RESUMO
In prophase of the first meiotic division, chromatin forms a compact spherical cluster called the karyosome within the enlarged oocyte nucleus in Drosophila melanogaster. Similar clustering of chromatin has been widely observed in oocytes in many species including humans. It was previously shown that the proper karyosome formation is required for faithful chromosome segregation, but knowledge about its formation and maintenance is limited. To identify genes involved in karyosome formation, we carried out a large-scale cytological screen using Drosophila melanogaster oocytes. This screen comprised 3916 genes expressed in ovaries, of which 106 genes triggered reproducible karyosome defects upon knockdown. The karyosome defects in 24 out of these 106 genes resulted from activation of the meiotic recombination checkpoint, suggesting possible roles in DNA repair or piRNA processing. The other genes identified in this screen include genes with functions linked to chromatin, nuclear envelope, and actin. We also found that silencing of genes with mitochondrial functions, including electron transport chain components, induced a distinct karyosome defect typically with de-clustered chromosomes located close to the nuclear envelope. Furthermore, mitochondrial dysfunction not only impairs karyosome formation and maintenance, but also delays synaptonemal complex disassembly in cells not destined to become the oocyte. These karyosome defects do not appear to be mediated by apoptosis. This large-scale unbiased study uncovered a set of genes required for karyosome formation and revealed a new link between mitochondrial dysfunction and chromatin organization in oocytes.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Humanos , Animais , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Interferência de RNA , Oócitos/metabolismo , Cromossomos/genética , Cromossomos/metabolismo , Meiose , Cromatina/genética , Cromatina/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Segregação de CromossomosRESUMO
By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to efficiently adapt to changes in their environment. To better understand how Escherichia coli acclimatizes to nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover Hfq-associated RNA-RNA interactions at specific growth stages. We demonstrate that Hfq CLASH robustly captures bona fide RNA-RNA interactions. We identified hundreds of novel sRNA base-pairing interactions, including many sRNA-sRNA interactions and involving 3'UTR-derived sRNAs. We rediscovered known and identified novel sRNA seed sequences. The sRNA-mRNA interactions identified by CLASH have strong base-pairing potential and are highly enriched for complementary sequence motifs, even those supported by only a few reads. Yet, steady state levels of most mRNA targets were not significantly affected upon over-expression of the sRNA regulator. Our results reinforce the idea that the reproducibility of the interaction, not base-pairing potential, is a stronger predictor for a regulatory outcome.
Assuntos
Metabolismo Energético , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Adaptação Fisiológica , Bases de Dados Genéticas , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Modelos Genéticos , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismoRESUMO
Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner.