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El mantenimiento de la integridad del genoma es un requisito indispensable para garantizar el correcto funcionamiento de la célula, así como una transmisión fidedigna de la información genética a la descendencia. Las fuentes de daño genotóxico pueden ser exógenas o derivadas del propio metabolismo celular. Para prevenir la inestabilidad genética, que en humanos está asociada al envejecimiento, tumorigénesis y enfermedades genéticas, las células cuentan con mecanismos que evitan o resuelven amenazas que potencialmente comprometen la estabilidad del genoma. La transcripción es un proceso esencial para el funcionamiento celular, pero también puede ser una fuente de inestabilidad genética. Durante el proceso de transcripción, tienen lugar cambios topológicos en el ADN que lo hacen más susceptible de ser dañado. Además, pueden formarse unas estructuras conocidas como bucles R ( R loops ) que constan de un híbrido ADN ARN y una cadena sencilla de ADN desplazada. Aunque cumplen funciones fisiológicas, la aparición no programada de bucles R puede dar lugar a roturas en el ADN, ya sea por la exposición de una cadena sencilla de ADN, que es más vulnerable a los agentes genotóxicos, o por el bloqueo de la horquilla de replicación, que da lugar a roturas en el ADN derivadas del estrés replicativo. Debido a que el mal funcionamiento de factores implicados en la prevención o resolución de los bucles R ha sido relacionado con diversas patologías, la identificación de nuevos factores relacionados con su metabolismo es de interés para el avance de la Biomedicina. Con el objetivo de identificar nuevas proteínas esenciales implicadas en la prevención de inestabilidad genómica asociada a bucles R, desarrollamos en esta tesis un escrutinio genético de daño en el ADN en una colección de más de 1200 mutantes termosensibles en el organismo modelo S accharomyces cerevisiae . Para facilitar la búsqueda, utilizamos la expresión controlada de la enzima citidina deaminasa humana (AID), que exac

La capacidad de los hongos filamentosos de adaptarse a cambios ambientales a cambios ambientales ha conducido al desarrollo de mecanismos sofisticados, como la producción de metabolitos secundarios que, a pesar de no ser esenciales para estos organismos, usualmente les supone una ventaja adaptativa. Dentro del género Fusarium, se encuentran diversas estirpes reconocidas como grandes patógenos de plantas, causando pérdidas económicas significativas a nivel global. Además de su virulencia y producción de micotoxinas, estos hongos producen metabolitos con interés industrial, como los carotenoides. F. fujikuroi produce un carotenoide particular llamado neurosporaxantina, que ha demostrado tener un gran poder antioxidante en estudios in vitro, y actividad provitamina A y buena absorción en ratones. Lo que le otorga un gran interés biotecnológico. Aunque la ruta bioquímica y los genes estructurales de esta producción están descritos, aspectos del mecanismo regulatorio aún permanecen sin resolver. Esta tesis contribuye principalmente a profundizar en la comprensión de reguladores involucrados en la síntesis de carotenoides y en el metabolismo secundario del hongo F. fujikuroi. Se esclarece el rol del complejo White-Collar en F. fujikuroi, confirmando su mecanismo de acción como un heterodímero en este hongo. Se profundiza en el estudio de la proteína CarS, previamente identificada en mutantes súper productores de carotenoides. La sobreexpresión de carS confirma su papel como represor de la síntesis de carotenoides, provocando una represión absoluta de los genes involucrados en la síntesis y de la propia producción de este metabolito. Además, la generación de perfiles metabólicos a partir de datos de GC-MS y HPLC contribuyó a comprender el impacto de CarS y la luz en el metaboloma de este hongo. Finalmente, se aborda el estudio de un gen que codifica un factor de transcripción, ubicado junto a los genes de la carotenogenesis en el genoma. Su deleción y sobreexpresión no tuv, Filamentous fungi are subject to many environmental changes that compromise their survival in nature. The ability to sense and transduce environmental signals has given them an advantage to prosper in most diverse ecological niches. For this reason, these organisms have developed molecular systems to respond adequately to a changing environment. An excellent example is found in the sophisticated regulatory mechanisms that fungi use to control the production of secondary metabolites, which includes complex photosensory systems and mechanisms specifically involved in nutrients availability sensing. Beyond its own adaptation, these machineries have an impact on plants and human lives. An outstanding example are Fusarium fungi, which comprise many phytopathogenic species responsible for agriculture damage and huge economic losses around the world. Because of its extreme complexity, the secondary metabolism of F. fujikuroi has been focus of study for several decades. The many secondary metabolites produced by this fungus includes gibberellin phytohormones, responsible of elongating symptoms of the bakanae plant disease, mycotoxins, such as fumonisin and fusarins, and pigments, such as bikaverin, fusaric acid, and carotenoids. Due to the industrial interest of carotenoids, we focus our attention on the regulation of their biosynthesis. The main carotenoid produced by this fungus is neurosporaxanthin, an unusual xanthophyll that has shown interesting antioxidant properties in vitro and good absorption and provitamin A activities in mice. The production of carotenoids is regulated primarily by light, but is also affected by other environmental signals, such as nitrogen starvation, oxidative stress, or heat stress. Currently, all structural genes and biosynthetic steps of the carotenoid pathway have been described, and some regulatory proteins have been investigated. However, most aspects of the regulatory mechanisms remain to be understood. This thesis investigates some asp

Ribosomes are not totally globular machines. Instead, they comprise prominent structural protrusions and a myriad of tentacle-like projections, which are frequently made up of ribosomal RNA expansion segments and N- or C-terminal extensions of ribosomal proteins. This is more evident in higher eukaryotic ribosomes. One of the most characteristic protrusions, present in small ribosomal subunits in all three domains of life, is the so-called beak, which is relevant for the function and regulation of the ribosome’s activities. During evolution, the beak has transitioned from an all ribosomal RNA structure (helix h33 in 16S rRNA) in bacteria, to an arrangement formed by three ribosomal proteins, eS10, eS12 and eS31, and a smaller h33 ribosomal RNA in eukaryotes. In this review, we describe the different structural and functional properties of the eukaryotic beak. We discuss the state-of-the-art concerning its composition and functional significance, including other processes apparently not related to translation, and the dynamics of its assembly in yeast and human cells. Moreover, we outline the current view about the relevance of the beak’s components in human diseases, especially in ribosomopathies and cancer.

Genome integrity relies on the accuracy of DNA metabolism, but as appreciated for more than four decades, transcription enhances mutation and recombination frequencies. More recent research provided evidence for a previously unforeseen link between RNA and DNA metabolism, which is often related to the accumulation of DNA–RNA hybrids and R-loops. In addition to physiological roles, R-loops interfere with DNA replication and repair, providing a molecular scenario for the origin of genome instability. Here, we review current knowledge on the multiple RNA factors that prevent or resolve R-loops and consequent transcription–replication conflicts and thus act as modulators of genome dynamics.

DNA N6-adenine methylation (6mA) has recently gained importance as an epigenetic modification in eukaryotes. Its function in lineages with high levels, such as early-diverging fungi (EDF), is of particular interest. Here, we investigated the biological significance and evolutionary implications of 6mA in EDF, which exhibit divergent evolutionary patterns in 6mA usage. The analysis of two Mucorales species displaying extreme 6mA usage reveals that species with high 6mA levels show symmetric methylation enriched in highly expressed genes. In contrast, species with low 6mA levels show mostly asymmetric 6mA. Interestingly, transcriptomic regulation throughout development and in response to environmental cues is associated with changes in the 6mA landscape. Furthermore, we identify an EDF-specific methyltransferase, likely originated from endosymbiotic bacteria, as responsible for asymmetric methylation, while an MTA-70 methylation complex performs symmetric methylation. The distinct phenotypes observed in the corresponding mutants reinforced the critical role of both types of 6mA in EDF.

DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Strikingly, the loss of EXO1 or DNA2 results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. This phenomenon aligns with the observation that BRCA1 alone contributes to the expansion of ssDNA gaps. Remarkably, BRCA1-deficient cells become addicted to the overexpression of EXO1, DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers.

Small RNAS (sRNAs) participate in regulatory RNA interference (RNAi) mechanisms in a wide range of eukaryotic organisms, including fungi. The fungus Fusarium fujikuroi, a model for the study of secondary metabolism, contains a complete set of genes for RNAi pathways. We have analyzed by high-throughput sequencing the content of sRNAs in total RNA samples of F. fujikuroi grown in synthetic medium in the dark or after 1 h of illumination, using libraries below 150 nt, covering sRNAs and their precursors. For comparison, a parallel analysis with Fusarium oxysporum was carried out. The sRNA reads showed a higher proportion of 5′ uracil in the RNA samples of the expected sizes in both species, indicating the occurrence of genuine sRNAs, and putative miRNA-like sRNAs (milRNAS) were identified with prediction software. F. fujikuroi carries at least one transcriptionally expressed Ty1/copia-like retrotransposable element, in which sRNAs were found in both sense and antisense DNA strands, while in F. oxysporum skippy-like elements also show sRNA formation. The finding of sRNA in these mobile elements indicates an active sRNA-based RNAi pathway. Targeted deletion of dcl2, the only F. fujikuroi Dicer gene with significant expression under the conditions tested, did not produce appreciable phenotypic or transcriptomic alterations.

Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial. Here, we observe that the BRCA1/BARD1 ubiquitin E3 activity is not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identify substrates for BRCA1/BARD1. We find that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results provide additional insight about the importance of BRCA1/BARD1 E3 activity in Homologous Recombination.

The genome of living cells is constantly challenged by DNA lesions that interfere with cellular processes such as transcription and replication. A manifold of mechanisms act in concert to ensure adequate DNA repair, gene expression, and genome stability. Bulky DNA lesions, such as those induced by UV light or the DNA-damaging agent 4-nitroquinoline oxide, act as transcriptional and replicational roadblocks and thus represent a major threat to cell metabolism. When located on the transcribed strand of active genes, these lesions are handled by transcription-coupled nucleotide excision repair (TC-NER), a yet incompletely understood NER sub-pathway. Here, using a genetic screen in the yeast Saccharomyces cerevisiae, we identified histone variant H2A.Z as an important component to safeguard transcription and DNA integrity following UV irradiation. In the absence of H2A.Z, repair by TC-NER is severely impaired and RNA polymerase II clearance reduced, leading to an increase in double-strand breaks. Thus, H2A.Z is needed for proficient TC-NER and plays a major role in the maintenance of genome stability upon UV irradiation.

Salmonella enterica serovar Typhimurium expresses two type III secretion systems, T3SS1 and T3SS2, which are encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. These are essential virulent factors that secrete more than 40 effectors that are translocated into host animal cells. This study focuses on three of these effectors, SlrP, SspH1, and SspH2, which are members of the NEL family of E3 ubiquitin ligases. We compared their expression, regulation, and translocation patterns, their role in cell invasion and intracellular proliferation, their ability to interact and ubiquitinate specific host partners, and their effect on cytokine secretion. We found that transcription of the three genes encoding these effectors depends on the virulence regulator PhoP. Although the three effectors have the potential to be secreted through T3SS1 and T3SS2, the secretion of SspH1 and SspH2 is largely restricted to T3SS2 due to their expression pattern. We detected a role for these effectors in proliferation inside fibroblasts that is masked by redundancy. The generation of chimeric proteins allowed us to demonstrate that the N-terminal part of these proteins, containing the leucine-rich repeat motifs, confers specificity towards ubiquitination targets. Furthermore, the polyubiquitination patterns generated were different for each effector, with Lys48 linkages being predominant for SspH1 and SspH2. Finally, our experiments support an anti-inflammatory role for SspH1 and SspH2.

Los hongos filamentosos han sido utilizados como organismos modelos para el estudio de diferentes procesos biológicos como la regulación por la luz y el ritmo circadiano, el control de la esporulación o las interacciones patógeno-planta, entre otros. El interés de estos hongos radica en sus capacidades para producir una gran diversidad de metabolitos secundarios. En este sentido, Fusarium fujikuroi destaca por la riqueza de su metabolismo secundario. Este trabajo se ha centrado en potenciar la producción de metabolitos de interés industrial, como los carotenoides y las giberelinas, a través de la regulación y modificación del metabolismo secundario de F. fujikuroi. También se ha analizado la producción de estos metabolitos en un biorreactor de tanque agitado. Los datos muestran que la sobreexpresión del dominio catalítico de la HMG-CoA reductasa en F. fujikuroi provoca un aumento de la producción de giberelinas, aunque no afecta a la producción de carotenoides. El análisis de la producción de carotenoides y giberelinas del mutante carS-/OE:thmgR sugiere que la alteración del flujo metabólico de los precursores de los terpenoides afecta a la producción de estos. La optimización de los parámetros de fermentación para la producción de giberelinas en el biorreactor revela que la velocidad de agitación favorece la solubilidad del oxígeno disuelto durante la producción de giberelinas, logrando aumentar la producción en un 550% y reduciendo el tiempo de fermentación. Además, existe un correlación positiva entre la disminución del pH del cultivo y un aumento de la producción de giberelinas. Estos resultados muestran el potencial de escalada del bioproceso utilizando estirpes modificadas para la producción de giberelinas mediante la valorización de subproductos agroindustriales como medios de cultivo Por otra parte, los mutantes superproductoras en fondo mutante ?carX, obtenidos mediante mutagénesis con luz UV, acumulan hasta un 73% de ?-caroteno tras cultivarlos en medio op

BACKGROUND: Several studies have described a potential anti-tumour effect of cannabinoids (CNB). CNB receptor 2 (CB2) is mostly present in hematopoietic stem cells (HSC). The present study evaluates the anti-leukaemic effect of CNB. METHODS: Cell lines and primary cells from acute myeloid leukaemia (AML) patients were used and the effect of the CNB derivative WIN-55 was evaluated in vitro, ex vivo and in vivo. RESULTS: We demonstrate a potent antileukemic effect of WIN-55 which is abolished with CB antagonists. WIN-treated mice, xenografted with AML cells, had better survival as compared to vehicle or cytarabine. DNA damage-related genes were affected upon exposure to WIN. Co-incubation with the PARP inhibitor Olaparib prevented WIN-induced cell death, suggesting PARPmediated apoptosis which was further confirmed with the translocation of AIF to the nucleus observed in WIN-treated cells. Nicotinamide prevented WIN-related apoptosis, indicating NAD+ depletion. Finally, WIN altered glycolytic enzymes levels as well as the activity of G6PDH. These effects are reversed through PARP1 inhibition. CONCLUSIONS: WIN-55 exerts an antileukemic effect through Parthanatos, leading to translocation of AIF to the nucleus and depletion of NAD+, which are reversed through PARP1 inhibition. It also induces metabolic disruptions. These effects are not observed in normal HSC.

Los ribosomas son los orgánulos celulares que llevan a cabo el llamado proceso de traducción, en el cual las proteínas se sintetizan utilizando la información codificada en los RNA mensajeros (mRNAs). Los ribosomas están formados por dos subunidades ribosómicas, una grande, denominada 60S en eucariotas, y otra pequeña, denominada 40S en eucariotas, que a su vez están compuestas por proteínas ribosómicas y RNA ribosómicos (rRNAs). En esta Tesis, se ha utilizado el organismo modelo Saccharomyces cerevisiae para estudiar el proceso de biogénesis de los ribosomas, ya que este proceso está altamente conservado a lo largo de la evolución. En S. cerevisiae, la síntesis de ribosomas comienza en el nucléolo, continúa a través del nucleoplasma y finaliza en el citoplasma, donde los ribosomas maduros llevan a cabo la traducción. Los rRNAs se sintetizan como precursores (pre-rRNAs) que deben ser procesados y sufrir varias modificaciones para producir los diferentes rRNAs maduros (25S, 5.8S y 5S, que forman parte de la subunidad ribosómica 60S, y 18S, que forma parte de la subunidad ribosómica 40S). A lo largo del proceso de maduración de los ribosomas, se ensamblan 79 proteínas ribosómicas (46 para formar la subunidad ribosómica 60S y 33 para formar la subunidad ribosómica 40S), y se requieren más de 300 de los denominados factores de actuación en trans o factores de ensamblaje ribosómico. Estos factores garantizan la velocidad, la direccionalidad y la precisión de la síntesis de los ribosomas, se asocian a las subunidades ribosómicas nacientes y se disocian de ellas siguiendo un orden jerárquico, y no están presentes en los ribosomas maduros. A pesar del gran número de factores de ensamblaje ribosómico identificados hasta la fecha, algunos siguen siendo desconocidos y muchos están poco caracterizados. En esta Tesis, hemos estudiado la proteína de levadura Pol5, la cual fue primero identificada como la quinta DNA polimerasa esencial, y después como miembro de un complejo implic

The phytopathogenic fungus Fusarium fujikuroi has a rich secondary metabolism which includes the synthesis of very different metabolites in response to diverse environmental cues, such as light or nitrogen. Here, we focused our attention on fusarins, a class of mycotoxins whose synthesis is downregulated by nitrogen starvation. Previous data showed that mutants of genes involved in carotenoid regulation (carS, encoding a RING finger protein repressor), light detection (wcoA, White Collar photoreceptor), and cAMP signaling (AcyA, adenylate cyclase) affect the synthesis of different metabolites. We studied the effect of these mutations on fusarin production and the expression of the fus1 gene, which encodes the key polyketide synthase of the pathway. We found that the three proteins are positive regulators of fusarin synthesis, especially WcoA and AcyA, linking light regulation to cAMP signaling. Genes for two other photoreceptors, the cryptochrome CryD and the Vivid flavoprotein VvdA, were not involved in fusarin regulation. In most cases, there was a correspondence between fusarin production and fus1 mRNA, indicating that regulation is mainly exerted at the transcriptional level. We conclude that fusarin synthesis is subject to a complex control involving regulators from different signaling pathways.

In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is achieved dynamically, coordinated with dense and constantly changing transcriptional activity. H-NS, a major bacterial nucleoid structuring protein, is of special interest due to its interplay with RNA polymerase. H-NS:DNA nucleoprotein filaments inhibit transcription initiation by RNA polymerase. However, the discovery that genes silenced by H-NS can be activated by transcription originating from neighboring regions has suggested that elongating RNA polymerases can disassemble H-NS:DNA filaments. In this study, we present evidence that transcription-induced counter-silencing does not require transcription to reach the silenced gene; rather, it exerts its effect at a distance. Counter-silencing is suppressed by introducing a DNA gyrase binding site within the intervening segment, suggesting that the long-range effect results from transcription-driven positive DNA supercoils diffusing toward the silenced gene. We propose a model wherein H-NS:DNA complexes form in vivo on negatively supercoiled DNA, with H-NS bridging the two arms of the plectoneme. Rotational diffusion of positive supercoils generated by neighboring transcription will cause the H-NS-bound negativelysupercoiled plectoneme to “unroll” disrupting the H-NS bridges and releasing H-NS.

Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are intrinsically difficult to transcribe are those that tend to form R-loops, three-stranded nucleic acid str uct ures f ormed b y a DNA-RNA hybrid and the displaced ssDNA. Here we compared the transcription of an endogenous gene with and without an R-loop-forming sequence inserted. We show that, in agreement with previous in vivo and in vitro analyses, transcription elongation is delayed by R-loops in y east. Importantly, w e demonstrate that the Rat1 transcription terminator factor facilitates transcription throughout such str uct ures by inducing premature termination of arrested RNAPIIs. We propose that RNase H degrades the RNA moiety of the hybrid, pro viding an entry site for Rat1. T hus, w e ha v e unco v ered an unanticipated function of Rat1 as a transcription restoring f actor opening up the possibility that it ma y also promote transcription through other genomic DNA str uct ures intrinsically difficult to transcribe. If R-loop-mediated transcriptional stress is not relie v ed b y Rat1, it will cause genomic inst abilit y, probably through the increase of transcription-replication conflicts, a deleterious situation that could lead to cancer.

RECQL5 is a member of the conserved RecQ family of DNA helicases involved in the maintenance of genome stability that is specifically found in higher eukaryotes and associates with the elongating RNA polymerase II. To expand our understanding of its function we expressed human RECQL5 in the yeast Saccharomyces cerevisiae, which does not have a RECQL5 ortholog. We found that RECQL5 expression leads to cell growth inhibition, increased genotoxic sensitivity and transcription-associated hyperrecombination. Chromatin immunoprecipitation and transcriptomic analysis of yeast cells expressing human RECQL5 shows that this is recruited to transcribed genes and although it causes only a weak impact on gene expression, in particular at G + C-rich genes, it leads to a transcription termination defect detected as readthrough transcription. The data indicate that the interaction between RNAPII and RECQL5 is conserved from yeast to humans. Unexpectedly, however, the RECQL5-ID mutant, previously shown to have reduced the association with RNAPII in vitro, associates with the transcribing polymerase in cells. As a result, expression of RECQL5-ID leads to similar although weaker phenotypes than wild-type RECQL5 that could be transcription-mediated. Altogether, the data suggests that RECQL5 has the intrinsic ability to function in transcription-dependent and independent genome dynamics in S. cerevisiae.

During genome duplication, replication forks (RFs) can be stalled by different obstacles or by depletion of replication factors or nucleotides. A limited number of histone post-translational modifications at stalled RFs are involved in RF protection and restart. Provided the recent observation that the SIN3A histone deacetylase complex reduces transcription-replication conflicts, we explore the role of the SIN3A complex in protecting RFs under stressed conditions. We observe that Sin3A protein is enriched at replicating DNA in the presence of hydroxyurea. In this situation, Sin3A-depleted cells show increased RF stalling, H3 acetylation, and DNA breaks at stalled RFs. Under Sin3A depletion, RF recovery is impaired, and DNA damage accumulates. Importantly, these effects are partially dependent on the MUS81 endonuclease, which promotes DNA breaks and MRE11-dependent DNA degradation of such breaks. We propose that chromatin deacetylation triggered by the SIN3A complex limits MUS81 cleavage of stalled RFs, promoting genome stability when DNA replication is challenged.

DNA is an essential molecule for the proper functioning of the cell that is constantly exposed to exogenous and endogenous damage. One of the most dangerous injuries in DNA are doublestrand breaks. There are two main pathways to repair the break. The choice between one pathway or another is highly regulated and a decisive step in this decision is a process known as resection of the break ends. This mechanism consists of DNA degradation around the break, leaving two 3' ends or tails of single-stranded DNA. This leads to the repair of the damage through homologous recombination, a pathway that is generally error-free. There are many diseases that are associated with problems in DNA repair. Recently, some of the genes which mutations are related to Aicardi-Goutières syndrome (AGS) have been associated with defects in the repair process. This is an inflammatory disease that typically develops during early stages of life and currently has no cure. Although some studies provide some information, the connection between this syndrome and DNA repair is still poorly understood. In this Thesis we have focused on verifying whether the different mutants associated with this disease (TREX1, SAMHD1, the RNase H2 complex, ADAR1, IFIH1 and LSM11) have a role in repair. We have confirmed that all of them present a defect on resection. After this, we have dedicated our efforts to find treatments that allow us to reverse this repair phenotype, with the prospect that they could also rescue the inflammatory-associated response observed in patients with this syndrome., El ADN es una molécula esencial para el correcto funcionamiento de la célula que está constantemente expuesta a daños exógenos y endógenos. Una de las lesiones más peligrosas son los cortes de doble cadena en el ADN. Hay dos vías principales a través de las cuales puede repararse la rotura. La elección entre una ruta u otra está altamente regulada y uno paso decisivo para esta decisión es un proceso conocido como resección de los extremos de la rotura. Este mecanismo consiste en la degradación del ADN alrededor de la rotura, dejando dos extremos 3’ de cadena sencilla. Esto determina que el daño sea reparado por recombinación homóloga, una ruta que por lo general no genera errores. Son muchas las enfermedades que se asocian con problemas en la reparación del ADN. Recientemente, se han asociado algunos de los genes cuyas mutaciones se relacionan con el síndrome de Aicardi-Goutières (AGS) con defectos en el proceso de reparación. Ésta es una enfermedad inflamatoria que se desarrolla por lo general durante las primeras etapas de vida y para la cual no hay cura actualmente. Aunque algunos estudios proporcionan algo de información, aún es poco lo que se sabe acerca de la relación entre este síndrome y la reparación del ADN. En esta Tesis nos hemos centrado en comprobar si los diferentes mutantes asociados a esta enfermedad (TREX1, SAMHD1, el complejo RNasa H2, ADAR1, IFIH1 y LSM11) tienen un papel en reparación. Hemos confirmado que todos ellos presentan un defecto en resección. Tras esto, hemos dedicado nuestros esfuerzos a buscar tratamientos que permitan revertir este fenotipo, con la perspectiva de que también pudieran rescatar el proceso inflamatorio observado en pacientes con este síndrome.

Developmental transitions in fungi are key to their success in colonizing new habitats and adapting to stressful environmental conditions. Developmental transitions are regulated by environmental factors such as light. Neurospora crassa is an ascomycete heterothallic filamentous fungus and a model organism for research on several aspects of fungal biology, including the study of light sensing and morphogenesis during development. The life cycle of N. crassa includes asexual development with the formation of vegetative conidia that are easily dispersed, and sexual development, a complex process involving the formation of sexual reproductive structures (perithecia), were the ascospores (meiotic products) are formed. The velvet complex is a fungal-specific protein complex that participates in the regulation of gene expression in response to environmental signals such as light, as well as developmental processes, pathogenesis, and secondary metabolism. In this thesis, we have characterized the role of the velvet complex (the velvet proteins VE-1 and VE-2, and the methyltransferase LAE-1) during different stages of the life cycle of N. crassa. We first started studying the role of this complex during asexual development. Mutations in ve-1 or ve-2, but not in lae-1 led to shorter height of aerial tissue and an increased development of macroconidia. Additionally, when ve-1 or ve-2 mutations were combined with mutations in the transcription factor gene fl, which is an activator of macroconidiation, lead to increased microconidiation. VE-2 and LAE-1 were detected during vegetative growth and conidiation, unlike VE-1 which was mostly observed in samples obtained from submerged vegetative hyphae. We propose that VE-1 is the limiting component of the velvet complex during conidiation and has a major role in the transcriptional regulation of conidiation. Characterization of the role of VE-1 by RNA seq experiments during mycelial growth and asexual development (conidiation) allow

Centrosomes act as the major microtubule (MT) organizing centers (MTOC) in most animal cells. Structural or numerical defects in this organelle are associated with chromosome missegregation, polarity defects and motility or signaling alterations related to the absence of cilia or flagella, leading to several diseases like ciliopathies, brain disorders and cancer. In the last years, numerous publications have reported a possible role for the centrosome in the DNA damage response (DDR), with clear implications in cancer. However, it remains unclear if and how the centrosome itself plays a role specifically in the regulation of the DNA double strand breaks (DSBs) repair pathways. In this thesis we explore this aspect. Specifically, we show that centrosome presence in the cell is of utmost importance for homologous recombination (HR) and DNA end resection to take place properly. Plus, we uncover specific centrosomal factors involved in these processes. Among them, we state subdistal appendages (SDAs) components are relevant in DNA end resection and that this relies on CEP170 localization at this centrosomal structure. According to our results, CEP170 role in DNA end resection is produced from the centrosome likely by a signaling that involved its phosphorylation at Ser637 upon DNA damage induction. Finally, we suggest that some mediators such as BRCA1 and Centrin2 may be involved in CEP170 control over DNA end resection., Los centrosomas funcionan como los centros organizadores de microtubulos principales en la mayoría de las células animales. Cambios en la estructura o el número de este orgánulo están asociados con errores de segregación de cromosomas, defectos en polaridad y alteraciones en la motilidad y la señalización relacionadas con la ausencia de cilios o flagelos, dando lugar a muchas enfermedades como ciliopatas, enfermedades cerebrales y cáncer. En los últimos años, numerosas publicaciones han reportado una posible conexión entre el centrosoma en la respuesta al daño al ADN, con claras implicaciones en cáncer. Sin embargo, no está claro si el centrosoma por si mismo tiene un papel en la regulación de las vías de reparación de cortes de DNA de doble cadena ni cómo lo hace. En esta tesis, exploramos dicho aspecto. Concretamente, mostramos que la presencia del centrosoma en la célula es de gran importancia para que se produzca de forma correcta la recombinación homóloga y resección del ADN. Además, descubrimos una serie de factores centrosomales involucrados en dichos procesos, Entre ellos, destacan en la resección del ADN los componentes de los apéndices subdistales, cuya función depende de la localización de CEP170 en estas estructuras centrosomales. Según nuestros resultados, la función de CEP170 en la resección del ADN depende de una señalización que involucra la fosforilación de CEP170 en Ser637 en condición de daño al ADN. Finalmente, nuestros datos sugieren que algunos mediadores como BRCA1 y Centrin2 podrían estar involucrado en el control que ejerce CEP170 sobre la resección del ADN.

The genus Fusarium is a heterogeneous group of phytopathogenic fungi that causes diseases in a large diversity of economically important plants. In addition, Fusarium species produce a wide variety of secondary metabolites, several of which have been investigated in Fusarium fujikuroi. Our group investigates in this fungus the molecular mechanisms that regulate the synthesis of carotenoids, a class of lipophilic terpenoid responsible for the yellow, orange, or reddish pigmentation characteristic of many organisms. In F. fujikuroi carotenoid production is up-regulated by light through the photoreceptor WcoA, whereas a putative E3 ubiquitin ligase, the CarS RING Finger protein, is a down-regulator of the biosynthesis. Both regulatory proteins act modulating the mRNA levels of the genes of the carotenoid pathway (car genes). Loss of carS function results in carotenoid overproduction while its overexpression produces an albino phenotype. Previous assays in our group identified potential target proteins for CarS, one of them CwhA, ortholog of Cwh43 of Saccharomyces cerevisiae. Other strategies have been carried out to search for new potential regulators of the car genes, and recently two proteins of the HMG-box family were identified by a biotin-mediated pulldown method as able to bind to car genes promoters. In this Thesis, we investigated the mechanisms of action of the CarS protein and the regulatory connection with the CwhA and HmbC proteins, the latter one of the HMG-box proteins mentioned above. In chapter I, in vitro ubiquitination assays demonstrated the E3 ligase activity of CarS and the capacity of this protein to ubiquitinate purified WcoA in vitro, supporting regulatory connection between CarS and WcoA. Unfortunately, because of its very low amounts, CarS protein was not sufficiently detected in in F. fujikuroi extracts by western blot, which did not encourage to carry immunoprecipitation assays. In Chapter II, the biological role of CwhA was investigated thr

Las lesiones que se producen en el ADN suponen un desafío continuo para la integridad genómica, siendo las roturas de doble cadena del ADN (DSBs) la lesión más citotóxica para la célula. Numerosos estudios han mostrado recientemente que una fuente fisiológica de DSBs es la actividad de las topoisomerasas de ADN, enzimas que eliminan el estrés torsional del ADN asociado a procesos relacionados con el metabolismo del ADN. Durante su actividad, las topoisomerasas inducen cortes transitorios en el ADN, pero estos cortes pueden volverse irreversibles y provocar DSBs generando inestabilidad genómica e incluso la muerte celular. Al comienzo de esta tesis, los mecanismos que permitían a las células eucariotas reparar estas DSBs eran prácticamente desconocidos. Esta es una pregunta importante ya que las DSBs asociadas a la transcripción son una fuente endógena importante de translocaciones cromosómicas, eventos clave en el origen y desarrollo de muchos tumores sólidos y leucemias. Adicionalmente, estas DSBs pueden constituir eventos clave en otros tipos de enfermedades como algunos síndromes neurodegenerativos. En esta tesis, hemos caracterizado el mecanismo molecular por el cual se forman y reparan las DSBs inducidas por las topoisomerasas de ADN 1 y 2 (TOP1 y TOP2) y sus consecuencias en la integridad genómica. Para ello, hemos estudiado la reparación de estas roturas inducidas por topoisomerasas en células quiescentes, así como su impacto en la inestabilidad genómica asociada a la transcripción. Además, también hemos diseñado un escrutinio genético en células quiescentes destinado a identificar factores implicados en la formación y en la reparación de estas DSBs inducidas por TOP1. Nuestros resultados han permitido aclarar las bases moleculares de la formación y la reparación de las DSBs originadas en el ADN por la actividad de TOP1 y TOP2 durante la transcripción, de manera independiente de la replicación. Es importante destacar que nuestros resultados tienen importantes

El scaling en la morfogénesis se refiere al proceso por el cual los tejidos y órganos mantienen proporciones y patrones invariantes a medida que crecen en tamaño. El ojo es una estructura muy conservada entre los vertebrados y estudiar su mecanismo de scaling tiene el potencial de generar un impacto significativo en varias disciplinas, abarcando desde una mejor comprensión de sistemas biológicos hasta la medicina regenerativa. Este trabajo cumple con el inicio de una investigación a mayor escala para estudiar y generar un modelo de scaling del ojo en zebrafish. Estudiamos el papel de los morfógenos Wnt2 y Wnt8b, los cuales se sospecha que cumplen una función en el scaling. Nos centramos además en la creación de la F0 de una línea mutante para el gen rx3 de zebrafish incapaz de desarrollar ojos con el fin de utilizarla en un escenario experimental. Los mutantes rx3 homocigotos mueren a las 2 semanas de vida no siendo viables para la generación de línea de interés. Se plantea, por tanto, la elección de los individuos heterocigotos portadores de la mutación para la creación de la línea. En el estudio de los morfógenos, nuestros resultados demostraron la implicación de Wnt8b y Wnt2 en la formación del ojo confirmando el papel del último en la diferenciación de la población celular de RPE. Además, con el fin de caracterizar con mayor exactitud propiedades biofísicas de Wnt2 y Wnt8b planteamos la generación de constructos de ADN codificantes para proteínas fusión Wnt-GFP. La creación del constructo Wnt8b-GFP resultó exitosa; sin embargo, su expresión no se da de forma adecuada probablemente debido a fallos en el plegamiento ante lo cual proponemos el uso de un linker distinto para la unión de los dominios Wnt y GFP.

DNA double-strand break (DSB) repair is initiated by DNA end resection. CtIP acts in short-range resection to stimulate MRE11–RAD50–NBS1 (MRN) to endonucleolytically cleave 5′-terminatedDNAto bypass protein blocks. CtIP also promotes the DNA2 helicase–nuclease to accelerate long-range resection downstream from MRN. Here, using AlphaFold2, we identified CtIP-F728E-Y736E as a separation-of-function mutant that is still proficient in conjunction with MRN but is not able to stimulate ssDNA degradation by DNA2. Accordingly, CtIP-F728E-Y736E impairs physical interaction with DNA2. Cellular assays revealed that CtIP-F728E-Y736E cells exhibit reduced DSB-dependent chromatin-bound RPA, impaired long-range resection, and increased sensitivity to DSB-inducing drugs. Previously, CtIP was shown to be targeted by PLK1 to inhibit long-range resection, yet the underlying mechanism was unclear. We show that the DNA2-interacting region in CtIP includes the PLK1 target site at S723. The integrity of S723 in CtIP is necessary for the stimulation of DNA2, and phosphorylation of CtIP by PLK1 in vitro is consequently inhibitory, explaining why PLK1 restricts long-range resection. Our data support a model in which CDK-dependent phosphorylation of CtIP activates resection by MRN in S phase, and PLK1-mediated phosphorylation of CtIP disrupts CtIP stimulation of DNA2 to attenuate long-range resection later at G2/M.

Transcription in Saccharomyces cerevisiae is associated with elevated mutation and this partially reflects enhanced damage of the corresponding DNA. Spontaneous deamination of cytosine to uracil leads to CG>TA mutations that provide a strand-specific read-out of damage in strains that lack the ability to remove uracil from DNA. Using the CAN1 forward mutation reporter, we found that C>T and G>A mutations, which reflect deamination of the non-transcribed and transcribed DNA strands, respectively, occurred at similar rates under low-transcription conditions. By contrast, the rate of C>T mutations was 3-fold higher than G>A mutations under high-transcription conditions, demonstrating biased deamination of the non-transcribed strand (NTS). The NTS is transiently single-stranded within the ∼15 bp transcription bubble, or a more extensive region of the NTS can be exposed as part of an R-loop that can form behind RNA polymerase. Neither the deletion of genes whose products restrain R-loop formation nor the over-expression of RNase H1, which degrades R-loops, reduced the biased deamination of the NTS, and no transcription-associated R-loop formation at CAN1 was detected. These results suggest that the NTS within the transcription bubble is a target for spontaneous deamination and likely other types of DNA damage.

Unscheduled R loops can be a source of genome instability, a hallmark of cancer cells. Although targeted proteomic approaches and cellular analysis of specific mutants have uncovered factors potentially involved in R-loop homeostasis, we report a more open screening of factors whose depletion causes R loops based on the ability of activation-induced cytidine deaminase (AID) to target R loops. Immunofluorescence analysis of gH2AX caused by small interfering RNAs (siRNAs) covering 3,205 protein-coding genes identifies 59 potential candidates, from which 13 are analyzed further and show a significant increase of R loops. Such candidates are enriched in factors involved in chromatin, transcription, and RNA biogenesis and other processes. A more focused study shows that the DDX47 helicase is an R-loop resolvase, whereas the MeCP2 methyl-CpG-binding protein uncovers a link between DNA methylation and R loops. Thus, our results suggest that a plethora of gene dysfunctions can alter cell physiology via affecting R-loop homeostasis by different mechanisms.

Bacterial biofilms are surface-attached communities that are difficult to eradicate due to a high tolerance to antimicrobial agents. The use of non-biocidal surface-active compounds to prevent the initial adhesion and aggregation of bacterial pathogens is a promising alternative to antibiotic treatments and several antibiofilm compounds have been identified, including some capsular polysaccharides released by various bacteria. However, the lack of chemical and mechanistic understanding of the activity of these polymers limits their use to control biofilm formation. Here, we screen a collection of 31 purified capsular polysaccharides and first identify seven new compounds with non-biocidal activity against Escherichia coli and/or Staphylococcus aureus biofilms. We measure and theoretically interpret the electrophoretic mobility of a subset of 21 capsular polysaccharides under applied electric field conditions, and we show that active and inactive polysaccharide polymers display distinct electrokinetic properties and that all active macromolecules share high intrinsic viscosity features. Despite the lack of specific molecular motif associated with antibiofilm properties, the use of criteria including high density of electrostatic charges and permeability to fluid flow enables us to identify two additional capsular polysaccharides with broad-spectrum antibiofilm activity. Our study therefore provides insights into key biophysical properties discriminating active from inactive polysaccharides. The characterization of a distinct electrokinetic signature associated with antibiofilm activity opens new perspectives to identify or engineer non-biocidal surface-active macromolecules to control biofilm formation in medical and industrial settings.

The integrity of the genome is crucial for the survival of all living organisms. However, genomes need to adapt to survive certain pressures, and for this purpose use several mechanisms to diversify. Chromosomal instability (CIN) is one of the main mechanisms leading to the creation of genomic heterogeneity by altering the number of chromosomes and changing their structures. In this review, we will discuss the different chromosomal patterns and changes observed in speciation, in evolutional biology as well as during tumor progression. By nature, the human genome shows an induction of diversity during gametogenesis but as well during tumorigenesis that can conclude in drastic changes such as the whole genome doubling to more discrete changes as the complex chromosomal rearrangement chromothripsis. More importantly, changes observed during speciation are strikingly similar to the genomic evolution observed during tumor progression and resistance to therapy. The different origins of CIN will be treated as the importance of double-strand breaks (DSBs) or the consequences of micronuclei. We will also explain the mechanisms behind the controlled DSBs, and recombination of homologous chromosomes observed during meiosis, to explain how errors lead to similar patterns observed during tumorigenesis. Then, we will also list several diseases associated with CIN, resulting in fertility issues, miscarriage, rare genetic diseases, and cancer. Understanding better chromosomal instability as a whole is primordial for the understanding of mechanisms leading to tumor progression.

Ribosome synthesis is a complex process that involves a large set of protein trans-acting factors, among them DEx(D/H)-box helicases. These are enzymes that carry out remodelling activities onto RNAs by hydrolysing ATP. The nucleolar DEGD-box protein Dbp7 is required for the biogenesis of large 60S ribosomal subunits. Recently, we have shown that Dbp7 is an RNA helicase that regulates the dynamic base-pairing between the snR190 small nucleolar RNA and the precursors of the ribosomal RNA within early pre-60S ribosomal particles. As the rest of DEx(D/H)-box proteins, Dbp7 has a modular organization formed by a helicase core region, which contains conserved motifs, and variable, non-conserved N- and C-terminal extensions. The role of these extensions remains unknown. Herein, we show that the N-terminal domain of Dbp7 is necessary for efficient nuclear import of the protein. Indeed, a basic bipartite nuclear localization signal (NLS) could be identified in its N-terminal domain. Removal of this putative NLS impairs, but does not abolish, Dbp7 nuclear import. Both N- and C-terminal domains are required for normal growth and 60S ribosomal subunit synthesis. Furthermore, we have studied the role of these domains in the association of Dbp7 with pre-ribosomal particles. Altogether, our results show that the N- and C-terminal domains of Dbp7 are important for the optimal function of this protein during ribosome biogenesis.

El carcinoma hepatocellular (CHC) es reconocido como el principal cáncer primario de hígado y uno de los cánceres con mayor incidencia y mortalidad a nivel mundial. Éste se desarrolla en un contexto de enfermedad hepática crónica donde la aparición, así como la progresión de la enfermedad, está influenciada por numerosos factores de riesgo. Desafortunadamente, el alto grado de heterogeneidad genética intratumoral dificulta el desarrollo de terapias verdaderamente eficaces. Sorafenib fue el primer fármaco aprobado por la FDA (Food and Drug Administration) para su uso como tratamiento en pacientes avanzados de CHC. A pesar de que otras terapias hayan sido recientemente aprobadas para su uso como primera línea de tratamiento en dichos pacientes, Sorafenib sigue siendo la opción recomendada para un porcentaje significativo de ellos. Este fármaco es un inhibidor multiquinasa con efectos anti-proliferativos, anti-angiogénicos y pro-apoptóticos. Desgraciadamente, Sorafenib cuenta con unos beneficios clínicos muy limitados ya que por un lado produce numerosos efectos secundarios y por otro, el desarrollo de resistencia al tratamiento es muy frecuente en esta enfermedad. Otras terapias basadas en diferentes inhibidores multiquinasa han sido estudiadas en ensayo clínico, pero desgraciadamente, han mostrado una eficacia inferior o igual al Sorafenib, sugiriendo que la efectividad clínica del fármaco podrías estar basada en efectos adicionales a su acción inhibidora de la actividad quinasa. El proceso de síntesis de proteínas, también conocido como traducción, consiste en la formación de nuevas cadenas polipeptídicas a partir de la información contenida en una molécula de RNA mensajero (mRNA). Dicho proceso es mediado por el ribosoma que, junto con numerosas proteínas llamadas factores de traducción, sintetizan las proteínas de novo en función de la demanda celular. El proceso de traducción constituye el principal punto de regulación de la expresión génica, donde los niveles de

Ribosomes are ribonucleoprotein complexes in charge of protein translation. Translation is an essential process in all organisms that involves decoding the information contained in mRNAs (messenger RNAs) to generate proteins. Similarly, the process of ribosome biogenesis represents a critical pathway for all cells. Ribosomes are comprised of two different subunits, one large and one small, also named 60S and 40S, respectively, in eukaryotes, among them Saccharomyces cerevisiae, which is the model organism employed in this thesis. As ribosome biogenesis is well conserved among different eukaryotes, S. cerevisiae is a perfectly appropriate organism for this study. Ribosomes of S. cerevisiae are composed of 79 ribosomal proteins and 4 ribosomal RNAs (rRNAs), three of them in the large (25S, 5.8S y 5S) and one in the small ribosomal subunit (18S). The biogenesis of ribosomes is an extraordinarily complex process, in which in addition of the four rRNAs and the 79-80 ribosomal proteins, take part about 100 small nucleolar RNAs (snoRNAs) and more than 300 assembly protein factors, which are not present in the mature subunits and therefore are also known as trans-acting factors. The synthesis of ribosomes is a sequential process highly regulated and organized that starts in the nucleolus and orderly progresses through the nucleoplasm; almost mature ribosomal particles are exported to the cytoplasm, where the last steps of maturation take place, previously to their involvement in the translation process. The role of the different components participating in the biogenesis of the ribosomal subunits has been broadly studied through last decades. However, there are numerous assembly factors and some ribosomal proteins which still await characterization. This was the case for the ribosomal proteins eL22 and eL15 during the assembly of the 60S ribosomal subunit before the study performed in this doctoral thesis. With respect to the eL22 ribosomal protein, in this thesis, we have, Los ribosomas son complejos ribonucleoproteicos encargados de la traducción de proteínas. La traducción es un proceso esencial en todos los organismos mediante el cual, la información contenida en el mRNA (RNA mensajero) se descodifica generando proteínas. El proceso de biogénesis ribosómica presenta igualmente una importancia crucial en todas las células. Los ribosomas se componen de dos subunidades diferenciadas, una llamada subunidad grande y otra llamada pequeña, también denominadas 60S y 40S en Saccharomyces cerevisiae, el organismo modelo usado en esta tesis. Como la biogénesis de los ribosomas está muy conservada entre los distintos eucariotas, S. cerevisiae es un organismo perfectamente adecuado para este estudio. Los ribosomas de S. cerevisiae están compuestos por 79 proteínas ribosómicas y 4 RNAs ribosómicos (rRNAs), tres de ellos en la subunidad grande (25S, 5.8S y 5S) y uno en la pequeña (18S). La biogénesis de los ribosomas es un proceso extraordinariamente complejo en el que intervienen los cuatro rRNAs y las 79-80 proteínas ribosómicas, del orden de 100 RNAs pequeños nucleolares (snoRNAs) y más de 300 factores proteicos de ensamblaje, que al no formar parte de las subunidades maduras también son llamados factores de actuación en trans. La síntesis de ribosomas es un proceso secuencial altamente regulado y organizado que empieza en el nucleolo y progresa ordenadamente a través del nucleoplasma; las partículas pre-ribosómicas prácticamente completas son exportadas al citoplasma donde los últimos pasos de maduración tienen lugar antes de ser reclutadas al proceso de traducción. El papel de los distintos componentes que participan en la biogénesis de las subunidades ribosómicas ha sido ampliamente estudiado en las ultimas décadas. Sin embargo, aún quedan numerosos factores de ensamblaje y algunas proteínas ribosómicas por ser caracterizadas en detalle. Este era el caso de las proteínas ribosómicas eL22 y eL15 durante el ensamblaje de la subunidad 60S ante

Tumour progression and therapy tolerance are highly regulated and complex processes largely dependent on the plasticity of cancer cells and their capacity to respond to stress. The higher plasticity of cancer cells highlights the need for identifying targetable molecular pathways that challenge cancer cell survival. Here, we show that N7-guanosine methylation (m7G) of tRNAs, mediated by METTL1, regulates survival to stress conditions in cancer cells. Mechanistically, we find that m7G in tRNAs protects them from stress-induced cleavage and processing into 5’ tRNA fragments. Our analyses reveal that the loss of tRNA m7G methylation activates stress response pathways, sensitising cancer cells to stress. Furthermore, we find that the loss of METTL1 reduces tumour growth and increases cytotoxic stress in vivo. Our study uncovers the role of m7G methylation of tRNAs in stress responses and highlights the potential of targeting METTL1 to sensitise cancer cells to chemotherapy.

Many eukaryotic membrane-dependent functions are often spatially and temporally regulated by membrane microdomains (FMMs), also known as lipid rafts. These domains are enriched in polyisoprenoid lipids and scaffolding proteins belonging to the stomatin, prohibitin, flotillin, and HflK/C (SPFH) protein superfamily that was also identified in Gram-positive bacteria. In contrast, little is still known about FMMs in Gram-negative bacteria. In Escherichia coli K-12, 4 SPFH proteins, YqiK, QmcA, HflK, and HflC, were shown to localize in discrete polar or lateral inner membrane locations, raising the possibility that E. coli SPFH proteins could contribute to the assembly of inner membrane FMMs and the regulation of cellular processes. Here, we studied the determinant of the localization of QmcA and HflC and showed that FMM-associated cardiolipin lipid biosynthesis is required for their native localization pattern. Using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress that is due to the absence of HflKC. Our study therefore provides further insights into the contribution of SPFH proteins to stress tolerance in E. coli. IMPORTANCE Eukaryotic cells often segregate physiological processes in cholesterol-rich functional membrane microdomains. These domains are also called lipid rafts and contain proteins of the stomatin, prohibitin, flotillin, and HflK/C (SPFH) superfamily, which are also present in prokaryotes but have been mostly studied in Gram-positive bacteria. Here, we showed that the cell localization of the SPFH proteins QmcA and HflKC in the Gram-negative bacterium E. coli is altered in the absence of cardiolipin lipid synthesis. This suggests that cardiolipins contribute to E. coli membrane microdomain assembly. Using a broad phenotypic analysis, we also showed that HflKC contribute to E. coli tolerance to aminoglycosides and oxidative stress. Our study, therefore, provide

In the fungus Fusarium fujikuroi, carotenoid production is up-regulated by light and down-regulated by the CarS RING finger protein, which modulates the mRNA levels of carotenoid pathway genes (car genes). To identify new potential regulators of car genes, we used a biotin-mediated pull-down procedure to detect proteins capable of binding to their promoters. We focused our attention on one of the proteins found in the screening, belonging to the High-Mobility Group (HMG) family that was named HmbC. The deletion of the hmbC gene resulted in increased carotenoid production due to higher mRNA levels of car biosynthetic genes. In addition, the deletion resulted in reduced carS mRNA levels, which could also explain the partial deregulation of the carotenoid pathway. The mutants exhibited other phenotypic traits, such as alterations in development under certain stress conditions, or reduced sensitivity to cell wall degrading enzymes, revealed by less efficient protoplast formation, indicating that HmbC is also involved in other cellular processes. In conclusion, we identified a protein of the HMG family that participates in the regulation of carotenoid biosynthesis. This is probably achieved through an epigenetic mechanism related to chromatin structure, as is frequent in this class of proteins.

The centrosome is a cytoplasmic organelle with roles in microtu-bule organization that has also been proposed to act as a hub forcellular signaling. Some centrosomal components are required forfull activation of the DNA damage response. However, whether thecentrosome regulates specific DNA repair pathways is not known.Here, we show that centrosome presence is required to fully acti-vate recombination, specifically to completely license its initialstep, the so-called DNA end resection. Furthermore, we identify acentriolar structure, the subdistal appendages, and a specific fac-tor, CEP170, as the critical centrosomal component involved in theregulation of recombination and resection. Cells lacking centro-somes or depleted for CEP170are, consequently, hypersensitive toDNA damaging agents. Moreover, low levels of CEP170in multiplecancer types correlate with an increase of the mutation burdenassociated with specific mutational signatures and a better prog-nosis, suggesting that changes in CEP170can act as a mutationdriver but could also be targeted to improve current oncologicaltreatments.

It has become increasingly clear in the last few years that gene expression in eukaryotes is not a linear process from mRNA synthesis in the nucleus to translation and degradation in the cytoplasm, but works as a circular one where the mRNA level is controlled by crosstalk between nuclear transcription and cytoplasmic decay pathways. One of the consequences of this crosstalk is the approximately constant level of mRNA. This is called mRNA buffering and happens when transcription and mRNA degradation act at compensatory rates. However, if transcription and mRNA degradation act additively, enhanced gene expression regulation occurs. In this work, we analyzed new and previously published genomic datasets obtained for several yeast mutants related to either transcription or mRNA decay that are not known to play any role in the other process. We show that some, which were presumed only transcription factors (Sfp1) or only decay factors (Puf3, Upf2/3), may represent examples of RNA-binding proteins (RBPs) that make specific crosstalk to enhance the control of the mRNA levels of their target genes by combining additive effects on transcription and mRNA stability. These results were mathematically modeled to see the effects of RBPs when they have positive or negative effects on mRNA synthesis and decay rates. We found that RBPs can be an efficient way to buffer or enhance gene expression responses depending on their respective effects on transcription and mRNA stability.

Genome instability is a feature of cancer cells, transcription being an important source of DNA damage. This is in large part associated with R-loops, which hamper replication, especially at head-on transcription-replication conflicts (TRCs). Here we show that TRCs trigger a DNA Damage Response (DDR) involving the chromatin network to prevent genome instability. Depletion of the key chromatin factors INO80, SMARCA5 and MTA2 results in TRCs, fork stalling and R-loop-mediated DNA damage which mostly accumulates at S/G2, while histone H3 Ser10 phosphorylation, a mark of chromatin compaction, is enriched at TRCs. Strikingly, TRC regions show increased mutagenesis in cancer cells with signatures of homologous recombination deficiency, transcription-coupled nucleotide excision repair (TC-NER) and of the AID/ APOBEC cytidine deaminases, being predominant at head-on collisions. Thus, our results support that the chromatin network prevents R-loops and TRCs from genomic instability and mutagenic signatures frequently associated with cancer.

Double-strand breaks (DSBs) are the most harmful DNA lesions, with a strong impact on cell proliferation and genome integrity. Depending on cell cycle stage, DSBs are preferentially repaired by non-homologous end joining or homologous recombination (HR). In recent years, numerous reports have revealed that DSBs enhance DNA–RNA hybrid formation around the break site. We call these hybrids “break-induced RNA–DNA hybrids” (BIRDHs) to differentiate them from sporadic R-loops consisting of DNA–RNA hybrids and a displaced single-strand DNA occurring co-transcriptionally in intact DNA. Here, we review and discuss the most relevant data about BIRDHs, with a focus on two main questions raised: (i) whether BIRDHs form by de novo transcription after a DSB or by a pre-existing nascent RNA in DNA regions undergoing transcription and (ii) whether they have a positive role in HR or are just obstacles to HR accidentally generated as an intrinsic risk of transcription. We aim to provide a comprehensive view of the exciting and yet unresolved questions about the source and impact of BIRDHs in the cell.

EHMT1 is an epigenetic factor with histone methyltransferase activity that appears mutated in Kleefstra syndrome, a neurodevelopmental genetic disorder characterized by developmental delay, intellectual disability, and autistic-like features. Despite recent progress in the study of the function of this gene and the molecular etiology of the disease, our knowledge of how EHMT1 haploinsufficiency causes Kleefstra syndrome is still very limited. Here, we show that EHMT1 depletion in RPE1 cells leads to alterations in the morphology and distribution of different subcellular structures, such as the Golgi apparatus, the lysosomes and different cell adhesion components. EHMT1 downregulation also increases centriolar satellites detection, which may indicate a role for EHMT1 in centrosome functioning. Furthermore, the migration process is also altered in EHMT1 depleted cells, which show reduced migration capacity. We consider that the described phenotypes could open new possibilities for understanding the functional impact of EHMT1 haploinsufficiency in Kleefstra syndrome, helping to elucidate the link between epigenetic regulation and the underlying cellular mechanisms that result in this neurodevelopmental disorder. This knowledge could be relevant not only for the treatment of this syndrome, but also for other neurodevelopmental conditions that could share similar deregulated cellular pathways.

Type III secretion systems are found in many Gram-negative pathogens and symbionts of animals and plants. Salmonella enterica has two type III secretion systems associated with virulence, one involved in the invasion of host cells and another involved in maintaining an appropriate intracellular niche. SrfJ is an effector of the second type III secretion system. In this study, we explored the biochemical function of SrfJ and the consequences for mammalian host cells of the expression of this S. enterica effector. Our experiments suggest that SrfJ is a glucosylceramidase that alters the lipidome and the transcriptome of host cells, both when expressed alone in epithelial cells and when translocated into macrophages in the context of Salmonella infection. We were able to identify seventeen lipids with higher levels and six lipids with lower levels in the presence of SrfJ. Analysis of the forty-five genes, the expression of which is significantly altered by SrfJ with a fold-change threshold of two, suggests that this effector may be involved in protecting Salmonella from host immune defenses.

Microbial cell individuality is receiving increasing interest in the scientific community. Individual cells within clonal populations exhibit noticeable phenotypic heterogeneity. The advent of fluorescent protein technology and advances in single-cell analysis has revealed phenotypic cell variant in bacterial populations. This heterogeneity is evident in a wide range of phenotypes, for example, individual cells display variable degrees of gene expression and survival under selective conditions and stresses, and can exhibit differing propensities to host interactions. Last few years, numerous cell sorting approaches have been employed for resolving the properties of bacterial subpopulations. This review provides an overview of applications of cell sorting to analyze Salmonella lineage-specific traits, including bacterial evolution studies, gene expression analysis, response to diverse cellular stresses and characterization of diverse bacterial phenotypic variants.

Proliferative heterogeneity has been widely described in clonal cultures, but the mechanisms underlying this phenomenon are still poorly understood. On another hand, cell cycle regulation is tightly linked to proliferative capacity and previous transcriptomic studies revealed that yeast cells with less proliferative capacity display higher levels of the G1-S transition inhibitor Whi5. This regulator has been also linked to replicative aging in late steps of the mother cell lifespan. Considering this data, this thesis work has the following objectives: 1. To stablish a reliable method to study the influence of replicative age on proliferation heterogeneity. 2. Determining the role of Whi5 in proliferative heterogeneity. 3. Decipher the role of cell cycle regulation in coupling replicative age and proliferative heterogeneity In this work, we combined single cell microencapsulation with confocal microscopy to study heterogeneity in clonal cultures. Using this new method we found that a significant proportion of slow-growing microcolonies are founded by mother cells with a short number of division cycles. Using a Whi5 induble strain, we also found that this reduction in the proliferation capacity of microcolonies founded by young mother cells is related to the expression levels of Whi5. We can resume our results with the following conclusions: 1. Combination of cell microencapsulation and confocal microscopy is a reliable method to analyze cell lineages. 2. Replicative age substantially contributes to proliferative heterogeneity in clonal populations. 3. Decrease in proliferative capacity of young mother cells arises after the first mitotic division. 4. The progeny of a young mother cell can non-genetically inherit its decreased proliferative capacity. 5. The cell cycle regulator Whi5 participates in one of the mechanisms that links replicative age and cell proliferative capacity. Our results indicate that stably reduced proliferation is not exclusively linked to aged c

Las células han desarrollado una serie de mecanismos que utilizan de forma coordinada para mantener la integridad del genoma y permitir una correcta transmisión del material genético a su descendencia. Sin embargo, deben hacer frente a una amplia gama de daños en el genoma generados por agentes genotóxicos tanto internos como externos, y que en algunos casos pueden provocar roturas en el ADN. Las roturas de cadena sencilla son las más comunes, pero las de doble cadena son más citotóxicas. Todo ello puede desencadenar inestabilidad genética que se manifiesta en forma de mutaciones o reordenamientos cromosómicos. Si bien es cierto que puede suponer un motor para la evolución, la inestabilidad genética es una patología celular asociada al envejecimiento prematuro, el cáncer y patologías neurológicas. La transcripción es un proceso celular que permite copiar la información requerida desde el ADN al ARN. Este proceso es esencial para la supervivencia celular pero puede comprometer la integridad del genoma. Ello se debe en parte a cambios topológicos en el ADN que lo pueden hacer más vulnerable. Puede suponer un obstáculo para la replicación y aumentar los conflictos transcripciónreplicación que pueden desembocar en roturas de ADN. Los bucles R son una de las causas más estudiadas de la inestabilidad genética asociada a transcripción. Estas estructuras se componen de un híbrido de ADN-ARN y una cadena simple de ADN desplazada. Estos bucles R aunque pueden tener funciones fisiológicas, también pueden suponer un riesgo para la estabilidad del genoma. Por ello las células poseen factores y mecanismos para prevenir y/o resolver estas estructuras, entre las que se encuentran proteínas del metabolismo del ARN. Recientemente, se ha descrito la presencia de bucles R en las roturas de ADN, aunque si tienen o no alguna función de reparación del ADN no se ha resulto aún. Dada la conexión entre la transcripción y el ARN con la inestabilidad genética y el cáncer, nos preguntamos si fa

Whereas both sperm and egg contribute nuclear genetic material to the zygote in metazoan organisms, the inheritance of other cellular constituents is unequal between the 2 gametes. Thus, 2 copies of the centriole are contributed solely by the sperm to the zygote in most species. Centrioles can have a stereotyped distribution in some asymmetric divisions, but whether sperm-contributed centrioles are distributed in a stereotyped manner in the resulting embryo is not known. Here, we address this question in Caenorhabditis elegans using marked mating experiments, whereby the presence of the 2 sperm-contributed centrioles is monitored in the embryo using the stable centriolar component SAS-4::GFP, as well as GFP::SAS-7. Our analysis demonstrates that the distribution of sperm-contributed centrioles is stochastic in 4-cell stage embryos. Moreover, using sperm from zyg-1 mutant males that harbor a single centriole, we show that the older sperm-contributed centriole is likewise distributed stochastically in the resulting embryo. Overall, we conclude that, in contrast to the situation during some asymmetric cell divisions, centrioles contributed by the male germ line are distributed stochastically in embryos of C. elegans

To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance