Artículos (Genética)

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The Role of Ribosomal Proteins eL15 and eL36 in the Early Steps of Yeast 60S Ribosomal Subunit Assembly
(Elsevier, 2023-12-15) Fernández Fernández, José; Martín Villanueva, Sara; Pérez Fernández, Jorge; Cruz Díaz, Jesús de la; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; Deutsche Forschungsgemeinschaft / German Research Foundation (DFG); Junta de Andalucía; Universidad de Sevilla
Ribosomal proteins have important roles in maintaining the structure and function of mature ribosomes, but they also drive crucial rearrangement reactions during ribosome biogenesis. The contribution of most, but not all, ribosomal proteins to ribosome synthesis has been previously analyzed in the yeast Saccharomyces cerevisiae. Herein, we characterize the role of yeast eL15 during 60S ribosomal subunit formation. In vivo depletion of eL15 results in a shortage of 60S subunits and the appearance of half-mer polysomes. This is likely due to defective processing of the 27SA3 to the 27SBS pre-rRNA and impaired subsequent processing of both forms of 27SB pre-rRNAs to mature 25S and 5.8S rRNAs. Indeed, eL15 depletion leads to the efficient turnover of the de novo formed 27S pre-rRNAs. Additionally, depletion of eL15 blocks nucleocytoplasmic export of pre-60S particles. Moreover, we have analyzed the impact of depleting either eL15 or eL36 on the composition of early pre-60S particles, thereby revealing that the depletion of eL15 or eL36 not only affects each other's assembly into pre-60S particles but also that of neighboring ribosomal proteins, including eL8. These intermediates also lack most ribosome assembly factors required for 27SA3 and 27SB pre-rRNA processing, named A3- and B-factors, respectively. Importantly, our results recapitulate previous ones obtained upon eL8 depletion. We conclude that assembly of eL15, together with that of eL8 and eL36, is a prerequisite to shape domain I of 5.8S/25S rRNA within early pre-60S particles, through their binding to this rRNA domain and the recruitment of specific groups of assembly factors.
Role of the 40S beak ribosomal protein eS12 in ribosome biogenesis and function in Saccharomyces cerevisiae
(Taylor & Francis, 2020-06-07) Martín Villanueva, Sara; Fernández Fernández, José; Rodríguez Galán, Olga; Fernández Boraita, Julia; Villalobo Polo, Eduardo; Cruz Díaz, Jesús de la; Universidad de Sevilla. Departamento de Microbiología; Universidad de Sevilla. Departamento de Genética; Ministerio de Economía y Competitividad (MINECO). España; Junta de Andalucía
In eukaryotes, the beak structure of 40S subunits is formed by the protrusion of the 18S rRNA helix 33 and three ribosomal proteins: eS10, eS12 and eS31. The exact role of these proteins in ribosome biogenesis is not well understood. While eS10 is an essential protein encoded by two paralogous genes in Saccharomyces cerevisiae, eS12 and eS31 are not essential proteins encoded by the single-copy genes RPS12 and UBI3, respectively. Here, we have analysed the contribution of yeast eS12 to ribosome biogenesis and compared it with that of eS31. Polysome analysis reveals that deletion of either RPS12 or UBI3 results in equivalent 40S deficits. Analysis of pre-rRNA processing indicates that eS12, akin to eS31, is required for efficient processing of 20S pre-rRNA to mature 18S rRNA. Moreover, we show that the 20S pre-rRNA accumulates within cytoplasmic pre-40S particles, as deduced from FISH experiments and the lack of nuclear retention of 40S subunit reporter proteins, in rps12∆ and ubi3∆ cells. However, these particles containing 20S pre-rRNA are not efficiently incorporated into polyribosomes. We also provide evidence for a genetic interaction between eS12 or eS31 and the late-acting 40S assembly factors Enp1 and Ltv1, which appears not to be linked to the dynamics of their association with or release from pre-40S particles in the absence of either eS12 or eS31. Finally, we show that eS12- and eS31-deficient ribosomes exhibit increased levels of translational misreading. Altogether, our data highlight distinct important roles of the beak region during ribosome assembly and function.
HMCES corrupts replication fork stability during base excision repair in homologous recombination–deficient cells
(Amer Assoc Advancement Science, 2025) Peña Gómez, Mª José; Rodríguez-Martín, Yaiza; del Rio Oliva, Marta; Wijesekara Hanthi, Yodhara; Berrada, Sara; Freire, Raimundo; Valle Rosado, Iván; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Agencia Estatal de Investigación. España; European Union (UE); Fondazione AIRC per la Ricerca sul Cancro (AIRC). Italy
Sorafenib-associated translation reprogramming in hepatocellular carcinoma cells
(Taylor and Francis Ltd., 2025-03-17) Contreras Bernal, Laura; Rodríguez Gil, Alfonso; Muntané Relat, Jordi; Cruz Díaz, Jesús de la; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Universidad de Sevilla. Departamento de Genética; European Cooperation in Science and Technology; Ministerio de Ciencia e Innovación (MICIN). España; European Commission (EC); Instituto de Salud Carlos III; Junta de Andalucía; Universidad de Sevilla
Sorafenib (Sfb) is a multikinase inhibitor regularly used for the management of patients with advanced hepatocellular carcinoma (HCC) that has been shown to increase very modestly life expectancy. We have shown that Sfb inhibits protein synthesis at the level of initiation in cancer cells. However, the global snapshot of mRNA translation following Sorafenib-treatment has not been explored so far. In this study, we performed a genome-wide polysome profiling analysis in Sfb-treated HCC cells and demonstrated that, despite global translation repression, a set of different genes remain efficiently translated or are even translationally induced. We reveal that, in response to Sfb inhibition, translation is tuned, which strongly correlates with the presence of established mRNA cis-acting elements and the corresponding protein factors that recognize them, including DAP5 and ARE-binding proteins. At the level of biological processes, Sfb leads to the translational down-regulation of key cellular activities, such as those related to the mitochondrial metabolism and the collagen synthesis, and the translational up-regulation of pathways associated with the adaptation and survival of cells in response to the Sfb-induced stress. Our findings indicate that Sfb induces an adaptive reprogramming of translation and provides valuable information that can facilitate the analysis of other drugs for the development of novel combined treatment strategies based on Sfb therapy.
Editorial: Functional implications of Piwi proteins and piRNAs in stem cell maintenance and development
(Frontiers Media SA, 2025-03-17) O’Sullivan, Fernanda Loureiro Almeida; Das, Ani V.; Rojas Ríos, Patricia; Universidad de Sevilla. Departamento de Genética; Junta de Andalucía
Radiotherapy Resistance Driven by Asparagine Endopeptidase through ATR Pathway Modulation in Breast Cancer
(BioMed Central, 2025) Morillo Huesca, Macarena; G. López Cepero, Ignacio; Conesa Bakkali, Ryan; Tomé, Mercedes; Watts, Colin; Huertas Sánchez, Pablo; Moreno Bueno, Gema; Durán, Raúl V.; Martínez Fábregas, Jonathan; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Universidad de Sevilla. Departamento de Genética; European Union (UE). H2020; Ministerio de Ciencia e Innovación (MICIN). España; Universidad de Sevilla; Instituto de Salud Carlos III
Background: Tumor resistance represents a major challenge in the current oncology landscape. Asparagine endopeptidase (AEP) overexpression correlates with worse prognosis and reduced overall survival in most human solid tumors. However, the underlying mechanisms of the connection between AEP and reduced overall survival in cancer patients remain unclear. Methods: High-throughput proteomics, cellular and molecular biology approaches and clinical data from breast cancer (BC) patients were used to identify novel, biologically relevant AEP targets. Immunoblotting and qPCR analyses were used to quantify protein and mRNA levels. Flow cytometry, confocal microscopy, chemical inhibitors, siRNA- and shRNA-silencing and DNA repair assays were used as functional assays. In-silico analyses using the TCGA BC dataset and immunofluorescence assays in an independent cohort of invasive ductal (ID) BC patients were used to validate the clinical relevance of our findings. Results: Here we showed a dual role for AEP in genomic stability and radiotherapy resistance in BC patients by suppressing ATR and PPP1R10 levels. Reduced ATR and PPP1R10 levels were found in BC patients expressing high AEP levels and correlated with worst prognosis. Mechanistically, AEP suppresses ATR levels, reducing DNA damage-induced cell death, and PPP1R10 levels, promoting Chek1/P53 cell cycle checkpoint activation, allowing BC cells to efficiently repair DNA. Functional studies revealed AEP-deficiency results in genomic instability, increased DNA damage signaling, reduced Chek1/P53 activation, impaired DNA repair and cell death, with phosphatase inhibitors restoring the DNA damage response in AEP-deficient BC cells. Furthermore, AEP inhibition sensitized BC cells to the chemotherapeutic reagents cisplatin and etoposide. Immunofluorescence assays in an independent cohort of IDBC patients showed increased AEP levels in ductal cells. These analyses showed that higher AEP levels in radioresistant IDBC patients resulted in ATR nuclear eviction, revealing AEPhigh/ATRlow protein levels as an efficient predictive biomarker for the stratification of radioresistant patients. Conclusion: The newly identified AEP/ATR/PPP1R10 axis plays a dual role in genomic stability and radiotherapy resistance in BC. Our work provides new clues to the underlying mechanisms of tumor resistance and strong evidence validating the AEP/ATR axis as a novel predictive biomarker and therapeutic target for the stratification and treatment of radioresistant BC patients.
The CDK12–BRCA1 signaling axis mediates dinaciclib-associated radiosensitivity through p53-mediated cellular senescence
(Willey, 2024-12-03) García Flores, Natalia; Fernández Aroca, Diego A.; Garnés García, Cristina; Domínguez Calvo, Andrés; Jiménez Suárez, Jaime; Sabater, Sebastià; Huertas Sánchez, Pablo; Sánchez Prieto, Ricardo; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER); Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Universidad de Castilla-La Mancha; Gobierno de Castilla-La Mancha
Pan-cyclin-dependent-kinase (CDK) inhibitors are a new class of targeted therapies that can act on multiple CDKs, with dinaciclib being one of the most promising compounds. Although used as a monotherapy, an interesting approach could be to combine it with radiotherapy. Here, we show that dinaciclib increases radiosensitivity in some experimental models of lung and colon cancer (A549 or HCT 116) but not in others (H1299 or HT-29). Dinaciclib did not alter serine-protein kinase ATM signalling or cell cycle profiling after ionising-radiation exposure, which have been described for other CDK inhibitors. Interestingly, in terms of apoptosis, although the combination renders a clear increase, no potentiation of the ionising-radiation-induced apoptosis was observed. Mechanistically, inhibition of CDK12 by dinaciclib diminishes BRCA1 expression, which decreases homologous recombination (HR) and probably promotes the nonhomologous end joining repair process (NHEJ), which ultimately promotes the induction of ionising-radiation-associated cellular senescence in a TP53-dependent manner, explaining the lack of effect observed in some experimental models. In conclusion, our report proposes a molecular mechanism, based on the signalling axis CDK12–BRCA1, involved in this newly identified therapeutic effect of dinaciclib, although other players implicated in HR should not be discarded. In addition, our data provide a rationale for more selective and personalised chemo/radiotherapy treatment according to the genetic background of the tumour.
Role of inorganic phosphate concentrations in in vitro activity of fosfomycin
(Elsevier, 2022-02) Ortiz Padilla, Miriam; Portillo Calderón, Inés María; Maldonado, Natalia; Rodríguez Martínez, José Manuel; De Gregorio Iaria, Belén; Merino Bohórquez, Vicente; Rodríguez-Baño, Jesús; Pascual Hernández, Álvaro; Docobo Pérez, Fernando; Universidad de Sevilla. Departamento de Microbiología; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Farmacología; Universidad de Sevilla. Departamento de Medicina; Instituto de Biomedicina de Sevilla (IBIS); Instituto de Salud Carlos III; European Union (UE); Universidad de Sevilla. BIO116: Genética Bacteriana; Universidad de Sevilla. CTS210: Resistencia a Antimicrobianos
Objectives: The objective of this study was to evaluate the in vitro activity of fosfomycin under different physiological concentrations of inorganic phosphate (Pi). Methods: The wild-type BW25113 strain, four isogenic mutants (ΔglpT, ΔuhpT, ΔglpT-uhpT, and ΔphoB) and six clinical isolates of Escherichia coli with different fosfomycin susceptibilities were used. EUCAST breakpoints were used. Susceptibility was evaluated by agar dilution using standard Mueller–Hinton agar (Pi concentration of 1 mM similar to human plasma concentration) and supplemented with Pi (13 and 42 mM, minimum and maximum urinary Pi concentrations) and/or glucose-6-phosphate (25 mg/L). Fosfomycin transporter promoter activity was assayed using PglpT::gfpmut2 or PuhpT::gfpmut2 promoter fusions in standard Mueller–Hinton Broth (MHB), supplemented with Pi (13 or 42 mM) ± glucose-6-phosphate. Fosfomycin activity was quantified, estimating fosfomycin EC50 under different Pi concentrations (1, 13 and 42 mM + glucose-6-phosphate) and in time–kill assays using fosfomycin concentrations of 307 (maximum plasma concentration (Cmax)), 1053 and 4415 mg/L (urine Cmax range), using MHB with 28 mM Pi (mean urine Pi concentration) + 25 mg/L glucose-6-phosphate. Results: All the strains showed decreased susceptibility to fosfomycin linked to increased Pi concentrations: 1–4 log2 dilution differences from 1 to 13 mM, and 1–8 log2 dilution differences at 42 mM Pi. Changes in phosphate concentration did not affect the expression of fosfomycin transporters. By increasing Pi concentrations higher fosfomycin EC50 bacterial viability was observed, except against ΔglpT-uhpT. The increase in Pi reduced the bactericidal effect of fosfomycin. Discussion: Pi variations in physiological fluids may reduce fosfomycin activity against E. coli. Elevated Pi concentrations in urine may explain oral fosfomycin failure in non-wild-type but fosfomycin-susceptible E. coli strains.
Activity of Fosfomycin and Amikacin against Fosfomycin-Heteroresistant Escherichia coli Strains in a Hollow-Fiber Infection Model
(ASM Journals, 2021-04-19) Portillo Calderón, I.; Ortiz Padilla, Miriam; De Gregorio Iaria, Belén; Merino Bohórquez, Vicente; Blázquez, J.; Rodríguez-Baño, Jesús; Rodríguez Martínez, José Manuel; Pascual Hernández, Álvaro; Docobo Pérez, Fernando; Universidad de Sevilla. Departamento de Microbiología; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Farmacología; Universidad de Sevilla. Departamento de Medicina; Instituto de Biomedicina de Sevilla (IBIS); Universidad de Sevilla. BIO116: Genética Bacteriana; Universidad de Sevilla. CTS210: Resistencia a Antimicrobianos
We evaluated human-like the efficacy of intravenous doses of fosfomycin of 8 g every 8 h (8 g/Q8h) and of amikacin (15 mg/kg/Q24h) in monotherapy and in combination against six fosfomycin-heteroresistant Escherichia coli isolates using a hollow-fiber infection model (HFIM). Six fosfomycin-heteroresistant E. coli isolates (four with strong mutator phenotype) and the control strain E. coli ATCC 25922 were used. Mutant frequencies for rifampin (100 mg/liter), fosfomycin (50 and 200 mg/liter), and amikacin (32 mg/liter) were determined. Fosfomycin and amikacin MICs were assessed by agar dilution (AD), gradient strip assay (GSA), and broth microdilution (BMD). Fosfomycin and amikacin synergies were studied by checkerboard and time-kill assays at different concentrations. The efficacies of fosfomycin (8 g/Q8h) and amikacin (15 mg/kg/Q24h) alone and in combination were assessed using an HFIM. Five isolates were determined to be resistant to fosfomycin by AD and BMD, but all were determined to be susceptible by GSA. All isolates were determined to be susceptible to amikacin. Antibiotic combinations were synergistic in two isolates, and no antagonism was detected. In time-kill assays, all isolates survived under fosfomycin at 64 mg/liter, although at 307 mg/liter only the normomutators and two hypermutators survived. Four isolates survived under 16 mg/liter amikacin, and none survived at 45 mg/liter. No growth was detected under combination conditions. In HFIM, fosfomycin and amikacin monotherapies failed to sterilize bacterial cultures; however, the combination of fosfomycin and amikacin yielded a rapid eradication. There may be a risk of treatment failure of fosfomycin-heteroresistant E. coli isolates using either amikacin or fosfomycin in monotherapy. These results support that the amikacin-fosfomycin combination can rapidly decrease bacterial burden and prevent the emergence of resistant subpopulations against fosfomycin-heteroresistant strains.
VE-1 Regulation of MAPK Signaling Controls Sexual Development in Neurospora Crassa
(American Society for Microbiology, 2024) Cea Sánchez, Sara; Martín Villanueva, Sara; Gutiérrez Pozo, Gabriel; Cánovas López, David; Corrochano Peláez, Luis María; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; National Institutes of Health (NIH)
Sexual reproduction in fungi allows genetic recombination and increases genetic diversity, allowing adaptation and survival. The velvet complex is a fungal-specific protein assembly that regulates development, pathogenesis, and secondary metabolism in response to environmental cues, such as light. In Neurospora crassa, this complex comprises VE-1, VE-2, and LAE-1. Deletion of ve-1 or ve-2, but not lae-1, leads to increased conidiation (asexual spore formation) and reduced sexual development. Mutants lacking ve-1 and/or ve-2 are female sterile and male fertile, indicating that a VE-1/VE-2 complex regulates the development of female structures. During sexual development, we observed differential regulation of 2,117 genes in dark and 4,364 genes in light between the wild type and the ∆ve-1 strain. The pheromone response and cell wall integrity pathways were downregulated in the ∆ve-1 mutant, especially in light. Additionally, we found reduced levels of both total and phosphorylated MAK-1 and MAK-2 kinases. In vitro experiments demonstrated the binding of VE-1 and VE-2 to the promoters of mak-1 and mak-2, suggesting a direct regulatory role of VE-1/VE-2 in the transcriptional control of MAPK genes to regulate sexual development. Deletion of the photosensor gene white-collar 1 prevented the light-dependent inhibition of sexual development in the ∆ve-1 mutant by increasing transcription of the pheromone response and cell wall integrity pathway genes to the levels in the dark. Our results support the proposal that the regulation of the MAP kinase pathways by the VE-1/ VE-2 complex is a key element in transcriptional regulation that occurs during sexual development. IMPORTANCE Sexual reproduction generates new gene combinations and novel phenotypic traits and facilitates evolution. Induction of sexual development in fungi is often regulated by environmental conditions, such as the presence of light and nutrients. The velvet protein complex coordinates internal cues and environmental signals to regulate development. We have found that VE-1, a component of the velvet complex, regulates transcription during sexual development in the fungus Neurospora crassa. VE-1 regulates the transcription of many genes, including those involved in mitogen-activated protein kinase (MAPK) signaling pathways that are essential in the regulation of sexual development, and regulates the activity of the MAPK pathway. Our findings provide valuable insights into how fungi respond to environmental signals and integrate them into their reproductive processes.
PIWI proteins and piRNAs: key regulators of stem cell biology
(Frontiers Media, 2025-02-06) Claro Linares, Fernando; Rojas Ríos, Patricia; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla; Junta de Andalucía
In this mini review, we discussed the functional roles of PIWI proteins and their associated small RNAs, piRNAs, in regulating gene expression within stem cell biology. Guided by piRNAs, these proteins transcriptionally and posttranscriptionally repress transposons using mechanisms such as the pingpong amplification cycle and phasing to protect germline genomes. Initially identified in Drosophila melanogaster, the piRNA pathway regulate germline stem cell self-renewal and differentiation via cell-autonomous and non-cellautonomous mechanisms. Precisely, in GSCs, PIWI proteins and piRNAs regulate gene expression by modulating chromatin states and directly influencing mRNA translation. For instance, the PIWI protein Aubergine loaded with piRNAs promotes and represses translation of certain mRNAs to balance self-renewal and differentiation. Thus, the piRNA pathway exhibits dual regulatory roles in mRNA stability and translation, highlighting its context-dependent functions. Moreover, PIWI proteins are essential in somatic stem cells to support the regenerative capacity of highly regenerative species, such as planarians. Similarly, in Drosophila intestinal stem cells, the PIWI protein Piwi regulates metabolic pathways and genome integrity, impacting longevity and gut homeostasis. In this case, piRNAs appear absent in the gut, suggesting piRNAindependent regulatory mechanisms. Together, PIWI proteins and piRNAs demonstrate evolutionary conservation in stem cell regulation, integrating TE silencing and gene expression regulation at chromatin and mRNA levels in somatic and germline lineages. Beyond their canonical roles, emerging evidence reveal their broader significance in maintaining stem cell properties and organismal health under physiological and pathological conditions.
Mutational analysis of the inactivating factors, IF7 and IF17 from Synechocystis sp. PCC 6803: Critical role of arginine amino acid residues for glutamine synthetase inactivation
(John Wiley & Sons, 2011-11) Saelices, Lorena; Galmozzi, Carla V.; Florencio Bellido, Francisco Javier; Muro Pastor, María Isabel; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
The Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by a process that involves protein-protein interaction with two inactivating factors (IF7 and IF17). IF7 is a natively unfolded, 65-residue-long protein, homologous to the carboxy-terminal region of IF17. Both proteins have abundance of positively charged amino acid residues and a high isoelectric point. In this study, we analyse the IF amino acid residues involved in GS inactivation by a mutational approach, both in vitro and in vivo. The results clearly indicate that the GS-IF complex formation must be determined mainly by electrostatic interactions. We have identified three conserved arginine residues of IF7 and IF17 that are essential for the interaction of these proteins with GS. All these residues map in the homologous region of IFs. Furthermore, in vitro analysis of a truncated IF17 protein without the 82-residue-long amino-terminal part, together with the analysis of a Synechocystis strain expressing a chimeric protein, containing this amino-terminal part of IF17 fused to IF7, demonstrates that amino-terminal region of IF17 mostly confers a higher stability to this protein.
The inactivating factor of glutamine synthetase IF17 Is an intrinsically disordered protein, which folds upon binding to its target
(American Chemical Society, 2011-10-12) Saelices, Lorena; Galmozzi, Carla V.; Florencio Bellido, Francisco Javier; Muro Pastor, María Isabel; Neira, José L.; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Generalitat Valenciana; Junta de Andalucía
In cyanobacteria, ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase and glutamate synthase (GOGAT). The activity of Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) is controlled by a post-transcriptional process involving protein-protein interactions with two inactivating factors: the 65-residue-long protein (IF7) and the 149-residue-long one (IF17). The sequence of the C terminus of IF17 is similar to IF7; IF7 is an intrinsically disordered protein (IDP). In this work, we study the structural propensities and affinity for GS of IF17 and a chimera protein, IF17N/IF7 (constructed by fusing the first 82 residues of IF17 with the whole IF7) by fluorescence, CD, and NMR. IF17 and IF17N/IF7 are IDPs with residual non-hydrogen-bonded structure, probably formed by α-helical, turn-like, and PPII conformations; several theoretical predictions support these experimental findings. IF17 seems to fold upon binding to GS, as suggested by CD thermal denaturations and steady-state far-UV spectra. The apparent affinity of IF17 for GS, as measured by fluorescence, is slightly smaller (K D ∼1 μM) than that measured for IF7 (∼0.3 μM). The K Ds determined by CD are similar to those measured by fluorescence, but slightly larger, suggesting possible conformational rearrangements in the IFs and/or GS upon binding. Further, the results with IFN17/IF7 suggest that (i) binding of IF17 to the GS is modulated not only by its C-terminal region but also by its N-terminus and (ii) there are weakly structured (that is, "fuzzy") complexes in the ternary GS-IF system.
Evolution of a bistable genetic system in fluctuating and nonfluctuating environments
(National Academy of Sciences, 2024-08-30) Fernández Fernández, Rocío; Rodríguez Olivenza, David; Weyer, Esther; Singh, Abhyudai; Casadesús Pursals, Josep; Sánchez Romero, María Antonia; Universidad de Sevilla. Departamento de Microbiología y Parasitología; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Agencia Estatal de Investigación. España; European Union (UE)
Epigenetic mechanisms can generate bacterial lineages capable of spontaneously switching between distinct phenotypes. Currently, mathematical models and simulations propose epigenetic switches as a mechanism of adaptation to deal with fluctuating environments. However, bacterial evolution experiments for testing these predictions are lacking. Here, we exploit an epigenetic switch in Salmonella enterica, the opvAB operon, to show clear evidence that OpvAB bistability persists in changing environments but not in stable conditions. Epigenetic control of transcription in the opvAB operon produces OpvABOFF (phage-sensitive) and OpvABON (phage-resistant) cells in a reversible manner and may be interpreted as an example of bet-hedging to preadapt Salmonella populations to the encounter with phages. Our experimental observations and computational simulations illustrate the adaptive value of epigenetic variation as an evolutionary strategy for mutation avoidance in fluctuating environments. In addition, our study provides experimental support to game theory models predicting that phenotypic heterogeneity is advantageous in changing and unpredictable environments.
Genome-wide Function of THO/TREX in Active Genes Prevents R-loop-dependent Replication Obstacles
(EMBO Press, 2011) Gómez González, Belén; García Rubio, María Luisa; Bermejo, Rodrigo; Gaillard, Hélène; Shirahige, Katsuhiko; Marín, Antonio; Foiani, Marco; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription-associated recombination. Whether or not it has a ubiquitous role in the genome is unknown. Chromatin immunoprecipitation (ChIP)-chip studies reveal that the Hpr1 component of THO and the Sub2 RNA-dependent ATPase have genome-wide distributions at active ORFs in yeast. In contrast to RNA polymerase II, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a gradual 5-2 3-2 gradient. This is accompanied by a genome-wide impact of THO-"Sub2 deletions on expression of highly expressed, long and high G+C-content genes. Importantly, ChIP-chips reveal an over-recruitment of Rrm3 in active genes in THO mutants that is reduced by RNaseH1 overexpression. Our work establishes a genome-wide function for THO-"Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription-mediated replication obstacles, including R-loops.
XGene expression is circular: Factors for mRNA degradation also foster mRNA synthesis
(Elsevier, 2013-05-23) Haimovich, Gal; Medina, Daniel A.; Causse, Sebastien Z.; Garber, Manuel; Millán Zambrano, Gonzalo; Barkai, Oren; Chávez de Diego, Sebastián; Pérez-Ortín, José E.; Darzacq, Xavier; Choder, Mordechai; Universidad de Sevilla. Departamento de Genética; Israel Science Foundation; Ministry of Health, and Rappaport Foundation; Ministerio de Ciencia e Innovación (MICIN). España; Generalitat Valenciana; Junta de Andalucía; European Union (UE); Center for Excellence in Genome Science
Maintaining proper mRNA levels is a key aspect in the regulation of gene expression. The balance between mRNA synthesis and decay determines these levels. We demonstrate that most yeast mRNAs are degraded by the cytoplasmic 5′-to-3′ pathway (the "decaysome"), as proposed previously. Unexpectedly, the level of these mRNAs is highly robust to perturbations in this major pathway because defects in various decaysome components lead to transcription downregulation. Moreover, these components shuttle between the cytoplasm and the nucleus, in a manner dependent on proper mRNA degradation. In the nucleus, they associate with chromatin - preferentially ∼30 bp upstream of transcription start-sites - and directly stimulate transcription initiation and elongation. The nuclear role of the decaysome in transcription is linked to its cytoplasmic role in mRNA decay; linkage, in turn, seems to depend on proper shuttling of its components. The gene expression process is therefore circular, whereby the hitherto first and last stages are interconnected.
Histone post-translational modifications — cause and consequence of genome function
(Springer Nature, 2022-03-25) Millán Zambrano, Gonzalo; Burton, Adam; Bannister, Andrew J.; Schneider, Robert; Universidad de Sevilla. Departamento de Genética; German Research Foundation (DFG); Cancer Research. United Kingdom; Wellcome Trust; Ministerio de Ciencia e Innovación (MICIN). España; European Union (UE)
Much has been learned since the early 1960s about histone post-translational modifications (PTMs) and how they affect DNA-templated processes at the molecular level. This understanding has been bolstered in the past decade by the identification of new types of histone PTM, the advent of new genome-wide mapping approaches and methods to deposit or remove PTMs in a locally and temporally controlled manner. Now, with the availability of vast amounts of data across various biological systems, the functional role of PTMs in important processes (such as transcription, recombination, replication, DNA repair and the modulation of genomic architecture) is slowly emerging. This Review explores the contribution of histone PTMs to the regulation of genome function by discussing when these modifications play a causative (or instructive) role in DNA-templated processes and when they are deposited as a consequence of such processes, to reinforce and record the event. Important advances in the field showing that histone PTMs can exert both direct and indirect effects on genome function are also presented.
A matter of packaging: Influence of nucleosome positioning on heterologous gene expression
(Springer Nature, 2011-11-21) Muñoz Centeno, María de la Cruz; Millán Zambrano, Gonzalo; Chávez de Diego, Sebastián; Universidad de Sevilla. Departamento de Genética; Ministerio de Educación y Ciencia (MEC). España; Junta de Andalucía
The organization of DNA into the various levels of chromatin compaction is the main obstacle that restricts the access of transcriptional machinery to genes. Genome-wide chromatin analyses have shown that there are common chromatin organization patterns for most genes but have also revealed important differences in nucleosome positioning throughout the genome. Such chromatin heterogeneity is one of the reasons why recombinant gene expression is highly dependent on integration sites. Different solutions have been tested for this problem, including artificial targeting of chromatin-modifying factors or the addition of DNA elements, which efficiently counteract the influence of the chromatin environment. An influence of the chromatin configuration of the recombinant gene itself on its transcriptional behavior has also been established. This view is especially important for heterologous genes since the general parameters of chromatin organization change from one species to another. The chromatin organization of bacterial DNA proves particularly dramatic when introduced into eukaryotes. The nucleosome positioning of recombinant genes is the result of the interaction between the machinery of the hosting cell and the sequences of both the recombinant genes and the promoter regions. We discuss the key aspects of this phenomenon from the heterologous gene expression perspective.
R‐loops cause replication impairment and genome instability during meiosis
(EMBO Press, 2012) Castellano Pozo, Maikel; García Muse, Tatiana; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; European Union (UE); Junta de Andalucía
R‐loops are harmful structures with a negative impact on transcription and recombination during mitosis, but no information exists for meiosis. We used Saccharomyces cerevisiae and Caenorhabditis elegans THO mutants as a tool to determine the consequences of R‐loops in meiosis. We found that both S. cerevisiae and C. elegans THO mutants show defective meiosis and an impairment of premeiotic replication as well as DNA‐damage accumulation. Importantly, RNase H partially suppressed the replication impairment and the DNA‐damage accumulation. We conclude that R‐loops can form during meiosis causing replication impairment with deleterious results.
R Loops Are Linked to Histone H3 S10 Phosphorylation and Chromatin Condensation
(Cell Press, 2013) Castellano Pozo, Maikel; Santos Pereira, José María; García Rondón, Ana; Barroso Ceballos, Sonia Inés; Andújar, Eloísa; Pérez Alegre, Mónica; García Muse, Tatiana; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Ministerio de Economía y Competitividad (MINECO). España; Junta de Andalucía; European Union (UE)
R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics.

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