RESUMO
During oogenesis in the Drosophila ovary, numerous translational regulators promote the self-renewal or differentiation of stem cells. An intriguing question is how these regulators combine to execute translational regulation. Here, we study mechanisms for the posttranscriptional regulation of nos, a critical stem cell self-renewal factor in the Drosophila ovary; specifically, regulators that promote differentiation of the stem cell daughter. Previous studies showed that Bam, Bgcn, Mei-P26, and Sxl form a complex and repress nos expression through the nos 3'UTR. To further elucidate mechanistic processes of Nos translational regulation, we reconstituted nos repression in cultured Drosophila cells. We identify Ago1 and Brat as new members, and show that Ago1 acts through miRNA binding sites in the proximal region of the nos 3'UTR, whereas Sxl acts via an Sxl binding sequence in the distal region. Combining these findings with published reports, we propose that additional factors Bam, Bgcn, Mei-P26, and Brat are recruited to nos mRNAs through interaction with Ago1 and Sxl. These findings elucidate mechanisms of nos regulation by diverse translational repressors.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células-Tronco de Oogônios/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Linhagem Celular , Linhagem da Célula/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Feminino , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Calcium-permeable and thermosensitive transient receptor potential (TRP) channels mediate the nociceptive transduction of noxious temperature in Drosophila nociceptors. However, the underlying molecular mechanisms are not completely understood. Here we find that Subdued, a calcium-activated chloride channel of the Drosophila anoctamin family, functions in conjunction with the thermo-TRPs in thermal nociception. Genetic analysis with deletion and the RNAi-mediated reduction of subdued show that subdued is required for thermal nociception in nociceptors. Further genetic analysis of subdued mutant and thermo-TRP mutants show that they interact functionally in thermal nociception. We find that Subdued expressed in heterologous cells mediates a strong chloride conductance in the presence of both heat and calcium ions. Therefore, our analysis suggests that Subdued channels may amplify the nociceptive neuronal firing that is initiated by thermo-TRP channels in response to thermal stimuli.
Assuntos
Canais de Cloreto/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Nociceptividade , Nociceptores/fisiologia , Animais , Comportamento Animal , Cloretos/química , Clonagem Molecular , Células HEK293 , Humanos , Mutação , Neurônios/metabolismo , Dor , Interferência de RNA , Canais de Potencial de Receptor Transitório/fisiologiaRESUMO
Pumilio (Pum) is an RNA-binding protein and translational repressor important to diverse biological processes. In the Drosophila ovary, Pum is expressed in female germline stem cells (GSCs), wherein it acts as an intrinsic stem cell maintenance factor via repressing target mRNAs that are as yet mostly unknown. Pum recognizes the Pum binding sequence (PBS) in the mRNA 3'UTR through its C-terminus Puf domain. Translational repression is mediated through its N-terminal domain, but the molecular mechanism remains largely unknown. We previously showed that Bag-of-marbles, a critical differentiation-promoting factor of female GSCs, physically interacts with the N-terminus of Pum. We further showed that this interaction is critical to Bam inhibition of Pum repressive action in cultured cells, but the physiological relevance was not addressed. Here we design an in vivo GFP translational reporter bearing the PBS in its 3'UTR and show that GFP expression is reduced in cells wherein Pum is known to be active. Furthermore, we demonstrate in pum mutant ovary that this GFP repression requires Pum, and also that the sensor faithfully monitors Pum activity. Finally, we show that forced expression of Bam inhibits Pum-mediated repression, validating that Bam inhibits Pum activity in vivo.
RESUMO
In mammals, pain is regulated by the combination of an ascending stimulating and descending inhibitory pain pathway. It remains an intriguing question whether such pain pathways are of ancient origin and conserved in invertebrates. Here we report a new Drosophila pain model and use it to elucidate the pain pathways present in flies. The model employs transgenic flies expressing the human capsaicin receptor TRPV1 in sensory nociceptor neurons, which innervate the whole fly body, including the mouth. Upon capsaicin sipping, the flies abruptly displayed pain-related behaviors such as running away, scurrying around, rubbing vigorously, and pulling at their mouth parts, suggesting that capsaicin stimulated nociceptors in the mouth via activating TRPV1. When reared on capsaicin-containing food, the animals died of starvation, demonstrating the degree of pain experienced. This death rate was reduced by treatment both with NSAIDs and gabapentin, analgesics that inhibit the sensitized ascending pain pathway, and with antidepressants, GABAergic agonists, and morphine, analgesics that strengthen the descending inhibitory pathway. Our results suggest Drosophila to possess intricate pain sensitization and modulation mechanisms similar to mammals, and we propose that this simple, non-invasive feeding assay has utility for high-throughput evaluation and screening of analgesic compounds.
Assuntos
Capsaicina , Drosophila , Dor , Animais , Humanos , Analgésicos/uso terapêutico , Capsaicina/farmacologia , Drosophila/metabolismo , Nociceptores/metabolismo , Dor/tratamento farmacológico , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
Reducing the oxidative stress in neurons extends lifespan in Drosophila melanogaster, highlighting the crucial role of neuronal oxidative damage in lifespan determination. However, the source of the reactive oxygen species (ROS) that provoke oxidative stress in neurons is not clearly defined. Here, we identify dual oxidase (duox), a calcium-activated ROS-producing enzyme, as a lifespan determinant. Due to the lethality of duox homozygous mutants, we employed a duox heterozygote that exhibited normal appearance and movement. We found that duox heterozygous male flies, which were isogenized with control flies, demonstrated extended lifespan. Neuronal knockdown experiments further suggested that duox is crucial to oxidative stress in neurons. Our findings suggest duox to be a source of neuronal oxidative stress associated with animal lifespan.
Assuntos
Drosophila melanogaster , Longevidade , Animais , Drosophila melanogaster/metabolismo , Oxidases Duais/metabolismo , Hidrogênio , Peróxido de Hidrogênio , Longevidade/fisiologia , Masculino , Neurônios/metabolismo , Estresse Oxidativo , Espécies Reativas de OxigênioRESUMO
In Drosophila larvae, nociceptive mdIV sensory neurons detect diverse noxious stimuli and prompt a nociceptive rolling response. Intriguingly, the same neurons also regulate stereotyped larval movement. The channels responsible for transducing these stimuli into electric signals are not yet fully identified. Here we undertook genetic and electrophysiological analysis of Ppk19, a member of the Deg/ENaC family of cationic channels. ppk19 mutants exhibited an impaired nociceptive rolling response upon mechanical force and acid, but no impairment in response to noxious temperature and gentle touch. Mutants also exhibited defective larval movement. RNAi against ppk19 in mdIV neurons likewise produced larvae with defects in mechanical and acid nociception and larval movement, but no impairment in detection of heat and gentle touch. Cultured cells transfected with ppk19 produced currents in acid and hypotonic solution, suggesting that ppk19 encodes an ion channel that responds to acid and cell swelling. Taken together, these findings suggest that Ppk19 acts in mdIV neurons as a proton- and mechano-gated ion channel to mediate acid- and mechano-responsive nociception and larval movement.
Assuntos
Drosophila melanogaster , Drosophila , Animais , Drosophila melanogaster/genética , Prótons , Canais Iônicos , Larva/fisiologia , Células Receptoras SensoriaisRESUMO
Hub cells comprise a niche for germline stem cells and cyst stem cells in the Drosophila testis. Hub cells arise from common somatic gonadal precursors in embryos, but the mechanism of their specification is still poorly understood. Here we find that RNA binding proteins Lin28 and Imp mediate transcript stability of Bowl, a known hub specification factor; Bowl transcripts were reduced in the testis of Lin28 and Imp mutants, and also when RNA-mediated interference against Lin28 or Imp was expressed in hub cells. In tissue culture Luciferase assays involving the Bowl 3'UTR, stability of Luc reporter transcripts depended on the Bowl 3'UTR and required Lin28 and Imp. Our findings suggest that proper Bowl function during hub cell specification requires Lin28 and Imp in the testis hub cells.
RESUMO
Age-related decline in stem cell function is observed in many tissues from invertebrates to humans. While cell intrinsic alterations impair stem cells, aging of the stem cell niche also significantly contributes to the loss of tissue homeostasis associated with reduced regenerative capacity. Hub cells, which constitute the stem cell niche in the Drosophila testis, exhibit age-associated decline in number and activities, yet underlying mechanisms are not fully understood. Here we show that Lin28, a highly conserved RNA binding protein, is expressed in hub cells and its expression dramatically declines in old testis. lin28 mutant testes exhibit hub cell loss and defective hub architecture, recapitulating the normal aging process. Importantly, maintained expression of Lin28 prolongs hub integrity and function in aged testes, suggesting that Lin28 decline is a driver of hub cell aging. Mechanistically, the level of unpaired (upd), a stem cell self-renewal factor, is reduced in lin28 mutant testis and Lin28 protein directly binds and stabilizes upd transcripts, in a let-7 independent manner. Altogether, our results suggest that Lin28 acts to protect upd transcripts in hub cells, and reduction of Lin28 in old testis leads to decreased upd levels, hub cell aging and loss of the stem cell niche.
Assuntos
Envelhecimento/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Ligação a RNA/metabolismo , Nicho de Células-Tronco , Testículo/metabolismo , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Masculino , Proteínas de Ligação a RNA/genética , Fatores de Transcrição STAT/metabolismo , Testículo/citologia , Fatores de Transcrição/metabolismoRESUMO
Puf family proteins are translational regulators essential to a wide range of biological processes, including cell fate specification, stem cell self-renewal, and neural function. Yet, despite being associated with hundreds of RNAs, the underlying mechanisms of Puf target specification remain to be fully elucidated. In Drosophila, Pumilio - a sole Puf family protein - is known to collaborate with cofactors Nanos (Nos) and Brain Tumor (Brat); however, their roles in target specification are not clearly defined. Here, we identify Bag-of-marbles (Bam) as a new Pum cofactor in repression of Mothers against dpp (mad) mRNAs, for which Nos is known to be dispensable. Notably, our data show that Nos (but not Bam) was required for Pum association with hunchback (hb) mRNAs, a well-known target of Pum and Nos. In contrast, Bam (but not Nos) was required for Pum association with mad mRNAs. These findings show for the first time that Pum target specificity is determined not independently but in collaboration with cofactors.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The mechanosensory neurons of Drosophila larvae are demonstrably activated by diverse mechanical stimuli, but the mechanisms underlying this function are not completely understood. Here we report a genetic, immunohistochemical, and electrophysiological analysis of the Ppk30 ion channel, a member of the Drosophila pickpocket (ppk) family, counterpart of the mammalian Degenerin/Epithelial Na+ Channel family. Ppk30 mutant larvae displayed deficits in proprioceptive movement and mechanical nociception, which are detected by class IV sensory (mdIV) neurons. The same neurons also detect heat nociception, which was not impaired in ppk30 mutant larvae. Similarly, Ppk30 mutation did not alter gentle touch mechanosensation, a distinct mechanosensation detected by other neurons, suggesting that Ppk30 has a functional role in mechanosensation in mdIV neurons. Consistently, Ppk30 was expressed in class IV neurons, but was not detectable in other larval skin sensory neurons. Mutant phenotypes were rescued by expressing Ppk30 in mdIV neurons. Electrophysiological analysis of heterologous cells expressing Ppk30 did not detect mechanosensitive channel activities, but did detect acid-induced currents. These data show that Ppk30 has a role in mechanosensation, but not in thermosensation, in class IV neurons, and possibly has other functions related to acid response.
Assuntos
Proteínas de Drosophila/genética , Nociceptividade , Propriocepção , Canais de Sódio/genética , Potenciais de Ação , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Mecanorreceptores/metabolismo , Mecanorreceptores/fisiologia , Canais de Sódio/metabolismoRESUMO
BACKGROUND: Nociceptive sensitization is an increase in pain perception in response to stimulus. Following brief irradiation of Drosophila larvae with UV, nociceptive sensitization occurs in class IV multiple dendritic (mdIV) neurons, which are polymodal sensory nociceptors. Diverse signaling pathways have been identified that mediate nociceptive sensitization in mdIV neurons, including TNF, Hedgehog, BMP, and Tachykinin, yet the underlying mechanisms are not completely understood. RESULTS: Here we report that duox heterozygous mutant larvae, which have normal basal nociception, exhibit an attenuated hypersensitivity response to heat and mechanical force following UV irradiation. Employing the ppk-Gal4 line, which is exclusively expressed in mdIV neurons, we further show that silencing duox in mdIV neurons attenuates UV-induced sensitization. CONCLUSIONS: Our findings reveal a novel role for duox in nociceptive sensitization of Drosophila larvae, and will enhance our understanding of the mechanisms underlying this process in Drosophila sensory neurons.
Assuntos
Drosophila melanogaster/fisiologia , Drosophila melanogaster/efeitos da radiação , Mutação/genética , Nociceptividade/efeitos da radiação , Raios Ultravioleta , Animais , Heterozigoto , Larva/genética , Larva/efeitos da radiação , TemperaturaRESUMO
The germline stem cells of the Drosophila ovary continuously produce eggs throughout the life- span. Intricate regulation of stemness and differentiation is critical to this continuous production. The translational regulator Nos is an intrinsic factor that is required for maintenance of stemness in germline stem cells. Nos expression is reduced in differentiating cells at the post-transcriptional level by diverse translational regulators. However, molecular mechanisms underlying Nos repression are not completely understood. Through three distinct protein-protein interaction experiments, we identified specific molecular interactions between translational regulators involved in Nos repression. Our findings suggest a model in which protein complexes assemble on the 3' untranslated region of Nos mRNA in order to regulate Nos expression at the post-transcriptional level.