Artículos (Genética)

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Properties of polyplexes formed between a cationic polymer derived from l-arabinitol and nucleic acids
(Royal Society of Chemistry, 2021-05-14) Pérez Alfonso, David; López López, Manuel; López-Cornejo, María del Pilar; Romero Azogil, Lucía; Benito Hernández, Elena María; García Martín, María de Gracia; García Calderón, Clara Beatriz; Valle Rosado, Iván; Romero Balestra, Fernando; Huertas Sánchez, Pablo; García Calderón, Margarita; Moyá Morán, María Luisa; Universidad de Sevilla. Departamento de Química Física; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Universidad de Sevilla. Departamento de Química Orgánica y Farmacéutica; Universidad de Sevilla. Departamento de Genética; Junta de Andalucía; Universidad de Sevilla; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; European Union (UE)
In this work a sugar-based cationic polymer derived from L-arabinitol, PUArab, was prepared and its interactions with the linear calf thymus DNA and with the circular plasmid pEGFP-C1 were investigated at different N/P ratios. The polyplexes were characterized by using several techniques. For both nucleic acids, a charge inversion was observed, together with a conformational change from a coiled structure to a more compacted one. However, the N/P ratio required to observe the DNA condensation depended on the nucleic acid architecture. PUArab presents low toxicity in several cell lines. The transfection efficiency, TE, of the PUArab/pEGFP-C1 polyplexes was investigated at several N/P ratios in order to study their potential as vectors in gene transfection.
The zinc-finger transcription factor Sfp1 imprints specific classes of mRNAs and links their synthesis to cytoplasmic decay
(eLife Sciences Publications, 2024-10-02) Kelbert, Moran; Jordán-Pla, Antonio; Miguel-Jiménez, Lola de; García-Martínez, José; Selitrennik, Michael; Guterman, Adi; Chávez de Diego, Sebastián; Choder, Mordechai; Universidad de Sevilla. Departamento de Genética; Israel Science Foundation (ISF). Israel; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Agencia Estatal de Investigación. España; Medical Research Council (MRC). Reino Unido
To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1's dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1's relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1's co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1's two modes of transcription regulation remains to be examined.
piRNAs are regulators of metabolic reprogramming in stem cells
(Springer Nature, 2024-09-27) Rojas Ríos, Patricia; Chartier, Aymeric; Enjolras, Camille; Cremaschi, Julie; Garret, Céline; Boughlita, Adel; Ramat, Anne; Simonelig, Martine; Universidad de Sevilla. Departamento de Genética; Centre national de la recherche scientifique (CNRS). France; Agence nationale de la recherche (ANR). France; Fondation pour la Recherche Médicale (FRM). France
Stem cells preferentially use glycolysis instead of oxidative phosphorylation and this metabolic rewiring plays an instructive role in their fate; however, the underlying molecular mechanisms remain largely unexplored. PIWI-interacting RNAs (piRNAs) and PIWI proteins have essential functions in a range of adult stem cells across species. Here, we show that piRNAs and the PIWI protein Aubergine (Aub) are instrumental in activating glycolysis in Drosophila female germline stem cells (GSCs). Higher glycolysis is required for GSC self-renewal and aub loss-of-function induces a metabolic switch in GSCs leading to their differentiation. Aub directly binds glycolytic mRNAs and Enolase mRNA regulation by Aub depends on its 5′UTR. Furthermore, mutations of a piRNA target site in Enolase 5′UTR lead to GSC loss. These data reveal an Aub/piRNA function in translational activation of glycolytic mRNAs in GSCs, and pinpoint a mechanism of regulation of metabolic reprogramming in stem cells based on small RNAs.
Evolutionary origin and population diversity of a cryptic hybrid pathogen
(Springer Nature, 2024-08-28) Steenwyk, Jacob L.; Knowles, Sonja; Bastos, Rafael W.; Balamurugan, Charu; Rinker, David; Mead, Matthew E.; Roberts, Christopher D.; Raja, Huzefa A.; Li, Yuanning; Colabardini, Ana Cristina; de Castro, Patricia Alves; Dos Reis, Thaila Fernanda; Gumilang, Adiyantara; Almagro-Molto, Maria; Alanio, Alexandre; Garcia-Hermoso, Dea; Delbaje, Endrews; Pontes, Lais; Pinzan, Camila Figueiredo; Schreiber, Angelica Zaninelli; Cánovas López, David; Sanchez Luperini, Rafael; Lagrou, Katrien; Torrado, Egidio; Rodrigues, Fernando; Oberlies, Nicholas H.; Zhou, Xiaofang; Goldman, Gustavo H.; Rokas, Antonis; Universidad de Sevilla. Departamento de Genética; National Science Foundation (NSF). United States; National Institute of Allergy and Infectious Diseases (NIAID); Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); European Union (UE). H2020; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Agencia Estatal de Investigación. España
Cryptic fungal pathogens pose disease management challenges due to their morphological resemblance to known pathogens. Here, we investigated the genomes and phenotypes of 53 globally distributed isolates of Aspergillus section Nidulantes fungi and found 30 clinical isolates—including four isolated from COVID-19 patients—were A. latus, a cryptic pathogen that originated via allodiploid hybridization. Notably, all A. latus isolates were misidentified. A. latus hybrids likely originated via a single hybridization event during the Miocene and harbor substantial genetic diversity. Transcriptome profiling of a clinical isolate revealed that both parental subgenomes are actively expressed and respond to environmental stimuli. Characterizing infection-relevant traits—such as drug resistance and growth under oxidative stress—revealed distinct phenotypic profiles among A. latus hybrids compared to parental and closely related species. Moreover, we identified four features that could aid A. latus taxonomic identification. Together, these findings deepen our understanding of the origin of cryptic pathogens.
Phytoplasma DNA Enrichment from Sugarcane White Leaves for Shotgun Sequencing Improvement
(Multidisciplinary Digital Publishing Institute (MDPI), 2024-10-28) Lohmaneeratana, Karan; Gutiérrez Pozo, Gabriel; Thamchaipenet, Arinthip; Wellinger, Ralf Erik; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla; Junta de Andalucía; Kasetsart University Research and Development Institute; National Research Council of Thailand
Sugarcane white leaf (SCWL) disease, caused by Candidatus Phytoplasma sacchari, poses a significant threat to sugarcane cultivation. An obligate parasite, phytoplasma is difficult to culture in laboratory conditions, making the isolation of its DNA from the massive amount of plant host DNA extremely challenging. Yet, the appropriate amount and quality of plant microbiome-derived DNA are key for high-quality DNA sequencing data. Here, a simple, cost-effective, alternative method for DNA isolation was applied using a guanidine-HCl-hydroxylated silica (GuHCl-Silica)-based method and microbiome DNA enrichment based on size-selective low-molecular-weight (LMW) DNA by PEG/NaCl precipitation. qPCR analysis revealed a significant enrichment of phytoplasma DNA in the LMW fraction. Additionally, the NEBNext Microbiome DNA enrichment kit was utilized to further enrich microbial DNA, demonstrating a remarkable increase in the relative abundance of phytoplasma DNA to host DNA. Shotgun sequencing of the isolated DNA gave high-quality data on the metagenome assembly genome (MAG) of Ca. Phytoplasma sacchari SCWL with completeness at 95.85 and 215× coverage. The results indicate that this combined approach of PEG/NaCl size selection and microbiome enrichment is effective for obtaining high-quality genomic data from phytoplasma, surpassing previous methods in efficiency and resource utilization. This low-cost method not only enhances the recovery of microbiome DNA from plant hosts but also provides a robust framework for studying plant pathogens in complex plant models.
Mechanism of BRCA1–BARD1 function in DNA end resection and DNA protection
(Springer Nature, 2024-09-11) Ceppi, Ilaria; Dello Stritto, Maria Rosaria; Mütze, Martin; Braunshier, Stefan; Mengoli, Valentina; Reginato, Giordano; Võ, Hồ Mỹ Phúc; Jimeno González, Sonia; Huertas Sánchez, Pablo; Cejka, Petr; Universidad de Sevilla. Departamento de Genética; Swiss National Science Foundation (SNFS); European Research Council (ERC); Ministerio de Ciencia e Innovación (MICIN). España; Agence Nationale de la Recherche
DNA double-strand break (DSB) repair by homologous recombination is initiated by DNA end resection, a process involving the controlled degradation of the 5′-terminated strands at DSB sites1,2. The breast cancer suppressor BRCA1–BARD1 not only promotes resection and homologous recombination, but it also protects DNA upon replication stress1,3–9. BRCA1–BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, but whether it functions in resection directly has been unclear10–16. Using purified recombinant proteins, we show here that BRCA1–BARD1 directly promotes long-range DNA end resection pathways catalysed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1–BARD1 stimulates DNA unwinding by the Werner or Bloom helicase. Together with MRE11–RAD50–NBS1 and phosphorylated CtIP, BRCA1–BARD1 forms the BRCA1–C complex17,18, which stimulates resection synergistically to an even greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1–C complex19–21, inhibits resection, showing that BRCA1–C is a functionally integrated ensemble. Whereas BRCA1–BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves a homologous recombination-independent function of the recombinase RAD51 (refs. 4–6,8). We show that in the presence of RAD51, BRCA1–BARD1 instead inhibits DNA degradation. On the basis of our data, the presence and local concentration of RAD51 might determine the balance between the pronuclease and the DNA protection functions of BRCA1–BARD1 in various physiological contexts.
Light Reception of Phycomyces Revisited: Several White Collar Proteins Confer Blue- and Red-light Sensitivity and Control Dynamic Range and Adaptation
(Springer Nature, 2024) Galland, P.; Corrochano Peláez, Luis María; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
The giant-fruiting body, sporangiophore, of the fungus Phycomyces blakesleeanus grows toward near-UV/blue-light (phototropism). The blue-light photoreceptor, MadA, should contain FAD bound to the LOV domain, and forms a complex with MadB. Both proteins are homologs of white collar proteins WC-1 and WC-2 from the fungus Neurospora crassa and should be localized in nuclei, where they function as a light-sensitive transcription factor complex. The photoreceptor properties of two further Wc proteins, WcoA and WcoB, remain unclear because of lack of mutants. We propose that WcoA and/or WcoB play essential roles in photoreception by enlarging the dynamic range that help explain complex stimulus–response relationships. Even though red light does not elicit photo-movement or -differentiation in Phycomyces, it affects the effectiveness of blue light which indicates an underlying photochromic receptor. Protein sequence searches show that other fungal red-light receptors are absent in Phycomyces. The solution to the red-light riddle is thus sought in the ability of Wc complexes to generate after blue-light irradiation a neutral flavosemiquinone radical that absorbs red light and functions as primary photochemical signal. Phototropism requires Ras-GAP (MadC) as part of the signal transduction cascade and, we propose, to allocate photoreceptors in the plasmalemma of the growing zone, which allows for receptor dichroism, range adjustment and contrast recognition for spatial orientation. Phototropic signal chains must entail transduction networks between Wc receptors and small G-proteins and their associated Ras-GAP and Ras-GEF proteins. The interactions among these proteins should occur in trans-Golgi vesicles and the plasmalemma of the growing zone. Graphical abstract: (Figure presented.)
Human DNA Topoisomerase I Poisoning Causes R Loop-mediated Genome Instability Attenuated by Transcription Factor IIS
(American Association for the Advancement of Science, 2024) Duardo, Renée C.; Marinello, Jessica; Russo, Marco; Morelli, Sara; Pepe, Simona; Guerra, Federico; Gómez González, Belén; Aguilera López, Andrés; Capranico, Giovanni; Universidad de Sevilla. Departamento de Genética; Associazione italiana per la Ricerca sul cancro (AIRC). Italy; Ministry of University and Research. Rome, Italy; Ministerio de Ciencia e Innovación (MICIN). España; European Research Council (ERC); European Union (UE)
DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms. DSBs are at highly transcribed genes in early replicating initiation zones and overlap with hybrids downstream of accumulated RNA polymerase II (RNAPII) at gene 5'-ends. A transcription factor IIS mutant impairing transcription elongation further increased RNAPII accumulation likely due to backtracking. Moreover, Top1ccs can trigger micronuclei when occurring during late G1 or early/mid S, but not during late S. As micronuclei and transcription-replication conflicts are attenuated by transcription factor IIS, our results support a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts leading to DSBs and micronuclei.
Role of J-domain Proteins in Yeast Physiology and Protein Quality Control
(Elsevier, 2024) Ruger Herreros, Carmen; Svoboda, Lucia; Mogk, Axel; Bukau, Bernd; Universidad de Sevilla. Departamento de Genética; Deutsche Forschungsgemeinschaft / German Research Foundation (DFG); Heidelberg Biosciences International Graduate School
The Hsp70 chaperone system is a central component of cellular protein quality control (PQC) by acting in a multitude of protein folding processes ranging from the folding of newly synthesized proteins to the disassembly and refolding of protein aggregates. This multifunctionality of Hsp70 is governed by J-domain proteins (JDPs), which act as indispensable co-chaperones that target specific substrates to Hsp70. The number of distinct JDPs present in a species always outnumbers Hsp70, documenting JDP function in functional diversification of Hsp70. In this review, we describe the physiological roles of JDPs in the Saccharomyces cerevisiae PQC system, with a focus on the abundant JDP generalists, Zuo1, Ydj1 and Sis1, which function in fundamental cellular processes. Ribosome-bound Zuo1 cooperates with the Hsp70 chaperones Ssb1/2 in folding and assembly of nascent polypeptides. Ydj1 and Sis1 cooperate with the Hsp70 members Ssa1 to Ssa4 to exert overlapping functions in protein folding and targeting of newly synthesized proteins to organelles including mitochondria and facilitating the degradation of aberrant proteins by E3 ligases. Furthermore, they act in protein disaggregation reactions, though Ydj1 and Sis1 differ in their modes of Hsp70 cooperation and substrate specificities. This results in functional specialization as seen in prion propagation and the underlying dominant role of Sis1 in targeting Hsp70 for shearing of prion amyloid fibrils.
Histone variant H2A.Z is needed for efficient transcription-coupled NER and genome integrity in UV challenged yeast cells
(Public Library of Science, 2024-09-10) Gaillard, Hélène; Ciudad, Toni; Aguilera López, Andrés; Wellinger, Ralf Erik; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Junta de Andalucía; Universidad de Sevilla; Ministerio de Economía y Competitividad (MINECO). España
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.
Spontaneous deamination of cytosine to uracil is biased to the non-transcribed DNA strand in yeast
(Elsevier, 2023-06) Williams, Jonathan D.; Zhu, Demi; García Rubio, María Luisa; Shaltz, Samantha; Aguilera López, Andrés; Jinks-Robertson, Sue; Universidad de Sevilla. Departamento de Genética; National Institutes of Health. United States; Ministerio de Ciencia e Innovación (MICIN). España
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.
RNA biogenesis and RNA metabolism factors as R-loop suppressors: a hidden role in genome integrity.
(Cold Spring Harbor Laboratory Press, 2024-07) Luna Varo, Rosa María; Gómez González, Belén; Aguilera López, Andrés; 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); Fundación Caixa de Investigación. España; Junta de Andalucía
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.
The Beak of Eukaryotic Ribosomes: Life, Work and Miracles
(MDPI, 2024-07-22) Martín Villanueva, Sara; Galmozzi, Carla V.; Ruger Herreros, Carmen; Kressler, Dieter; Cruz Díaz, Jesús de la; 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); Swiss National Science Foundation (SNFS)
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.
PLK1 Regulates CtIP and DNA2 Interplay in Long-range DNA end Resection.
(Cold Spring Harbor Laboratory Press, 2023) Ceppi, Ilaria; Cannavo, Elda; Bret, Helene; Camarillo Daza, María Rosa; Vivalda, Francesca; Thakur, Roshan Singh; Romero Franco, Amador; Sartori, Alessandro A.; Huertas Sánchez, Pablo; Guerois, Raphael; Cejka, Petr; Universidad de Sevilla. Departamento de Genética; European Research Council (ERC); Swiss National Science Foundation (SNFS); Ministerio de Ciencia e Innovación (MICIN). España; French government
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.
Symmetric and asymmetric DNA N6-adenine methylation regulates different biological responses in Mucorales
(Nature Research, 2024-07-18) Lax, Carlos; Mondo, Stephen J.; Osorio Concepción, Macario; Muszewska, Anna; Corrochano Luque, María; Gutiérrez Pozo, Gabriel; Cánovas López, David; Corrochano Peláez, Luis María; Garre, Victoriano; 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); Comunidad Autónoma de Murcia. España; Department of Energy Joint Genome Institute. U. S.; Office of Science of the US Department of Energy. U. S.
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.
The RNAi Machinery in the Fungus Fusarium fujikuroi Is Not Very Active in Synthetic Medium and Is Related to Transposable Elements
(Multidisciplinary Digital Publishing Institute (MDPI), 2024-05-16) Pardo Medina, Javier; Dahlmann, Tim A.; Nowrousian, Minou; Limón Mirón, María del Carmen; Ávalos Cordero, Francisco Javier; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España
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.
EXO1 and DNA2-mediated ssDNA gap expansion is essential for ATR activation and to maintain viability in BRCA1-deficient cells
(Oxford University Press, 2024-06-24) García Rodríguez, Néstor; Domínguez García, Iria; Domínguez Pérez, María del Carmen; Huertas Sánchez, Pablo; Universidad de Sevilla. Departamento de Genética; Junta de Andalucía; Ministerio de Ciencia e Innovación (MICIN). España
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.
Expression of human RECQL5 in Saccharomyces cerevisiae causes transcription defects and transcription-associated genome instability
(Springer Nature, 2024-05-26) Lafuente Barquero, Juan Francisco; Svejstrup, Jesper Q.; Luna Varo, Rosa María; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; European Research Council (ERC); Novo Nordisk Foundation; Danish National Research Foundation
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.
BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis
(Nature Research, 2024-05-20) Salas Lloret, Daniel; García Rodríguez, Néstor; Soto Hidalgo, Emily; González Vinceiro, Lourdes; Espejo Serrano, Carmen; Giebel, Lisanne; Huertas Sánchez, Pablo; González Prieto, Román; Universidad de Sevilla. Departamento de Biología Celular; Universidad de Sevilla. Departamento de Genética; Junta de Andalucía; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Agencia Estatal de Investigación. España; European Union (UE)
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.
Three Genes Involved in Different Signaling Pathways, carS, wcoA, and acyA, Participate in the Regulation of Fusarin Biosynthesis in Fusarium fujikuroi
(MDPI, 2024) Díaz Sánchez, Violeta; Castrillo, Marta; García Martínez, Jorge; Avalos, Javier; Limón Mirón, María del Carmen; Universidad de Sevilla. Departamento de Genética; Plan Propio de la Universidad de Sevilla; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España; Plan Andaluz de Investigación, Desarrollo e Innovación (Junta de Andalucía); European Regional Development Fund (ERDF)
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.

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