Unveiling adaptive mechanisms though experimental evolution: the role of duplicated genes and phenotypic plasticity in yeast, and the genetic variability in Coxsackievirus

Los seres vivos se enfrentan a condiciones ambientales cambiantes y habitualmente estresantes, agravadas por el cambio climático, que ponen a prueba su capacidad de supervivencia. El cambio en la composición genética de las poblaciones reside en las mutaciones, que son la fuente para la evolución y la adaptación a los cambios. La diversidad genética intrapoblacional está regulada por dos grandes fuerzas evolutivas que cambian la composición genética permitiendo así el acceso a nuevos fenotipos: la deriva genética y la selección natural. Por un lado, la deriva genética fija mutaciones en la población de manera aleatoria e independiente del efecto que suponga dicha mutación para la población. Por otro lado, la selección natural si bien favorece la fijación de mutaciones beneficiosas también elimina mutaciones perjudiciales en un determinado ambiente. Por lo tanto, el efecto de las mutaciones está fuertemente ligado al ambiente y, en consecuencia, aquellas poblaciones que exhiben una mayor diversidad genética serán capaces de evolucionar más rápido y adaptarse mejor. Además de la evolución por selección natural y deriva genética, la duplicación genética también es de especial importancia para la evolución, pues es la principal fuente de nuevo material genético y de innovaciones biológicas. Tanto es así que las grandes transiciones evolutivas, como la radiación de las plantas angiospermas o las grandes innovaciones morfológicas en animales se han relacionado con eventos de duplicación. Sin embargo, los mecanismos moleculares que permiten mantener los genes duplicados durante largos periodos de tiempo siguen siendo desconocidos. Para intentar ampliar el conocimiento respecto a estos mecanismos, y dado que los efectos de la evolución en la naturaleza tarda mucho tiempo en poder observarse, se necesitan sistemas biológicos que sean capaces de evolucionar rápido. En este contexto, te tiempo adecuado al experimentador, se han llevado a cabo estudios de evolución experimental con virus y microorganismos que han supuesto una herramienta muy valiosa en el estudio de la biología evolutiva. Por un lado, los virus presentan tasas de mutación extremadamente elevadas, especialmente los virus de ARN, lo que les confiere la capacidad de adaptarse de manera muy rápida a un cambio ambiental. Por otro lado, la levadura Saccharomyces cerevisiae, cuyo origen se debe a una duplicación genómica acaecida hace más de 100 millones de años, es un buen modelo para estudiar la duplicación genética y su papel en la adaptación. Además, más allá de ser útil para la evolución experimental y el estudio de la biología evolutiva, la elevada capacidad evolutiva de los virus de ARN supone un desafío importante para la medicina y en la prevención de enfermedades emergentes y, la levadura S.cerevisiae es una de las especies con mayor impacto económico en la industria biotecnológica. Por lo tanto, llevar a cabo un análisis exhaustivo del efecto de las mutaciones en poblaciones virales y estudiar en profundidad como la duplicación genética influye la adaptación y la innovación biológica en la levadura, es fundamental para generar un marco de conocimiento que permitirá maximizar el potencial biomédico y biotecnológico de los virus de ARN y de la levadura. Esta tesis doctoral trata de abordar dos cuestiones fundamentales en biología evolutiva: ¿Cuáles son los mecanismos moleculares que determinan la estabilidad de los genes duplicados en el genoma durante el tiempo suficiente para que sean capaces de adquirir relevancia evolutiva? Y, ¿Cómo contribuye la variabilidad genética a la evolución y a la adaptación a nuevos ambientes? Para intentar responder a estas preguntas hemos utilizado dos modelos experimentales diferentes: la levadura S. cerevisiae y el coxsackievirus B3 (CVB3). En la primera parte de esta tesis, con la levadura hemos visto que el nivel de expresión génica de los genes duplicados, así como la divergencia transcripcional y funcional, son fundamentales para la estabilidad de los genes duplicados en el genoma. Además, hemos observado que la plasticidad transcripcional de los genes duplicados juegan un papel clave en la adaptación a nuevos ambientes desfavorables, tales como condiciones de estrés oxidativo o altas concentraciones de etanol, glicerol o ácido láctico. Y en la segunda parte de la tesisi, utilizando el virus CVB3, hemos realizado una aproximación de Deep mutational scanning sobre las proteínas de la cápside viral y hemos generado poblaciones virales con una elevada variabilidad genética. Gracias a ello hemos podido evaluar el efecto de las mutaciones en la cápside caracterizando alrededor del 90% de los cambios de amino ácidos. Además, hemos empleado estas poblaciones virales altamente diversas y hemos estudiado como esta variabilidad genética contribuye a la adaptación contra la inactivación térmica. Nuestros resultados muestran que, incluso en virus de ARN con tasas de mutación extremadamente elevadas, un aumento de la diversidad genética de la población al inicio de la evolución experimental acelera el proceso evolutivo y facilita la adaptación al nuevo ambiente.Living beings face changing and usually stressful environmental conditions, aggravated by climate change, which tests their ability to survive. Changes in the genetic composition of the population, in the form of mutations, is the source for evolution and adaptation to environmental changes. This genetic diversity is driven by two major evolutionary forces that change the genetic composition in a population, allowing access to new phenotypes: genetic drift and natural selection. On one hand, genetic drift randomly fixes mutations in the population independent of their effect. On the other, natural selection either selects for beneficial mutations or purges deleterious mutations in a given environment. Hence, the effect of the mutations is closely linked to the environment and, as a consequence, populations exhibiting high genetic diversity can evolve faster and adapt better to environmental fluctuations. In addition to natural selection and genetic drift, gene duplication is also of great importance to evolution, as it is the main source of new genetic material. Not surprisingly, gene duplication has been related to major leaps in evolution, such as the radiation of angiosperm plants or large morphological innovations in animals. However, the molecular mechanisms that underlie the preservation of duplicated genes for long periods of time remain unknown. To better understand these mechanisms, experimental systems enabling rapid evolution are needed as the natural time scale for natural evolution can be extremely long. For this reason, experimental evolution approaches using viruses and microorganisms have become a valuable tool in evolutionary biology. On one hand, viruses show extremely high mutation rates, especially RNA viruses, conferring them the ability to rapidly adapt to a changing environment, thus representing an ideal model to study the effect of mutations. On the other hand, the yeast Saccharomyces cerevisiae, which has its origin in a whole genome duplication that occurred more than 100 million years ago, is a good model for studying gene duplication and its role in adaptation. Moreover, beyond their use as models for understanding evolutionary processes, the rapid evolutionary capacity of RNA viruses poses a challenge for treating and preventing infections and S. cerevisiae is currently one of the species with the largest biotechnological and economic impact. Therefore, a comprehensive analysis of the effect of mutations in large virus populations and, a deep knowledge about how genetic duplication influences adaptation and biological innovation is essential for gaining a better understanding of evolutionary processes and can help maximize our use of the full biomedical and biotechnological potential of RNA viruses and the budding yeast. This doctoral thesis aims to shed light on two fundamental evolutionary biology questions: What are the molecular mechanisms that determine the genomic stability of duplicated genes that are maintained in the genome for enough time to acquire evolutionary relevance? And, how does genetic variability contribute to evolution and adaptation? To address these questions we used two different models: the yeast S.cerevisiae and the coxsackievirus B3 (CVB3). In the first part of this thesis, we show that the level of gene expression of duplicated genes, as well as transcriptional and functional divergence, are key for the stability of duplicated genes. In addition, we find that the transcriptional plasticity of duplicated genes plays a key role in adaptation to new and stressful environments like high concentrations of ethanol, glycerol, and lactate or for oxidative stress conditions. In the second part, we performed a deep mutational scanning of the CVB3 capsid to generate highly genetically diverse populations and captured the mutational fitness effect of >90% of all possible single amino acid mutations in the viral capsid. We then used these highly diverse populations to study the contribution of genetic variability to adapt to thermal inactivation, observing that increasing the initial genetic variability in the population helps evolution even in RNA viruses with extremely high mutation rates

Increased RNA virus population diversity improves adaptability

The replication machinery of most RNA viruses lacks proofreading mechanisms. As a result, RNA virus populations harbor a large amount of genetic diversity that confers them the ability to rapidly adapt to changes in their environment. In this work, we investigate whether further increasing the initial population diversity of a model RNA virus can improve adaptation to a single selection pressure, thermal inactivation. For this, we experimentally increased the diversity of coxsackievirus B3 (CVB3) populations across the capsid region. We then compared the ability of these high diversity CVB3 populations to achieve resistance to thermal inactivation relative to standard CVB3 populations in an experimental evolution setting. We find that viral populations with high diversity are better able to achieve resistance to thermal inactivation at both the temperature employed during experimental evolution as well as at a more extreme temperature. Moreover, we identify mutations in the CVB3 capsid that confer resistance to thermal inactivation, finding significant mutational epistasis. Our results indicate that even naturally diverse RNA virus populations can benefit from experimental augmentation of population diversity for optimal adaptation and support the use of such viral populations in directed evolution efforts that aim to select viruses with desired characteristics.This work was funded by a Grant from the Spanish Ministerio de Ciencia, Innovación y Universidades to RG (BFU2017-86094-R). RG holds the Ramón y Cajal fellowship from the Spanish Ministry of Economy and Competitiveness (RYC-2015-17517) and FM an FPI grant from the Spanish Ministerio de Ciencia, Innovación y Universidades (BES-2016-076677)

The Role of Ancestral Duplicated Genes in Adaptation to Growth on Lactate, a Non-Fermentable Carbon Source for the Yeast Saccharomyces cerevisiae

[EN] The cell central metabolism has been shaped throughout evolutionary times when facing challenges from the availability of resources. In the budding yeast, Saccharomyces cerevisiae, a set of duplicated genes originating from an ancestral whole-genome and several coetaneous small-scale duplication events drive energy transfer through glucose metabolism as the main carbon source either by fermentation or respiration. These duplicates (~a third of the genome) have been dated back to approximately 100 MY, allowing for enough evolutionary time to diverge in both sequence and function. Gene duplication has been proposed as a molecular mechanism of biological innovation, maintaining balance between mutational robustness and evolvability of the system. However, some questions concerning the molecular mechanisms behind duplicated genes transcriptional plasticity and functional divergence remain unresolved. In this work we challenged S. cerevisiae to the use of lactic acid/lactate as the sole carbon source and performed a small adaptive laboratory evolution to this non-fermentative carbon source, determining phenotypic and transcriptomic changes. We observed growth adaptation to acidic stress, by reduction of growth rate and increase in biomass production, while the transcriptomic response was mainly driven by repression of the whole-genome duplicates, those implied in glycolysis and overexpression of ROS response. The contribution of several duplicated pairs to this carbon source switch and acidic stress is also discussed.This research was funded by Spanish National Plan for Scientific and Technical Research and Innovation from the Spanish Ministry of Economy and Competitiveness (MINECOFEDER), actually the Ministry of Science and Innovation (MCIN), Spanish Research Agency (AEI), MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe (FEDER "Una forma de hacer Europa") with grant number BFU2015-66073-P (to M.A.F.) and Generalitat Valenciana, Conselleria de Innovacion, Universidades y Sociedad Digital with grant number SEJI/2018/046 (to C.T.). F.M. was supported by a Spanish PhD Fellowship number FPI BES-2016-076677, from MCIN/AEI/10.13039/501100011033 and ESF "Investing in your future".Mattenberger, F.; Fares Riaño, MA.; Toft, C.; Sabater-Muñoz, B. (2021). The Role of Ancestral Duplicated Genes in Adaptation to Growth on Lactate, a Non-Fermentable Carbon Source for the Yeast Saccharomyces cerevisiae. International Journal of Molecular Sciences. 22(22):1-17. https://doi.org/10.3390/ijms222212293S117222

Expression properties exhibit correlated patterns with the fate of duplicated genes, their divergence, and transcriptional plasticity in Saccharomycotina

[EN] Gene duplication is an important source of novelties and genome complexity. What genes are preserved as duplicated through long evolutionary times can shape the evolution of innovations. Identifying factors that influence gene duplicability is therefore an important aim in evolutionary biology. Here, we show that in the yeast Saccharomyces cerevisiae the levels of gene expression correlate with gene duplicability, its divergence, and transcriptional plasticity. Genes that were highly expressed before duplication are more likely to be preserved as duplicates for longer evolutionary times and wider phylogenetic ranges than genes that were lowly expressed. Duplicates with higher expression levels exhibit greater divergence between their gene copies. Duplicates that exhibit higher expression divergence are those enriched for TATA-containing promoters. These duplicates also show transcriptional plasticity, which seems to be involved in the origin of adaptations to environmental stresses in yeast. While the expression properties of genes strongly affect their duplicability, divergence and transcriptional plasticity are enhanced after gene duplication. We conclude that highly expressed genes are more likely to be preserved as duplicates due to their promoter architectures, their greater tolerance to expression noise, and their ability to reduce the noise-plasticity conflict.We would like to thank members of Fares' Lab for a careful reading and discussion of the results in the manuscript. We are also grateful to colleagues at Trinity College for helpful discussions. This work was supported by a grant from the Spanish Ministerio de Economia y Competitividad (MINECO-FEDER; BFU2015-66073-P) to M.A.F. F.M. is supported by a PhD grant from the Spanish Ministerio de Economia y Competitividad (reference: BES-2016-076677). C.T. was supported by a grant Juan de la Cierva from the Spanish Ministerio de Economia y Competitividad (reference: JCA-2012-14056).Mattenberger, F.; Sabater-Muñoz, B.; Toft, C.; Sablok, G.; Fares Riaño, MA. (2017). Expression properties exhibit correlated patterns with the fate of duplicated genes, their divergence, and transcriptional plasticity in Saccharomycotina. DNA Research. 24(6):559-570. https://doi.org/10.1093/dnares/dsx025S559570246Ohno, S. (1999). Gene duplication and the uniqueness of vertebrate genomes circa 1970–1999. Seminars in Cell & Developmental Biology, 10(5), 517-522. doi:10.1006/scdb.1999.0332Lynch, M. (2000). The Evolutionary Fate and Consequences of Duplicate Genes. Science, 290(5494), 1151-1155. doi:10.1126/science.290.5494.1151Otto, S. P., & Whitton, J. (2000). Polyploid Incidence and Evolution. Annual Review of Genetics, 34(1), 401-437. doi:10.1146/annurev.genet.34.1.401Carretero-Paulet, L., & Fares, M. A. (2012). 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The Role of Ancestral Duplicated Genes in Adaptation to Growth on Lactate, a Non-Fermentable Carbon Source for the Yeast Saccharomyces cerevisiae

The cell central metabolism has been shaped throughout evolutionary times when facing challenges from the availability of resources. In the budding yeast, Saccharomyces cerevisiae, a set of duplicated genes originating from an ancestral whole-genome and several coetaneous small-scale duplication events drive energy transfer through glucose metabolism as the main carbon source either by fermentation or respiration. These duplicates (~a third of the genome) have been dated back to approximately 100 MY, allowing for enough evolutionary time to diverge in both sequence and function. Gene duplication has been proposed as a molecular mechanism of biological innovation, maintaining balance between mutational robustness and evolvability of the system. However, some questions concerning the molecular mechanisms behind duplicated genes transcriptional plasticity and functional divergence remain unresolved. In this work we challenged S. cerevisiae to the use of lactic acid/lactate as the sole carbon source and performed a small adaptive laboratory evolution to this non-fermentative carbon source, determining phenotypic and transcriptomic changes. We observed growth adaptation to acidic stress, by reduction of growth rate and increase in biomass production, while the transcriptomic response was mainly driven by repression of the whole-genome duplicates, those implied in glycolysis and overexpression of ROS response. The contribution of several duplicated pairs to this carbon source switch and acidic stress is also discussed

Chaperoning the <i>Mononegavirales</i>: Current Knowledge and Future Directions

The order Mononegavirales harbors numerous viruses of significant relevance to human health, including both established and emerging infections. Currently, vaccines are only available for a small subset of these viruses, and antiviral therapies remain limited. Being obligate cellular parasites, viruses must utilize the cellular machinery for their replication and spread. Therefore, targeting cellular pathways used by viruses can provide novel therapeutic approaches. One of the key challenges confronted by both hosts and viruses alike is the successful folding and maturation of proteins. In cells, this task is faced by cellular molecular chaperones, a group of conserved and abundant proteins that oversee protein folding and help maintain protein homeostasis. In this review, we summarize the current knowledge of how the Mononegavirales interact with cellular chaperones, highlight key gaps in our knowledge, and discuss the potential of chaperone inhibitors as antivirals

Cellular protein folding capacity and temperature uniquely shape mutational fitness effects

Trabajo presentado a la Virtual Conference Virus Genomics and Evolution, celebrada del 13 al 15 de septiembre de 2021.Peer reviewe

Globally defining the effects of mutations in a picornavirus capsid

The capsids of non-enveloped viruses are highly multimeric and multifunctional protein assemblies that play key roles in viral biology and pathogenesis. Despite their importance, a comprehensive understanding of how mutations affect viral fitness across different structural and functional attributes of the capsid is lacking. To address this limitation, we globally define the effects of mutations across the capsid of a human picornavirus. Using this resource, we identify structural and sequence determinants that accurately predict mutational fitness effects, refine evolutionary analyses, and define the sequence specificity of key capsid-encoded motifs. Furthermore, capitalizing on the derived sequence requirements for capsid-encoded protease cleavage sites, we implement a bioinformatic approach for identifying novel host proteins targeted by viral proteases. Our findings represent the most comprehensive investigation of mutational fitness effects in a picornavirus capsid to date and illuminate important aspects of viral biology, evolution, and host interactions.Ministerio de Economia, Industria y Competitividad, Gobierno de España: BFU2017-86094-R / RYC-2015-17517 / BES-2016-076677. In addition, the authors would like to acknowledge the use of the Principe Felipe Research Center (CIPF) server which was cofinanced by the European Union through the Operativa Program of the European Regional Development Fund (ERDF/FEDER) of the Comunitat Valenciana 2014–2020.Peer reviewe

Evolution of phenotypes: understanding diversity and the role of plasticity in adaptation to new environments

Trabajo presentado al Meeting of the Society for Molecular Biology and Evolution (SMBE), celebrado en Manchester (UK) del 21 al 25 de julio de 2019.The Baker's yeast Saccharomyces cerevisiaeis one of the most important biotechnologically microbes because of its ability to produce and tolerate high levels of ethanol. Due to these properties, much interest has been put in understanding the genetic underpinnings of alcohol tolerance to improve the quality and productions of the products. A reason study by Voordeckers et al (2015) has shown that adaptation to high ethanol tolerance involves point mutation, copy number variation, ploidy changes, and clonal interference. Nonetheless, the transcriptional re-wiring occurring during the response or adaptation to ethanol and its importance in comparison with the contribution to mutations in coding changes has not been explored. With both copy number variation and change of ploidy involved in the adaptation to ethanol, indicates that duplication plays an important role in the adaptation. We have already observed in our previous studies that ancient duplicates (small-scale and whole genome duplicates) play an important role in acute ethanol response (Mattenbergeret al. 2017). Here we use experimental evolution of S. cerevisiaeto uncover the transcriptional rewiring during ethanol adaptation and its link with gene duplication. First the populations of S. cerevisiaewere evolved for hundreds of generations for the purpose of diversification and later we evolved the populations with ethanol being the only carbon source. Little overlap was observed between enriched functional categories of up-regulated genes in response and adaptation to ethanol. In particular, we see a significantly larger proportion of duplicated genes responding to ethanol stress than singletons. Moreover, the fold change in duplicated genes was higher compared to singletons. All of which indicate that duplicated genes play a central role in the acute and chronic response to ethanol.Peer reviewe

Role of ancient duplicates in the metabolic switching in Saccharomyces cerevisiae

Resumen del póster presentado al Society for Molecular Biology and Evolution Meeting (SMBEv), celebrado de forma virtual del 3 al 8 de julio de 2021.Gene duplication events have been associated with increasing biological complexity throughout the tree of life, but also with illnesses, such as cancer. Early evolutionary theories indicated that duplicated genes could explore alternative functions due to the relaxation of selective constraints in one of the copies, as the other remains an ancestral-function backup. In unicellular eukaryotes like yeasts, it has been demonstrated that the fate and persistence of both duplicated copies in the genome depend on the duplication mechanism (whole-genome or small-scale events). Although it has been shown that smallscale duplicates tend to innovate and whole-genome duplicates specialize in ancestral functions, the implication of ancient duplicates¿ transcriptional plasticity and transcriptional divergence on environmental and metabolic responses remains largely obscure. Here we subject Saccharomyces cerevisiae to a metabolic switch by enforcing acute and chronic growth on a non-fermentative carbon source (ethanol) unrevealing the central role, the ancient duplicates have in metabolic shifts. In particular, the duplicates respond by transcriptional rewiring, depending on their transcriptional background. Our results shed light on the mechanisms that determine the role of duplicates, and on their continued evolvability.Peer reviewe