular ageing owing their amenability to systematic functional assays under controlled conditions. Even with yeast cells, however, ageing assays can be laborious and resource-intensive. Here we present improved experimental and computational methods to study chronological lifespan in Schizosaccharomyces pombe. We decoded the barcodes for 3206 mutants of the latest gene-deletion library, enabling the parallel profiling of [~]700 additional mutants compared to previous screens. We then applied a refined method of barcode sequencing (Bar-seq), addressing technical and statistical issues raised by persisting DNA in dead cells and sampling bottlenecks in aged cultures, to screen for mutants showing altered lifespan during stationary phase. This screen identified 341 long-lived mutants and 1246 short-lived mutants which point to many previously unknown ageing-associated genes, including 51 conserved but entirely uncharacterized genes. The ageing-associated genes showed coherent enrichments in processes also associated with human ageing, particularly with respect to ageing in non-proliferative brain cells. We also developed an automated colony-forming unit assay for chronological lifespan to facilitate medium- to high-throughput ageing studies by saving time and resources compared to the traditional assay. Results from the Bar-seq screen showed good agreement with this new assay, validating 33 genes not previously associated with cellular ageing. This study provides an effective methodological platform and identifies many new ageing-associated genes as a framework for analysing cellular ageing in yeast and beyond.

genomics↗

bioRxiv2020

Pyphe: A python toolbox for assessing microbial growth and cell viability in high-throughput colony screens

Microbial fitness screens are a key technique in functional genomics. We present an all-in-one solution, pyphe, for automating and improving data analysis pipelines associated with large-scale fitness screens, including image acquisition and quantification, data normalisation, and statistical analysis. Pyphe is versatile and processes fitness data from colony sizes, viability scores from phloxine B staining or colony growth curves, all obtained with inexpensive transilluminating flatbed scanners. We apply pyphe to show that the fitness information contained in late endpoint measurements of colony sizes is similar to maximum growth slopes from time series. We phenotype gene-deletion strains of fission yeast in 59,350 individual fitness assays in 70 conditions, revealing that colony size and viability provide complementary, independent information. Viability scores obtained from quantifying the redness of phloxine-stained colonies accurately reflect the fraction of live cells within colonies. Pyphe is user-friendly, open-source and fully-documented, illustrated by applications to diverse fitness analysis scenarios.

genomics↗

bioRxiv2019

A natural variant of the sole pyruvate kinase of fission yeast lowers glycolytic flux triggering increased respiration and oxidative-stress resistance but decreased growth

Cells balance glycolysis with respiration to support their energetic and biosynthetic needs in different environmental or physiological contexts. With abundant glucose, many cells prefer to grow by aerobic glycolysis, or fermentation in yeast. Using 161 natural isolates of fission yeast, we investigated the genetic basis and phenotypic effects of the fermentation-respiration balance. The laboratory and a few other strains were more dependent on respiration. This trait was associated with a missense variant in a highly conserved region of Pyk1. Pyk1 is the single pyruvate kinase in fission yeast, while most organisms possess isoforms with different activity. This variant reduced Pyk1 activity and glycolytic flux. Replacing the low-activity pyk1 allele in the laboratory strain with the common high-activity allele was sufficient to increase fermentation and decrease respiration. This metabolic reprogramming triggered systems-level adaptations in the transcriptome and proteome, and in cellular phenotypes, including increased growth and chronological lifespan, but decreased resistance to oxidative stress. Thus, low Pyk1 activity provided no growth advantage but stress tolerance, despite increased respiration. The genetic tuning of glycolytic flux by a single-nucleotide change might reflect an adaptive trade-off in a species lacking pyruvate-kinase isoforms.

genetics↗

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↗

bioRxiv2019

Physiological changes during cellular ageing in fission yeast drive non-random patterns of genome rearrangements

Aberrant repair of DNA double-strand breaks can recombine distant pairs of chromosomal breakpoints. Such chromosomal rearrangements are a hallmark of ageing and compromise the structure and function of genomes. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To gain a better understanding of genome rearrangements during cellular ageing, we focused on a single diagnostic measure - DNA breakpoint junctions - allowing us to interrogate the changing genomic landscape in non-dividing cells of fission yeast (Schizosaccharomyces pombe). Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints on the same or different chromosomes, including the mitochondrial chromosome. An unexpected mechanism of genomic instability caused more local hotspots: age-associated reduction in an RNA-binding protein could trigger R-loop formation at target loci. This finding suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and potential mechanisms of genome rearrangements in non-dividing cells, which can be triggered by ageing-related changes in gene-regulatory proteins.

genomics↗

bioRxiv2018

A family of transcription factors that limit lifespan: ETS factors have conserved roles in longevity

Increasing average population age, and the accompanying burden of ill health, is one of the public health crises of our time. Understanding the basic biology of the ageing process may help ameliorate the pathologies that characterise old age. Ageing can be modulated, often through changes in gene expression where regulation of transcription plays a pivotal role. Activities of Forkhead transcription factors (TFs) are known to extend lifespan, but detailed knowledge of the broader transcriptional networks that promote longevity is lacking. This study focuses on the E twenty-six (ETS) family of TFs. This family of TFs is large, conserved across metazoa, and known to play roles in development and cancer, but the role of its members in ageing has not been studied extensively. In Drosophila, an ETS transcriptional repressor, Aop, and an ETS transcriptional activator, Pnt, are known to genetically interact with Foxo and activating Aop is sufficient to extend lifespan. Here, it is shown that Aop and Foxo effect a related gene-expression programme. Additionally, Aop can modulate Foxos transcriptional output to moderate or synergise with Foxo activity depending on promoter context, both in vitro and in vivo. In vivo genome-wide mRNA expression analysis in response to Aop, Pnt or Foxo indicated, and further experiments confirmed, that combinatorial activities of the three TFs dictate metabolic status, and that direct reduction of Pnt activity is sufficient to promote longevity. The role of ETS factors in longevity was not limited to Pnt and Aop. Knockdown of Ets21c or Eip74EF in distinct cell types also extended lifespan, revealing that lifespan is limited by transcription from the ETS binding site in multiple cellular contexts. Reducing the activity of the C. elegans ETS TF Lin-1 also extended lifespan, a finding that corroborates established evidence of roles of this TF family in ageing. Altogether, these results reveal the ETS family of TFs as pervasive and evolutionarily conserved brokers of longevity.

genetics↗

bioRxiv2018

Fitness Landscape of the Fission Yeast Genome

BackgroundNon-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.\n\nResultsTowards 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.\n\nConclusionsWe 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.

genomics↗

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↗