RESUMEN
A ten-eleven translocation (TET) ortholog exists as a DNA N6-methyladenine (6mA) demethylase (DMAD) in Drosophila. However, the molecular roles of 6mA and DMAD remain unexplored. Through genome-wide 6mA and transcriptome profiling in Drosophila brains and neuronal cells, we found that 6mA may epigenetically regulate a group of genes involved in neurodevelopment and neuronal functions. Mechanistically, DMAD interacts with the Trithorax-related complex protein Wds to maintain active transcription by dynamically demethylating intragenic 6mA. Accumulation of 6mA by depleting DMAD coordinates with Polycomb proteins and contributes to transcriptional repression of these genes. Our findings suggest that active 6mA demethylation by DMAD plays essential roles in fly CNS by orchestrating through added epigenetic mechanisms.
Asunto(s)
Adenina/análogos & derivados , Expresión Génica/fisiología , Neuronas/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Adenina/metabolismo , Animales , Metilación de ADN/fisiología , Desmetilación , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Epigénesis Genética/fisiología , Perfilación de la Expresión Génica/métodos , Genoma/fisiologíaRESUMEN
The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.
Asunto(s)
Enfermedades Cerebelosas/genética , Proteínas del Citoesqueleto/genética , Drosophila melanogaster/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Proteínas de Unión al ARN/genética , Sustitución de Aminoácidos/genética , Animales , Sistemas CRISPR-Cas/genética , Enfermedades Cerebelosas/patología , Cerebelo/metabolismo , Cerebelo/patología , Modelos Animales de Enfermedad , Exosomas/genética , Humanos , Mutación/genética , Neuronas/patología , ARN/genéticaRESUMEN
The human ZC3H14 gene, which encodes a ubiquitously expressed polyadenosine zinc finger RNA-binding protein, is mutated in an inherited form of autosomal recessive, nonsyndromic intellectual disability. To gain insight into neurological functions of ZC3H14, we previously developed a Drosophila melanogaster model of ZC3H14 loss by deleting the fly ortholog, Nab2. Studies in this invertebrate model revealed that Nab2 controls final patterns of neuron projection within fully developed adult brains, but the role of Nab2 during development of the Drosophila brain is not known. Here, we identify roles for Nab2 in controlling the dynamic growth of axons in the developing brain mushroom bodies, which support olfactory learning and memory, and regulating abundance of a small fraction of the total brain proteome. The group of Nab2-regulated brain proteins, identified by quantitative proteomic analysis, includes the microtubule-binding protein Futsch, the neuronal Ig-family transmembrane protein turtle, the glial:neuron adhesion protein contactin, the Rac GTPase-activating protein tumbleweed, and the planar cell polarity factor Van Gogh, which collectively link Nab2 to the processes of brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycles, and synaptic development. Overall, these data indicate that Nab2 controls the abundance of a subset of brain proteins during the active process of wiring the pupal brain mushroom body and thus provide a window into potentially conserved functions of the Nab2/ZC3H14 RNA-binding proteins in neurodevelopment.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Neurogénesis , Proteoma/genética , Proteínas de Unión al ARN/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Contactinas/genética , Contactinas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Memoria , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteoma/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive muscular atrophy and respiratory failure. The G4C2 repeat expansion in the C9orf72 gene is the most prevalent genetic risk for ALS. Mutation carriers (C9ALS) display variability in phenotypes such as age-at-onset and duration, suggesting the existence of additional genetic factors. Here we introduce a three-step gene discovery strategy to identify genetic factors modifying the risk of both C9ALS and sporadic ALS (sALS) using limited samples. We first identified 135 candidate genetic modifiers of C9ALS using whole-genome sequencing (WGS) of extreme C9ALS cases diagnosed ~30 years apart. We then performed an unbiased genetic screen using a Drosophila model of the G4C2 repeat expansion with the genes identified from WGS analysis. This genetic screen identified the novel genetic interaction between G4C2 repeat-associated toxicity and 18 genetic factors, suggesting their potential association with C9ALS risk. We went on to test if 14 out of the 18 genes, those which were not known to be risk factors for ALS previously, are also associated with ALS risk in sALS cases. Gene-based-statistical analyses of targeted resequencing and WGS were performed. These analyses together reveal that rare variants in MYH15 represent a likely genetic risk factor for ALS. Furthermore, we show that MYH15 could modulate the toxicity of dipeptides produced from expanded G4C2 repeat. Our study presented here demonstrates the power of combining WGS with fly genetics to facilitate the discovery of fundamental genetic components of complex traits with a limited number of samples.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Expansión de las Repeticiones de ADN , Drosophila/genética , Cadenas Pesadas de Miosina/genética , Adulto , Anciano , Animales , Animales Modificados Genéticamente , Proteína C9orf72/metabolismo , Proteína C9orf72/toxicidad , Dipéptidos/metabolismo , Dipéptidos/toxicidad , Modelos Animales de Enfermedad , Drosophila/citología , Drosophila/crecimiento & desarrollo , Drosophila/ultraestructura , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Humanos , Masculino , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Mutación , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Factores de Riesgo , Secuenciación Completa del GenomaRESUMEN
A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.
Asunto(s)
Encéfalo/fisiología , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Núcleo Celular/metabolismo , Secuencia Conservada , Exones , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Proteínas Nucleares/genética , Proteínas de Unión a Poli(A) , Isoformas de Proteínas , ARN/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genéticaRESUMEN
The ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, nuclear, zinc finger polyadenosine RNA-binding protein, was recently linked to autosomal recessive, nonsyndromic intellectual disability. Although studies have been carried out to examine the function of putative orthologs of ZC3H14 in Saccharomyces cerevisiae, where the protein is termed Nab2, and Drosophila, where the protein has been designated dNab2, little is known about the function of mammalian ZC3H14. Work from both budding yeast and flies implicates Nab2/dNab2 in poly(A) tail length control, while a role in poly(A) RNA export from the nucleus has been reported only for budding yeast. Here we provide the first functional characterization of ZC3H14. Analysis of ZC3H14 function in a neuronal cell line as well as in vivo complementation studies in a Drosophila model identify a role for ZC3H14 in proper control of poly(A) tail length in neuronal cells. Furthermore, we show here that human ZC3H14 can functionally substitute for dNab2 in fly neurons and can rescue defects in development and locomotion that are present in dNab2 null flies. These rescue experiments provide evidence that this zinc finger-containing class of nuclear polyadenosine RNA-binding proteins plays an evolutionarily conserved role in controlling the length of the poly(A) tail in neurons.
Asunto(s)
Neuronas/metabolismo , Proteínas Nucleares/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Animales , Secuencia Conservada , Drosophila/genética , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas de Unión a Poli(A) , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
The Drosophila melanogaster gene archipelago (ago) encodes the F-box/WD-repeat protein substrate specificity factor for an SCF (Skp/Cullin/F-box)-type polyubiquitin ligase that inhibits tumor-like growth by targeting proteins for degradation by the proteasome. The Ago protein is expressed widely in the fly embryo and larva and promotes degradation of pro-proliferative proteins in mitotically active cells. However the requirement for Ago in post-mitotic developmental processes remains largely unexplored. Here we show that Ago is an antagonist of the physiologic response to low oxygen (hypoxia). Reducing Ago activity in larval muscle cells elicits enhanced branching of nearby tracheal terminal cells in normoxia. This tracheogenic phenotype shows a genetic dependence on sima, which encodes the HIF-1α subunit of the hypoxia-inducible transcription factor dHIF and its target the FGF ligand branchless (bnl), and is enhanced by depletion of the Drosophila Von Hippel Lindau (dVHL) factor, which is a subunit of an oxygen-dependent ubiquitin ligase that degrades Sima/HIF-1α protein in metazoan cells. Genetic reduction of ago results in constitutive expression of some hypoxia-inducible genes in normoxia, increases the sensitivity of others to mild hypoxic stimulus, and enhances the ability of adult flies to recover from hypoxic stupor. As a molecular correlate to these genetic data, we find that Ago physically associates with Sima and restricts Sima levels in vivo. Collectively, these findings identify Ago as a required element of a circuit that suppresses the tracheogenic activity of larval muscle cells by antagonizing the Sima-mediated transcriptional response to hypoxia.
Asunto(s)
Proteínas Argonautas , Proteínas de Unión al ADN , Proteínas de Drosophila , Drosophila melanogaster , Subunidad alfa del Factor 1 Inducible por Hipoxia , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hipoxia/genética , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Musculares/metabolismo , Células Musculares/fisiología , Oxígeno/metabolismo , Proteolisis , Tráquea/metabolismo , Tráquea/fisiología , Activación Transcripcional/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
archipelago (ago)/Fbw7 encodes a conserved protein that functions as the substrate-receptor component of a polyubiquitin ligase that suppresses tissue growth in flies and tumorigenesis in vertebrates. Ago/Fbw7 targets multiple proteins for degradation, including the G1-S regulator Cyclin E and the oncoprotein dMyc/c-Myc. Despite prominent roles in growth control, little is known about the signals that regulate Ago/Fbw7 abundance in developing tissues. Here we use the Drosophila eye as a model to identify developmental signals that regulate ago expression. We find that expression of ago mRNA and protein is induced by passage of the morphogenetic furrow (MF) and identify the hedgehog (hh) and Notch (N) pathways as elements of this inductive mechanism. Cells mutant for N pathway components, or hh-defective cells that express reduced levels of the Notch ligand Delta, fail to upregulate ago transcription in the region of the MF; reciprocally, ectopic N activation in eye discs induces expression of ago mRNA. A fragment of the ago promoter that contains consensus binding sites for the N pathway transcription factor Su(H) is bound by Su(H) and confers N-inducibility in cultured cells. The failure to upregulate ago in N pathway mutant cells correlates with accumulation of the SCF-Ago target Cyclin E in the area of the MF, and this is rescued by re-expression of ago. These data suggest a model in which N acts through ago to restrict levels of the pro-mitotic factor Cyclin E. This N-Ago-Cyclin E link represents a significant new cell cycle regulatory mechanism in the developing eye.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Proteínas F-Box/genética , Receptores Notch/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Ciclo Celular , Ciclina E/metabolismo , Cartilla de ADN/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ojo/citología , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Proteínas Hedgehog/genética , Mutación , Regiones Promotoras Genéticas , Subunidades de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Here we report a human intellectual disability disease locus on chromosome 14q31.3 corresponding to mutation of the ZC3H14 gene that encodes a conserved polyadenosine RNA binding protein. We identify ZC3H14 mRNA transcripts in the human central nervous system, and we find that rodent ZC3H14 protein is expressed in hippocampal neurons and colocalizes with poly(A) RNA in neuronal cell bodies. A Drosophila melanogaster model of this disease created by mutation of the gene encoding the ZC3H14 ortholog dNab2, which also binds polyadenosine RNA, reveals that dNab2 is essential for development and required in neurons for normal locomotion and flight. Biochemical and genetic data indicate that dNab2 restricts bulk poly(A) tail length in vivo, suggesting that this function may underlie its role in development and disease. These studies reveal a conserved requirement for ZC3H14/dNab2 in the metazoan nervous system and identify a poly(A) RNA binding protein associated with a human brain disorder.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Discapacidad Intelectual/genética , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Sistema Nervioso Central/fisiología , Mapeo Cromosómico , Cromosomas Humanos Par 14/genética , Estudios de Cohortes , Consanguinidad , Secuencia Conservada , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Evolución Molecular , Femenino , Vuelo Animal/fisiología , Técnicas de Silenciamiento del Gen , Genes Recesivos , Hipocampo/metabolismo , Humanos , Irán , Masculino , Modelos Animales , Datos de Secuencia Molecular , Linaje , Proteínas de Unión a Poli(A) , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Adulto Joven , Dedos de Zinc/genéticaRESUMEN
Rapidly dividing cells can eliminate slow growing neighbors through the apoptotic process of cell competition. This process ensures that only high fitness cells populate embryonic tissues and is proposed to underlie the ability of oncogene-transformed cells to progressively replace normal cells within a tissue. Patches of cells in the Drosophila wing disc overexpressing the oncogenic Taiman (Tai) transcriptional coactivator kill normal neighbors by secreting Spätzle ligands that trigger pro-apoptotic Toll signaling in receiving cells. However, extracellular signaling mechanisms responsible for elimination of slow growing cells by normal neighbors remain poorly defined. Here we show that slow growing cells with reduced Tai (Tailow) are killed by normal neighbors through a mechanism involving competition for the Wingless (Wg/Wnt) ligand. Elevated Wg signaling significantly rescues elimination of Tailow cells in multiple organs, suggesting that Tai may normally promote Wg activity. Examining distribution of Wg components reveals that Tai promotes extracellular spread of the Wg ligand from source cells across the wing disc, thus ensuring patterned expression of multiple Wg-regulated target genes. Tai controls Wg spread indirectly through the extracellular glypican Dally-like protein (Dlp), which binds Wg and promotes its extracellular diffusion and capture by receptors. Data indicate that Tai likely controls Dlp at two levels: transcription of dlp mRNA and Dlp intracellular trafficking. Overall, these data indicate that the Tai acts through Dlp to enable Wg transport and signaling, and that cell competition in the Tailow model arises due to inequity in the ability of epithelial cells to sequester limiting amounts of the Wg growth factor.
RESUMEN
Environmental insults, including mild head trauma, significantly increase the risk of neurodegeneration. However, it remains challenging to establish a causative connection between early-life exposure to mild head trauma and late-life emergence of neurodegenerative deficits, nor do we know how sex and age compound the outcome. Using a Drosophila model, we demonstrate that exposure to mild head trauma causes neurodegenerative conditions that emerge late in life and disproportionately affect females. Increasing age-at-injury further exacerbates this effect in a sexually dimorphic manner. We further identify Sex Peptide (SP) signaling as a key factor in female susceptibility to post-injury brain deficits. RNA sequencing highlights a reduction in innate immune defense transcripts specifically in mated females during late life. Our findings establish a causal relationship between early head trauma and late-life neurodegeneration, emphasizing sex differences in injury response and the impact of age-at-injury. Finally, our findings reveal that reproductive signaling adversely impacts female response to mild head insults and elevates vulnerability to late-life neurodegeneration.
RESUMEN
The Drosophila RNA-binding protein (RBP) Nab2 acts in neurons to regulate neurodevelopment and is orthologous to the human intellectual disability-linked RBP, ZC3H14. Nab2 governs axon projection in mushroom body neurons and limits dendritic arborization of class IV sensory neurons in part by regulating splicing events in â¼150 mRNAs. Analysis of the Sex-lethal (Sxl) mRNA revealed that Nab2 promotes an exon-skipping event and regulates m6A methylation on Sxl pre-mRNA by the Mettl3 methyltransferase. Mettl3 heterozygosity broadly rescues Nab2null phenotypes implying that Nab2 acts through similar mechanisms on other RNAs, including unidentified targets involved in neurodevelopment. Here, we show that Nab2 and Mettl3 regulate the removal of a 5'UTR (untranslated region) intron in the trio pre-mRNA. Trio utilizes two GEF domains to balance Rac and RhoGTPase activity. Intriguingly, an isoform of Trio containing only the RhoGEF domain, GEF2, is depleted in Nab2null nervous tissue. Expression of Trio-GEF2 rescues projection defects in Nab2null axons and dendrites, while the GEF1 Rac1-regulatory domain exacerbates these defects, suggesting Nab2-mediated regulation Trio-GEF activities. Collectively, these data indicate that Nab2-regulated processing of trio is critical for balancing Trio-GEF1 and -GEF2 activity and show that Nab2, Mettl3, and Trio function in a common pathway that shapes axon and dendrite morphology.
Asunto(s)
Axones , Dendritas , Proteínas de Drosophila , Drosophila melanogaster , Factores de Intercambio de Guanina Nucleótido , Proteínas de Unión al ARN , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Axones/metabolismo , Dendritas/metabolismo , Drosophila melanogaster/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Empalme del ARN , ARN Mensajero/metabolismo , ARN Mensajero/genética , Precursores del ARN/metabolismo , Precursores del ARN/genéticaRESUMEN
Some pathogens are able to establish themselves within the host because they have evolved mechanisms to disrupt host innate immunity. For example, a number of pathogens secrete preformed effector proteins via type III secretion apparatuses that influence innate immune or apoptotic signalling pathways. One group of effector proteins that usurp innate immune signalling is the YopJ-like family of bacterial effector proteins, which includes AopP from Aeromonas salmonicida. Aeromonas species are known to cause gastrointestinal disease in humans, and are associated mainly with subcutaneous wound infections and septicaemia in other metazoans, particularly fish. AopP has been reported to have inhibitory activity against the NF-κB pathway in cultured cells, although the pathological outcomes of AopP activity have not been examined. Here, we show that AopP has potent pro-apoptotic activity when expressed in cultured mammalian macrophage or epithelial cells, or when ectopically expressed in Drosophila melanogaster haemocytes or imaginal disk epithelial cells. Furthermore, apoptosis was significantly elevated upon concurrent AopP expression and TNF-α cellular stimulation. Together, our results demonstrate how the specificity of a YopJ-like protein towards signalling pathways directly governs cellular pathological outcome in disease.
Asunto(s)
Aeromonas salmonicida/patogenicidad , Apoptosis , Proteínas Bacterianas/metabolismo , Factores de Virulencia/metabolismo , Animales , Línea Celular , Supervivencia Celular , Drosophila melanogaster , Células Epiteliales/microbiología , Células Epiteliales/fisiología , Hemocitos/microbiología , Hemocitos/fisiología , Humanos , Inmunidad Innata , Macrófagos/microbiología , Macrófagos/fisiología , Análisis de SupervivenciaRESUMEN
The canonical function of the Hippo signaling pathway is the regulation of organ growth. How this pathway controls cell-fate determination is less well understood. Here, we identify a function of the Hippo pathway in cell-fate decisions in the developing Drosophila eye, exerted through the interaction of Yorkie (Yki) with the transcriptional regulator Bonus (Bon), an ortholog of mammalian transcriptional intermediary factor 1/tripartite motif (TIF1/TRIM) family proteins. Instead of controlling tissue growth, Yki and Bon promote epidermal and antennal fates at the expense of the eye fate. Proteomic, transcriptomic, and genetic analyses reveal that Yki and Bon control these cell-fate decisions by recruiting transcriptional and post-transcriptional co-regulators and by repressing Notch target genes and activating epidermal differentiation genes. Our work expands the range of functions and regulatory mechanisms under Hippo pathway control.
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Proteínas de Drosophila , Drosophila , Animales , Diferenciación Celular , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Vía de Señalización Hippo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mamíferos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The rapid succession of events during development poses an inherent challenge to achieve precise synchronization required for rigorous, quantitative phenotypic and genotypic analyses in multicellular model organisms. Drosophila melanogaster is an indispensable model for studying the development and function of higher order organisms due to extensive genome homology, tractability, and its relatively short lifespan. Presently, nine Nobel prizes serve as a testament to the utility of this elegant model system. Ongoing advancements in genetic and molecular tools allow for the underlying mechanisms of human disease to be investigated in Drosophila. However, the absence of a method to precisely age-match tissues during larval development prevents further capitalization of this powerful model organism. Drosophila spends nearly half of its life cycle progressing through three morphologically distinct larval instar stages, during which the imaginal discs, precursors of mature adult external structures (e.g., eyes, legs, wings), grow and develop distinct cell fates. Other tissues, such as the central nervous system, undergo massive morphological changes during larval development. While these three larval stages and subsequent pupal stages have historically been identified based on the number of hours post egg-laying under standard laboratory conditions, a reproducible, efficient, and inexpensive method is required to accurately age-match larvae within the third instar. The third instar stage is of particular interest, as this developmental stage spans a 48-hr window during which larval tissues switch from proliferative to differentiation programs. Moreover, some genetic manipulations can lead to developmental delays, further compounding the need for precise age-matching between control and experimental samples. This article provides a protocol optimized for synchronous staging of Drosophila third instar larvae by colorimetric characterization and is useful for age-matching a variety of tissues for numerous downstream applications. We also provide a brief discussion of the technical challenges associated with successful application of this protocol. © 2023 Wiley Periodicals LLC. Basic Protocol: Synchronization of third instar Drosophila larvae.
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Drosophila melanogaster , Drosophila , Animales , Humanos , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Larva/fisiología , Colorimetría , PupaRESUMEN
Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here, we present in detail the development of a procedure to incorporate stable isotope-labeled amino acids into the fly proteome. In the method of stable isotope labeling with amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC-labeled yeast grown with modified media, enabling near complete labeling in a single generation. Biological variation in the proteome among individual flies was evaluated in a series of null experiments. We further applied the SILAC fly method to profile proteins from a model of fragile X syndrome, the most common cause of inherited mental retardation in human. The analysis identified a number of altered proteins in the disease model, including actin-binding protein profilin and microtubulin-associated protein futsch. The change of both proteins was validated by immunoblotting analysis. Moreover, we extended the SILAC fly strategy to study the dynamics of protein ubiquitination during the fly life span (from day 1 to day 30), by measuring the level of ubiquitin along with two major polyubiquitin chains (K48 and K63 linkages). The results show that the abundance of protein ubiquitination and the two major linkages do not change significantly within the measured age range. Together, the data demonstrate the application of the SILAC principle in D. melanogaster, facilitating the integration of powerful fly genomics with emerging proteomics.
Asunto(s)
Proteínas de Drosophila/análisis , Drosophila melanogaster/química , Marcaje Isotópico/métodos , Proteoma/análisis , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Immunoblotting , Masculino , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Proteoma/química , Proteoma/metabolismo , Reproducibilidad de los Resultados , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Nab2 encodes the Drosophila melanogaster member of a conserved family of zinc finger polyadenosine RNA-binding proteins (RBPs) linked to multiple steps in post-transcriptional regulation. Mutation of the Nab2 human ortholog ZC3H14 gives rise to an autosomal recessive intellectual disability but understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part because no comprehensive identification of metazoan Nab2/ZC3H14-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships remain unidentified. Here, we present evidence that Nab2 genetically interacts with Ataxin-2 (Atx2), which encodes a neuronal translational regulator, and that these factors coordinately regulate neuronal morphology, circadian behavior, and adult viability. We then present the first high-throughput identifications of Nab2- and Atx2-associated RNAs in Drosophila brain neurons using RNA immunoprecipitation-sequencing (RIP-Seq). Critically, the RNA interactomes of each RBP overlap, and Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with a small fraction of all polyadenylated RNAs. The identities of shared associated transcripts (e.g., drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g., Arpc2 and tea) promise insight into neuronal functions of, and genetic interactions between, each RBP. Consistent with prior biochemical studies, Nab2-associated neuronal RNAs are overrepresented for internal A-rich motifs, suggesting these sequences may partially mediate Nab2 target selection. These data support a model where Nab2 functionally opposes Atx2 in neurons, demonstrate Nab2 shares associated neuronal RNAs with Atx2, and reveal Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest.
Asunto(s)
Drosophila melanogaster , AnimalesRESUMEN
RNA-binding proteins support neurodevelopment by modulating numerous steps in post-transcriptional regulation, including splicing, export, translation, and turnover of mRNAs that can traffic into axons and dendrites. One such RNA-binding protein is ZC3H14, which is lost in an inherited intellectual disability. The Drosophila melanogaster ZC3H14 ortholog, Nab2, localizes to neuronal nuclei and cytoplasmic ribonucleoprotein granules and is required for olfactory memory and proper axon projection into brain mushroom bodies. Nab2 can act as a translational repressor in conjunction with the Fragile-X mental retardation protein homolog Fmr1 and shares target RNAs with the Fmr1-interacting RNA-binding protein Ataxin-2. However, neuronal signaling pathways regulated by Nab2 and their potential roles outside of mushroom body axons remain undefined. Here, we present an analysis of a brain proteomic dataset that indicates that multiple planar cell polarity proteins are affected by Nab2 loss, and couple this with genetic data that demonstrate that Nab2 has a previously unappreciated role in restricting the growth and branching of dendrites that elaborate from larval body-wall sensory neurons. Further analysis confirms that Nab2 loss sensitizes sensory dendrites to the genetic dose of planar cell polarity components and that Nab2-planar cell polarity genetic interactions are also observed during Nab2-dependent control of axon projection in the central nervous system mushroom bodies. Collectively, these data identify the conserved Nab2 RNA-binding protein as a likely component of post-transcriptional mechanisms that limit dendrite growth and branching in Drosophila sensory neurons and genetically link this role to the planar cell polarity pathway. Given that mammalian ZC3H14 localizes to dendritic spines and controls spine density in hippocampal neurons, these Nab2-planar cell polarity genetic data may highlight a conserved path through which Nab2/ZC3H14 loss affects morphogenesis of both axons and dendrites in diverse species.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Animales , Axones/metabolismo , Polaridad Celular/genética , Dendritas/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Mamíferos , Proteómica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Drosophila melanogaster is an excellent model organism to study neurodegeneration. Assessing evident neurodegeneration within the fly brain involves the laborious preparation of thin-sectioned H&E-stained heads to visualize brain vacuole degeneration. Here, we present an advanced microscopy-based protocol, without the need for sectioning, to detect vacuole degeneration within whole fly brains by applying commonly used stains to reveal the brain parenchyma. This approach preserves the whole-brain architecture and enables rapid, reproducible, and quantitative analyses of vacuole-like degeneration associated with specific brain regions. For complete details on the use and execution of this protocol, please refer to Behnke et al. (2021).
Asunto(s)
Encéfalo/diagnóstico por imagen , Técnicas Histológicas/métodos , Enfermedades Neurodegenerativas/diagnóstico por imagen , Animales , Encéfalo/metabolismo , Proteínas de Drosophila , Drosophila melanogaster , Microscopía/métodos , Neuronas/metabolismoRESUMEN
Mild head trauma, including concussion, can lead to chronic brain dysfunction and degeneration but the underlying mechanisms remain poorly understood. Here, we developed a novel head impact system to investigate the long-term effects of mild head trauma on brain structure and function, as well as the underlying mechanisms in Drosophila melanogaster. We find that Drosophila subjected to repetitive head impacts develop long-term deficits, including impaired startle-induced climbing, progressive brain degeneration, and shortened lifespan, all of which are substantially exacerbated in female flies. Interestingly, head impacts elicit an elevation in neuronal activity and its acute suppression abrogates the detrimental effects in female flies. Together, our findings validate Drosophila as a suitable model system for investigating the long-term effects of mild head trauma, suggest an increased vulnerability to brain injury in female flies, and indicate that early altered neuronal excitability may be a key mechanism linking mild brain trauma to chronic degeneration.