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Precise spatiotemporal regulation of gene expression is of paramount importance for eukaryotic development. The maternal-to-zygotic transition (MZT) during early embryogenesis in Drosophila involves the gradual replacement of maternally contributed mRNAs and proteins by zygotic gene products. The zygotic genome is transcriptionally activated during the first 3 hours of development, in a process known as "zygotic genome activation" (ZGA), by the orchestrated activities of a few pioneer factors. Their decisive role during ZGA has been characterized in detail, whereas the contribution of chromatin factors to this process has been historically overlooked. In this review, we aim to summarize the current knowledge of how chromatin regulation impacts the first stages of Drosophila embryonic development. In particular, we will address the following questions: how chromatin factors affect ZGA and transcriptional silencing, and how genome architecture promotes the integration of these processes early during development. Remarkably, certain chromatin marks can be intergenerationally inherited, and their presence in the early embryo becomes critical for the regulation of gene expression at later stages. Finally, we speculate on the possible roles of these chromatin marks as carriers of epialleles during transgenerational epigenetic inheritance (TEI).
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In nearly every species of insect, embryonic development takes place outside of the mother's body and is entirely dependent on the elements that the mother had previously stored within the eggs. It is well known that the follicle cells (FCs) synthesize the eggshell (chorion) components during the process of choriogenesis, the final step of oogenesis before fertilization. These cells have developed a specialization in the massive production of chorion proteins, which are essential for the protection and survival of the embryo. Here, we investigate the function of Sec16, a protein crucial for the endoplasmic reticulum (ER) to Golgi traffic, in the oocyte development in the insect Rhodnius prolixus. We discovered that Sec16 is strongly expressed in vitellogenic females' ovaries, particularly in the choriogenic oocyte and it is mainly associated with the FCs. Silencing of Sec16 by RNAi caused a sharp decline in oviposition rates, F1 viability, and longevity in adult females. In the FCs, genes involved in the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy were massively upregulated, whereas the mRNAs of Rp30 and Rp45-which code for the two major chorion proteins - were downregulated as a result of Sec16 silencing, indicating general proteostasis disturbance. As a result, the outer surface ultrastructure of Sec16-silenced chorions was altered, with decreased thickness, dityrosine crosslinking, sulfur signals, and lower amounts of the chorion protein Rp30. These findings collectively demonstrate the critical role Sec16 plays in the proper functioning of the FCs, which impacts the synthesis and deposition of particular components of the chorion as well as the overall reproduction of this vector.
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INTRODUCTION: Cervical disease control might be challenging in advanced thyroid cancer (DTC). Indications for cervical external beam radiation therapy (EBRT) are controversial. PURPOSE: To identify clinical and molecular factors associated with control of cervical disease with EBRT. METHODS: Retrospective evaluation and molecular analysis of the primary tumor DTC patients who underwent cervical EBRT between 1995 and 2022 was performed. RESULTS: Eighty adults, median age of 61 years, were included. T4 disease was present in 43.7%, lymph node involvement in 42.5%, and distant metastasis in 47.5%. Those with cervical progression were older (62.5 vs. 57.3, p = 0.04) with more nodes affected (12.1 vs. 2.8, p = 0.04) and had EBRT performed later following surgery (76.6 vs. 64 months, p = 0.05). EBRT associated with multikinase inhibitors showed longer overall survival than EBRT alone (64.3 vs. 37.9, p = 0.018) and better local disease control. Performing EBRT before radioiodine (RAI) was associated with longer cervical progression-free survival (CPFS) than was RAI before (67.5 vs. 34.5, p < 0.01). EBRT ≥2 years after surgery was associated with worse CPFS (4.9 vs. 34, p = 0.04). The most common molecular alterations were ERBB2, BRAF, FAT1, RET and ROS1 and TERT mutation was predictive of worse disease control after EBRT (p = 0.04). CONCLUSION: Younger patients, with fewer affected nodes and treated earlier after surgery had better cervical disease control. Combination of EBRT with MKI improved OS. TERT mutation might indicate worse responders to EBRT; however, further studies are necessary to clarify the role of molecular testing in selecting candidates for cervical EBRT.
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Recurrencia Local de Neoplasia , Neoplasias de la Tiroides , Humanos , Femenino , Persona de Mediana Edad , Neoplasias de la Tiroides/radioterapia , Neoplasias de la Tiroides/mortalidad , Neoplasias de la Tiroides/patología , Masculino , Estudios Retrospectivos , Anciano , Adulto , Neoplasia Residual , Radioisótopos de Yodo/uso terapéutico , Tiroidectomía , Factores de TiempoRESUMEN
Autophagy and the ubiquitin-proteasome system (UPS) are important cellular mechanisms that coordinate protein degradation essential for proteostasis. P62/SQSTM1 is a receptor cargo protein able to deliver ubiquitinated targets to the proteasome proteolytic complex and/or to the autophagosome. In the insect vector of Chagas disease, Rhodnius prolixus, previous works have shown that the knockdown of different autophagy-related genes (ATGs) and ubiquitin-conjugating enzymes resulted in abnormal oogenesis phenotypes and embryo lethality. Here, we investigate the role of the autophagy/UPS adaptor protein p62 during the oogenesis and reproduction of this vector. We found that R. prolixus presents one isoform of p62 encoded by a non-annotated gene. The predicted protein presents the domain architecture anticipated for p62: PB1 (N-term), ZZ-finger, and UBA (C-term) domains, and phylogenetic analysis showed that this pattern is highly conserved within insects. Using parental RNAi, we found that although p62 is expressed in the ovary, midgut, and fat body of adult females, systemic silencing of this gene did not result in any apparent phenotypes under in-house conditions. The insects' overall levels of blood meal digestion, lifespan, yolk protein production, oviposition, and embryo viability were not altered when compared to controls. Because it is known that autophagy and UPS can undergo compensatory mechanisms, we asked whether the silencing of p62 was triggering adaptative changes in the expression of genes of the autophagy, UPS, and the unfolded protein response (UPR) and found that only ATG1 was slightly up regulated in the ovaries of silenced females. In addition, experiments to further investigate the role of p62 in insects previously silenced for the E1-conjugating enzyme (a condition known to trigger the upregulation of p62), also did not result in any apparent phenotypes in vitellogenic females.
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Complejo de la Endopetidasa Proteasomal , Rhodnius , Femenino , Animales , Proteína Sequestosoma-1 , Filogenia , Interferencia de ARN , UbiquitinaRESUMEN
Pigmentation in insects has been linked to mate selection and predator evasion, thus representing an important aspect for natural selection. Insect body color is classically associated to the activity of tyrosine pathway enzymes, and eye color to pigment synthesis through the tryptophan and guanine pathways, and their transport by ATP-binding cassette proteins. Among the hemiptera, the genetic basis for pigmentation in kissing bugs such as Rhodnius prolixus, that transmit Chagas disease to humans, has not been addressed. Here, we report the functional analysis of R. prolixus eye and cuticle pigmentation genes. Consistent with data for most insect clades, we show that knockdown for yellow results in a yellow cuticle, while scarlet and cinnabar knockdowns display red eyes as well as cuticle phenotypes. In addition, tyrosine pathway aaNATpreto knockdown resulted in a striking dark cuticle that displays no color pattern or UV reflectance. In contrast, knockdown of ebony and tan, that encode N-beta-alanyl dopamine hydroxylase branch tyrosine pathway enzymes, did not generate the expected dark and light brown phenotypes, respectively, as reported for other insects. We hypothesize that R. prolixus, which requires tyrosine pathway enzymes for detoxification from the blood diet, evolved an unusual strategy for cuticle pigmentation based on the preferential use of a color erasing function of the aaNATpreto tyrosine pathway branch. We also show that genes classically involved in the generation and transport of eye pigments regulate red body color in R. prolixus. This is the first systematic approach to identify the genes responsible for the generation of color in a blood-feeding hemiptera, providing potential visible markers for future transgenesis.
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Rhodnius , Animales , Pigmentación/genética , Rhodnius/genética , TirosinaRESUMEN
BACKGROUND: Glioblastoma is a fatal brain tumour with a poor patient survival outcome. Hypoxia has been shown to reprogram cells towards a stem cell phenotype associated with self-renewal and drug resistance properties. Activation of hypoxia-inducible factors (HIFs) helps in cellular adaptation mechanisms under hypoxia. Similarly, miRNAs are known to be dysregulated in GBM have been shown to act as critical mediators of the hypoxic response and to regulate key processes involved in tumorigenesis. METHODS: Glioblastoma (GBM) cells were exposed to oxygen deprivation to mimic a tumour microenvironment and different cell aspects were analysed such as morphological changes and gene expression of miRNAs and survival genes known to be associated with tumorigenesis. RESULTS: It was observed that miR-128a-3p, miR-34-5p, miR-181a/b/c, were down-regulated in 6 GBM cell lines while miR-17-5p and miR-221-3p were upregulated when compared to a non-GBM control. When the same GBM cell lines were cultured under hypoxic microenvironment, a further 4-10-fold downregulation was observed for miR-34-5p, miR-128a-3p and 181a/b/c while a 3-6-fold upregulation was observed for miR-221-3p and 17-5p for most of the cells. Furthermore, there was an increased expression of SOX2 and Oct4, GLUT-1, VEGF, Bcl-2 and survivin, which are associated with a stem-like state, increased metabolism, altered angiogenesis and apoptotic escape, respectively. CONCLUSION: This study shows that by mimicking a tumour microenvironment, miRNAs are dysregulated, stemness factors are induced and alteration of the survival genes necessary for the cells to adapt to the micro-environmental factors occurs. Collectively, these results might contribute to GBM aggressiveness.
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Neoplasias Encefálicas/genética , Glioblastoma/genética , MicroARNs/metabolismo , Hipoxia Tumoral/genética , Microambiente Tumoral/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Regulación hacia Abajo , Genotipo , Glioblastoma/metabolismo , Glioblastoma/patología , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Transcripción SOXB1/metabolismo , Survivin/metabolismo , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Triatomine assassin bugs comprise hematophagous insect vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Although the microbiome of these species has been investigated to some extent, only one virus infecting Triatoma infestans has been identified to date. Here, we describe for the first time seven (+) single-strand RNA viruses (RpV1-7) infecting Rhodnius prolixus, a primary vector of Chagas disease in Central and South America. We show that the RpVs belong to the Iflaviridae, Permutotetraviridae and Solemoviridae and are vertically transmitted from the mothers to the progeny via transovarial transmission. Consistent with this, all the RpVs, except RpV2 that is related to the entomopathogenic Slow bee paralysis virus, established persistent infections in our R. prolixus colony. Furthermore, we show that R. prolixus ovaries express 22-nucleotide viral siRNAs (vsiRNAs), but not viral piRNAs, that originate from the processing of dsRNA intermediates during viral replication of the RpVs. Interestingly, the permutotetraviruses and sobemoviruses display shared pools of vsiRNAs that might provide the basis for a cross-immunity system. The vsiRNAs are maternally deposited in the eggs, where they likely contribute to reduce the viral load and protect the developing embryos. Our results unveil for the first time a complex core virome in R. prolixus and begin to shed light on the RNAi-based antiviral defenses in triatomines.
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Enfermedad de Chagas/transmisión , Insectos Vectores/virología , Virus ARN/fisiología , Rhodnius/virología , Triatoma/virología , Trypanosoma cruzi/fisiología , Viroma , Animales , Femenino , Genoma Viral , Oogénesis , Virus ARN/clasificación , ARN Interferente Pequeño/genética , Conejos , TranscriptomaRESUMEN
Rhodnius prolixus is a Triatominae insect species and a primary vector of Chagas disease. The genome of R. prolixus has been recently sequenced and partially assembled, but few transcriptome analyses have been performed to date. In this study, we describe the stage-specific transcriptomes obtained from previtellogenic stages of oogenesis and from mature eggs. By analyzing ~ 228 million paired-end RNA-Seq reads, we significantly improved the current genome annotations for 9206 genes. We provide extended 5' and 3' UTRs, complete Open Reading Frames, and alternative transcript variants. Strikingly, using a combination of genome-guided and de novo transcriptome assembly we found more than two thousand novel genes, thus increasing the number of genes in R. prolixus from 15,738 to 17,864. We used the improved transcriptome to investigate stage-specific gene expression profiles during R. prolixus oogenesis. Our data reveal that 11,127 genes are expressed in the early previtellogenic stage of oogenesis and their transcripts are deposited in the developing egg including key factors regulating germline development, genome integrity, and the maternal-zygotic transition. In addition, GO term analyses show that transcripts encoding components of the steroid hormone receptor pathway, cytoskeleton, and intracellular signaling are abundant in the mature eggs, where they likely control early embryonic development upon fertilization. Our results significantly improve the R. prolixus genome and transcriptome and provide novel insight into oogenesis and early embryogenesis in this medically relevant insect.
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Enfermedad de Chagas/genética , Ovario/metabolismo , Rhodnius/genética , Transcriptoma/genética , Animales , Enfermedad de Chagas/parasitología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Genoma de los Insectos/genética , Humanos , Insectos Vectores/genética , Insectos Vectores/parasitología , Oogénesis/genética , Ovario/crecimiento & desarrollo , Rhodnius/parasitología , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidadRESUMEN
Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi, and is transmitted by triatomine insects during its blood meal. Proliferative epimastigotes forms thrive inside the insects in the presence of heme (iron protoporphyrin IX), an abundant product of blood digestion, however little is known about the metabolic outcome of this signaling molecule in the parasite. Trypanosomatids exhibit unusual gene transcription employing a polycistronic transcription mechanism through trans-splicing that regulates its life cycle. Using the Deep Seq transcriptome sequencing we characterized the heme induced transcriptome of epimastigotes and determined that most of the upregulated genes were related to glucose metabolism inside the glycosomes. These results were supported by the upregulation of glycosomal isoforms of PEPCK and fumarate reductase of heme-treated parasites, implying that the fermentation process was favored. Moreover, the downregulation of mitochondrial gene enzymes in the presence of heme also supported the hypothesis that heme shifts the parasite glycosomal glucose metabolism towards aerobic fermentation. These results are examples of the environmental metabolic plasticity inside the vector supporting ATP production, promoting epimastigotes proliferation and survival.
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Perfilación de la Expresión Génica , Hemo/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/metabolismo , Animales , Enfermedad de Chagas/metabolismo , Genes Mitocondriales , Glucosa/metabolismo , Insectos Vectores/parasitología , Microcuerpos/metabolismo , Transducción de Señal , Transcripción Genética , Triatominae/parasitología , Trypanosoma cruzi/genética , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
The piRNA pathway is a surveillance system that guarantees oogenesis and adult fertility in a range of animal species. The pathway is centered on PIWI clade Argonaute proteins and the associated small non-coding RNAs termed piRNAs. In this study, we set to investigate the evolutionary conservation of the piRNA pathway in the hemimetabolous insect Rhodnius prolixus. Our transcriptome profiling reveals that core components of the pathway are expressed during previtellogenic stages of oogenesis. Rhodnius' genome harbors four putative piwi orthologs. We show that Rp-piwi2, Rp-piwi3 and Rp-ago3, but not Rp-piwi1 transcripts are produced in the germline tissues and maternally deposited in the mature eggs. Consistent with a role in Rhodnius oogenesis, parental RNAi against the Rp-piwi2, Rp-piwi3 and Rp-ago3 results in severe egg laying and female adult fertility defects. Furthermore, we show that the reduction of the Rp-piwi2 levels by parental RNAi disrupts oogenesis by causing a dramatic loss of trophocytes, egg chamber degeneration and oogenesis arrest. Intriguingly, the putative Rp-Piwi2 protein features a polyglutamine tract at its N-terminal region, which is conserved in PIWI proteins encoded in the genome of other Triatomine species. Together with R. prolixus, these hematophagous insects are primary vectors of the Chagas disease. Thus, our data shed more light on the evolution of the piRNA pathway and provide a framework for the development of new control strategies for Chagas disease insect vectors.
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Regulación de la Expresión Génica , Insectos Vectores/genética , Insectos Vectores/fisiología , Oogénesis , ARN Interferente Pequeño/metabolismo , Rhodnius/genética , Rhodnius/fisiología , Animales , Femenino , Perfilación de la Expresión GénicaRESUMEN
Since the pioneering studies of Thomas Hunt Morgan and coworkers at the dawn of the twentieth century, Drosophila melanogaster and its sister species have tremendously contributed to unveil the rules underlying animal genetics, development, behavior, evolution, and human disease. Recent advances in DNA sequencing technologies launched Drosophila into the post-genomic era and paved the way for unprecedented comparative genomics investigations. The complete sequencing and systematic comparison of the genomes from 12 Drosophila species represents a milestone achievement in modern biology, which allowed a plethora of different studies ranging from the annotation of known and novel genomic features to the evolution of chromosomes and, ultimately, of entire genomes. Despite the efforts of countless laboratories worldwide, the vast amount of data that were produced over the past 15 years is far from being fully explored.In this chapter, we will review some of the bioinformatic approaches that were developed to interrogate the genomes of the 12 Drosophila species. Setting off from alignments of the entire genomic sequences, the degree of conservation can be separately evaluated for every region of the genome, providing already first hints about elements that are under purifying selection and therefore likely functional. Furthermore, the careful analysis of repeated sequences sheds light on the evolutionary dynamics of transposons, an enigmatic and fascinating class of mobile elements housed in the genomes of animals and plants. Comparative genomics also aids in the computational identification of the transcriptionally active part of the genome, first and foremost of protein-coding loci, but also of transcribed nevertheless apparently noncoding regions, which were once considered "junk" DNA. Eventually, the synergy between functional and comparative genomics also facilitates in silico and in vivo studies on cis-acting regulatory elements, like transcription factor binding sites, that due to the high degree of sequence variability usually impose increased challenges for bioinformatics approaches.
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Drosophila/genética , Evolución Molecular , Genoma de los Insectos , Genómica/métodos , Algoritmos , Animales , Biología Computacional , Anotación de Secuencia Molecular , Programas InformáticosRESUMEN
The hemiptera Rhodnius prolixus is a blood-feeding insect and a primary vector of Trypanosoma cruzi, the etiological agent of the Chagas disease. Over the past century, Rhodnius has been the subject of intense investigations, which have contributed to unveil important aspects of metabolism and physiology in insects. Recent technological innovations are helping dissect the genetic and molecular underpinnings of Rhodnius embryogenesis and organogenesis, thus fostering the use of this important species in the fields of developmental and evolutionary biology. Rhodnius represents also an excellent system to study development under stressful conditions, since the embryo must develop in the presence of a large amount of blood-derived reactive oxygen species. With a recently sequenced genome, small among other Hemiptera, and the identification of basic elements for all classical development pathways, functional studies in this species are revealing novel aspects of insect development and evolution. Here we review early studies on this model insect and how this paved the way for recent functional studies using the kissing bug.
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Insectos Vectores/crecimiento & desarrollo , Rhodnius/crecimiento & desarrollo , Animales , Desarrollo Embrionario , Evolución Molecular , Insectos Vectores/genética , Insectos Vectores/fisiología , Rhodnius/genética , Rhodnius/fisiologíaRESUMEN
Piwi-interacting RNAs (piRNAs) are central components of the piRNA pathway, which directs transposon silencing and guarantees genome integrity in the germ cells of several metazoans. In Drosophila, piRNAs are produced from discrete regions of the genome termed piRNA clusters, whose expression relies on the RDC complex comprised of the core proteins Rhino, Deadlock, and Cutoff. To date, the RDC complex has been exclusively implicated in the regulation of the piRNA loci. Here we further elucidate the function of Cutoff and the RDC complex by performing genome-wide ChIP-seq and RNA-seq assays in the Drosophila ovaries and analyzing these data together with other publicly available data sets. In agreement with previous studies, we confirm that Cutoff is involved in the transcriptional regulation of piRNA clusters and in the repression of transposable elements in germ cells. Surprisingly, however, we find that Cutoff is enriched at and affects the expression of other noncoding RNAs, including spliceosomal RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). At least in some instances, Cutoff appears to act at a transcriptional level in concert with Rhino and perhaps Deadlock. Finally, we show that mutations in Cutoff result in the deregulation of hundreds of protein-coding genes in germ cells. Our study uncovers a broader function for the RDC complex in the Drosophila germline development.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Ovario/crecimiento & desarrollo , ARN Interferente Pequeño/metabolismo , ARN no Traducido/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Inmunoprecipitación de Cromatina , Elementos Transponibles de ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Femenino , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Mutación , Ovario/química , Proteínas de Unión al ARN/genética , Análisis de Secuencia de ARN/métodosRESUMEN
Heterochromatin represents a significant portion of eukaryotic genomes and has essential structural and regulatory functions. Its molecular organization is largely unknown due to difficulties in sequencing through and assembling repetitive sequences enriched in the heterochromatin. Here we developed a novel strategy using chromosomal rearrangements and embryonic phenotypes to position unmapped Drosophila melanogaster heterochromatic sequence to specific chromosomal regions. By excluding sequences that can be mapped to the assembled euchromatic arms, we identified sequences that are specific to heterochromatin and used them to design heterochromatin specific probes ("H-probes") for microarray. By comparative genomic hybridization (CGH) analyses of embryos deficient for each chromosome or chromosome arm, we were able to map most of our H-probes to specific chromosome arms. We also positioned sequences mapped to the second and X chromosomes to finer intervals by analyzing smaller deletions with breakpoints in heterochromatin. Using this approach, we were able to map >40% (13.9 Mb) of the previously unmapped heterochromatin sequences assembled by the whole-genome sequencing effort on arm U and arm Uextra to specific locations. We also identified and mapped 110 kb of novel heterochromatic sequences. Subsequent analyses revealed that sequences located within different heterochromatic regions have distinct properties, such as sequence composition, degree of repetitiveness, and level of underreplication in polytenized tissues. Surprisingly, although heterochromatin is generally considered to be transcriptionally silent, we detected region-specific temporal patterns of transcription in heterochromatin during oogenesis and early embryonic development. Our study provides a useful approach to elucidate the molecular organization and function of heterochromatin and reveals region-specific variation of heterochromatin.
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Deleción Cromosómica , Mapeo Cromosómico/métodos , Hibridación Genómica Comparativa/métodos , Drosophila melanogaster/genética , Heterocromatina/genética , Animales , Cromosomas de Insectos/genética , Variaciones en el Número de Copia de ADN , Desarrollo Embrionario/genética , Femenino , Regulación de la Expresión Génica , Reordenamiento Génico , Heterocromatina/química , Masculino , Análisis por Micromatrices , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
In a broad range of organisms, Piwi-interacting RNAs (piRNAs) have emerged as core components of a surveillance system that protects the genome by silencing transposable and repetitive elements. A vast proportion of piRNAs is produced from discrete genomic loci, termed piRNA clusters, which are generally embedded in heterochromatic regions. The molecular mechanisms and the factors that govern their expression are largely unknown. Here, we show that Cutoff (Cuff), a Drosophila protein related to the yeast transcription termination factor Rai1, is essential for piRNA production in germline tissues. Cuff accumulates at centromeric/pericentromeric positions in germ-cell nuclei and strongly colocalizes with the major heterochromatic domains. Remarkably, we show that Cuff is enriched at the dual-strand piRNA cluster 1/42AB and is likely to be involved in regulation of transcript levels of similar loci dispersed in the genome. Consistent with this observation, Cuff physically interacts with the Heterochromatin Protein 1 (HP1) variant Rhino (Rhi). Our results unveil a link between Cuff activity, heterochromatin assembly and piRNA cluster expression, which is critical for stem-cell and germ-cell development in Drosophila.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Germinativas/metabolismo , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Animales , Secuencia de Bases , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Células Germinativas/citología , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Proteínas Nucleares/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Células Madre/citología , Células Madre/metabolismo , Transcripción GenéticaRESUMEN
Posttranscriptional regulation plays a crucial role in germline and early embryonic development, but the underlying mechanisms are only partially understood. Here we report the genetic and molecular analysis of the maternally and zygotically expressed microRNA miR-184 in Drosophila. Loss of miR-184 leads to multiple severe defects during oogenesis and early embryogenesis, culminating in the complete loss of egg production. Using both in vitro and in vivo assays, we characterize the relevant miR-184 targets and target sites for three of the observed phenotypes. miR-184 controls germline stem cell differentiation by tuning the DPP receptor Saxophone, dorsoventral patterning of the egg shell by regulating the gurken transport factor K10, and anteroposterior patterning of the blastoderm by tuning the transcriptional repressor Tramtrack69. Our study highlights the importance of microRNA-mediated regulation in the major developmental transitions of the female germline, and provides insights into several aspects of microRNA function.
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Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/fisiología , MicroARNs/metabolismo , Oogénesis/fisiología , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Tipificación del Cuerpo , Diferenciación Celular , Drosophila melanogaster/clasificación , Femenino , Células Germinativas/citología , Humanos , MicroARNs/genética , Datos de Secuencia Molecular , Oocitos/citología , Oocitos/fisiología , Células Madre/fisiologíaRESUMEN
The Doublesex (DSX) transcription factor regulates somatic sexual differentiation in Drosophila melanogaster. Female and male isoforms (DSXF and DSXM) are produced due to sex-specific RNA splicing. Here we show that in the distantly related dipteran Ceratitis capitata, the DSXM male-specific isoform is conserved and able to induce masculinization of both somatic and germline tissues when ectopically expressed in XX Drosophila transgenic individuals.
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Ceratitis capitata/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Proteínas de Insectos/genética , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Sitios de Unión/genética , Clonación Molecular , Cartilla de ADN/genética , Femenino , Genes de Insecto , Masculino , Datos de Secuencia Molecular , Fenotipo , Isoformas de Proteínas/genética , Homología de Secuencia de Aminoácido , Diferenciación Sexual/genética , Especificidad de la EspecieRESUMEN
RNAi is a widespread mechanism by which organisms regulate gene expression and defend their genomes against viruses and transposable elements. Here we report the identification of Drosophila zucchini (zuc) and squash (squ), which function in germline RNAi processes. Zuc and Squ contain domains with homologies to nucleases. Mutant females are sterile and show dorsoventral patterning defects during oogenesis. In addition, Oskar protein is ectopically expressed in early oocytes, where it is normally silenced by RNAi mechanisms. Zuc and Squ localize to the perinuclear nuage and interact with Aubergine, a PIWI class protein. Mutations in zuc and squ induce the upregulation of Het-A and Tart, two telomere-specific transposable elements, and the expression of Stellate protein in the Drosophila germline. We show that these defects are due to the inability of zuc and squ mutants to produce repeat-associated small interfering RNAs.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Embrión no Mamífero/metabolismo , Endonucleasas/metabolismo , Endorribonucleasas/metabolismo , Células Germinativas/metabolismo , Oogénesis/fisiología , ARN Interferente Pequeño/farmacología , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Elementos Transponibles de ADN/fisiología , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Endonucleasas/genética , Endorribonucleasas/genética , Femenino , Regulación Fúngica de la Expresión Génica , Productos del Gen gag/metabolismo , Células Germinativas/citología , Inmunoprecipitación , Masculino , Datos de Secuencia Molecular , Mutación , Oocitos/citología , Oocitos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
Gametogenesis is a highly regulated process in all organisms. In Drosophila, a meiotic checkpoint which monitors double-stranded DNA breaks and involves Drosophila ATR and Chk2 coordinates the meiotic cell cycle with signaling events that establish the axis of the egg and embryo. Checkpoint activity regulates translation of the transforming growth-factor-alpha-like Gurken signaling molecule which induces dorsal cell fates in the follicle cells [1-3]. We found that mutations in the Drosophila gene cutoff (cuff) affect germline cyst development and result in ventralized eggs as a result of reduced Grk protein expression. Surprisingly, cuff mutations lead to a marked increase in the transcript levels of two retrotransposable elements, Het-A and Tart. We found that small interfering RNAs against the roo element are still produced in cuff mutant ovaries. These results indicate that Cuff is involved in the rasiRNA pathway and most likely acts downstream of siRNA biogenesis. The eggshell and egg-laying defects of cuff mutants are suppressed by a mutation in chk2. We also found that mutations in aubergine (aub), another gene implicated in the rasiRNA pathway, are significantly suppressed by the chk2 mutation. Our results indicate that mutants in rasiRNA pathways lead to elevated transposition incidents in the germline, and that this elevation activates a checkpoint that causes a loss of germ cells and a reduction of Gurken protein in the remaining egg chambers.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Retroelementos/genética , Regulación hacia Arriba , Animales , Quinasa de Punto de Control 2 , Proteínas de Drosophila/metabolismo , Femenino , Fertilidad , Células Germinativas/citología , Meiosis , Mutación , Oogénesis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Proteínas de Unión al ARN/metabolismo , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
Transformer functions as a binary switch gene in the sex determination and sexual differentiation of Drosophila melanogaster and Ceratitis capitata, two insect species that separated nearly 100 million years ago. The TRA protein is required for female differentiation of XX individuals, while XY individuals express smaller, presumably nonfunctional TRA peptides and consequently develop into adult males. In both species, tra confers female sexual identity through a well-conserved double-sex gene. However, unlike Drosophila tra, which is regulated by the upstream Sex-lethal gene, Ceratitis tra itself is likely to control a feedback loop that ensures the maintenance of the female sexual state. The putative CcTRA protein shares a very low degree of sequence identity with the TRA proteins from Drosophila species. However, in this study we show that a female-specific Ceratitis Cctra cDNA encoding the putative full-length CcTRA protein is able to support the female somatic and germline sexual differentiation of D. melanogaster XX; tra mutant adults. Although highly divergent, CcTRA can functionally substitute for DmTRA and induce the female-specific expression of both Dmdsx and Dmfru genes. These data demonstrate the unusual plasticity of the TRA protein that retains a conserved function despite the high evolutionary rate. We suggest that transformer plays an important role in providing a molecular basis for the variety of sex-determining systems seen among insects.