RESUMO
Different signaling pathways are deployed in specific developmental contexts to generate sexually dimorphic traits. Recently, Sex-lethal (Sxl), the female determinant in Drosophila melanogaster, was shown to down-regulate Notch (N) signaling to accomplish sex-specific patterning. Paradoxically, however, both Sxl and N are ubiquitously expressed in all of the female cells. This raises a key question as to how, during monomorphic female development, N signaling escapes the negative impact of Sxl. Here, we uncover a regulatory loop involving Hrp48, an abundant Drosophila hnRNP, Sxl and N. Phenotypic consequences of the partial loss of hrp48 resemble that of N but are more pronounced in females than in males. Likewise, N levels are drastically diminished only in females. Interestingly, monomorphic female tissues including wing, eye and antennal discs display considerable increase in Sxl amounts. Finally, female-specific attenuation of N signaling is rescued upon simultaneous removal of Sxl. Thus, our data demonstrate that in monomorphic contexts, Hrp48 functions as a moderator of Sxl expression to achieve adequate levels of N receptor production and signaling. We propose that it is critical to modulate the activities of the master determinant underling sexual dimorphism, to ensure that it does not function inappropriately in monomorphic tissues and disrupt their development.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Receptores Notch/metabolismo , Alelos , Animais , Cruzamentos Genéticos , Feminino , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Masculino , Mitose , Modelos Biológicos , Fatores Sexuais , Transdução de Sinais , Asas de Animais/embriologiaRESUMO
Patterning of the anterior-posterior body axis of the Drosophila embryo requires production of Nanos protein selectively in the posterior. Spatially restricted Nanos synthesis is accomplished by translational repression of unlocalized nanos mRNA together with translational activation of posteriorly localized nanos. Repression of unlocalized nanos mRNA is mediated by a bipartite translational control element (TCE) in its 3' untranslated region. TCE stem-loop II functions during embryogenesis, through its interaction with the Smaug repressor. Stem-loop III represses unlocalized nanos mRNA during oogenesis, but trans-acting factors that carry out this function have remained elusive. Here we identify a Drosophila hnRNP, Glorund, that interacts specifically with stem-loop III. We establish that the ability of the TCE to repress translation in vivo reflects its ability to bind Glorund in vitro. These data, together with the analysis of a glorund null mutant, reveal a specific role for an hnRNP in repression of nanos translation during oogenesis.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Padronização Corporal , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oócitos/citologia , Oócitos/metabolismo , Oogênese/fisiologia , Ovário/citologia , Ovário/fisiologia , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Alinhamento de SequênciaRESUMO
Precise temporal and spatial regulation of gene expression during Drosophila oogenesis is essential for patterning the anterior-posterior and dorsal-ventral body axes. Establishment of the anterior-posterior axis requires posterior localization and translational control of both oskar and nanos mRNAs. Establishment of the dorsal-ventral axis depends on the precise restriction of gurken mRNA and protein to the dorsal-anterior corner of the oocyte. We have previously shown that Glorund, the Drosophila hnRNP F/H homolog, contributes to anterior-posterior axis patterning by regulating translation of nanos mRNA, through a direct interaction with its 3' untranslated region. To investigate the pleiotropy of the glorund mutant phenotype, which includes dorsal-ventral and nuclear morphology defects, we searched for proteins that interact with Glorund. Here we show that Glorund is part of a complex containing the hnRNP protein Hrp48 and the splicing factor Half-pint and plays a role both in mRNA localization and nurse cell chromosome organization, probably by regulating alternative splicing of ovarian tumor. We propose that Glorund is a component of multiple protein complexes and functions both as a translational repressor and splicing regulator for anterior-posterior and dorsal-ventral patterning.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Processamento Alternativo , Animais , Padronização Corporal/fisiologia , Cromossomos/metabolismo , Drosophila/embriologia , Proteínas de Drosophila/genética , Embrião não Mamífero/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Mutação , Oócitos/fisiologia , Oogênese/fisiologia , Ligação Proteica , RNA Mensageiro/genética , Fator de Crescimento Transformador alfa/genética , Regiões não TraduzidasRESUMO
Abscisic acid stress ripening (ASR1) is a highly charged low molecular weight plant specific protein that is regulated by salt- and water-stresses. The protein possesses a zinc-dependent DNA-binding activity (Kalifa et al., Biochem. J. 381 (2004) 373) and overexpression in transgenic plants results in an increased salt-tolerance (Kalifa et al., Plant Cell Environ. 27 (2004) 1459). There are no structure homologs of ASR1, thus the structural and functional domains of the protein cannot be predicted. Here, we map the protein domains involved in the binding of Zn(2+) and DNA. Using mild acid hydrolysis, and a series of ASR1 carboxy-terminal truncations we show that the zinc-dependent DNA-binding could be mapped to the central/carboxy-terminal domain. In addition, using MALDI-TOF-MS with a non-acidic matrix, we show that two zinc ions are bound to the amino-terminal domain. Other zinc ion(s) bind the DNA-binding domain. Binding of zinc to ASR1 induces conformational changes resulting in a decreased sensitivity to proteases.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Solanum lycopersicum , Zinco/metabolismo , Ácido Abscísico , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , DNA/química , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Mapeamento de Peptídeos , Proteínas de Plantas/genética , Estrutura Terciária de ProteínaRESUMO
Tomato (Lycopersicon esculantum) ASR1 (abscisic acid stress ripening protein), a small plant-specific protein whose cellular mode of action defies deduction based on its sequence or homology analyses, is one of numerous plant gene products with unknown biological roles that become over-expressed under water- and salt-stress conditions. Steady-state cellular levels of tomato ASR1 mRNA and protein are transiently increased following exposure of plants to poly(ethylene glycol), NaCl or abscisic acid. Western blot and indirect immunofluorescence analysis with anti-ASR1 antibodies demonstrated that ASR1 is present both in the cytoplasmic and nuclear subcellular compartments; approx. one-third of the total ASR1 protein could be detected in the nucleus. Nuclear ASR1 is a chromatin-bound protein, and can be extracted with 1 M NaCl, but not with 0.5% Triton X-100. ASR1, overexpressed in Escherichia coli and purified to homogeneity, possesses zinc-dependent DNA-binding activity. Competitive-binding experiments and SELEX (systematic evolution of ligands by exponential enrichment) analysis suggest that ASR1 binds at a preferred DNA sequence.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Plantas/genética , Cloreto de Sódio/metabolismo , Solanum lycopersicum/genética , Água/metabolismo , Dedos de Zinco/genética , Sítios de Ligação/genética , Fracionamento Celular/métodos , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Solanum lycopersicum/citologia , Proteínas de Plantas/metabolismo , Frações Subcelulares/químicaRESUMO
Almost all animals contain mitochondria of maternal origin only, but the exact mechanisms underlying this phenomenon are still vague. We investigated the fate of Drosophila paternal mitochondria after fertilization. We demonstrate that the sperm mitochondrial derivative (MD) is rapidly eliminated in a stereotypical process dubbed paternal mitochondrial destruction (PMD). PMD is initiated by a network of vesicles resembling multivesicular bodies and displaying common features of the endocytic and autophagic pathways. These vesicles associate with the sperm tail and mediate the disintegration of its plasma membrane. Subsequently, the MD separates from the axoneme and breaks into smaller fragments, which are then sequestered by autophagosomes for degradation in lysosomes. We further provide evidence for the involvement of the ubiquitin pathway and the autophagy receptor p62 in this process. Finally, we show that the ubiquitin ligase Parkin is not involved in PMD, implying a divergence from the autophagic pathway of damaged mitochondria.
Assuntos
Autofagia/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endocitose/fisiologia , Padrões de Herança/fisiologia , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Animais , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA , Proteínas de Drosophila/genética , Fertilização/fisiologia , Padrões de Herança/genética , Masculino , Proteínas Nucleares/genética , Espermatozoides/citologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Caspases are executioners of apoptosis but also participate in a variety of vital cellular processes. Here, we identified Soti, an inhibitor of the Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during Drosophila spermatid terminal differentiation (individualization). We further provide evidence that the giant inhibitor of apoptosis-like protein dBruce is a target for the Cullin-3-based complex, and that Soti competes with dBruce for binding to Klhl10, the E3 substrate recruitment subunit. We then demonstrate that Soti is expressed in a subcellular gradient within spermatids and in turn promotes proper formation of a similar dBruce gradient. Consequently, caspase activation occurs in an inverse graded fashion, such that the regions of the developing spermatid that are the last to individualize experience the lowest levels of activated caspases. These findings elucidate how the spatial regulation of caspase activation can permit caspase-dependent differentiation while preventing full-blown apoptosis.