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1.
Development ; 151(14)2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38881530

RESUMO

HemK2 is a highly conserved methyltransferase, but the identification of its genuine substrates has been controversial, and its biological importance in higher organisms remains unclear. We elucidate the role of HemK2 in the methylation of eukaryotic Release Factor 1 (eRF1), a process that is essential for female germline development in Drosophila melanogaster. Knockdown of hemK2 in the germline cells (hemK2-GLKD) induces apoptosis, accompanied by a pronounced decrease in both eRF1 methylation and protein synthesis. Overexpression of a methylation-deficient eRF1 variant recapitulates the defects observed in hemK2-GLKD, suggesting that eRF1 is a primary methylation target of HemK2. Furthermore, hemK2-GLKD leads to a significant reduction in mRNA levels in germline cell. These defects in oogenesis and protein synthesis can be partially restored by inhibiting the No-Go Decay pathway. In addition, hemK2 knockdown is associated with increased disome formation, suggesting that disruptions in eRF1 methylation may provoke ribosomal stalling, which subsequently activates translation-coupled mRNA surveillance mechanisms that degrade actively translated mRNAs. We propose that HemK2-mediated methylation of eRF1 is crucial for ensuring efficient protein production and mRNA stability, which are vital for the generation of high-quality eggs.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Oogênese , Biossíntese de Proteínas , Estabilidade de RNA , Animais , Oogênese/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Estabilidade de RNA/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Feminino , Metilação , Metiltransferases/metabolismo , Metiltransferases/genética , Fatores de Terminação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Apoptose/genética , RNA Helicases DEAD-box
2.
Commun Biol ; 6(1): 1287, 2023 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-38123715

RESUMO

Reproduction is highly dependent on environmental and physiological factors including nutrition, mating stimuli and microbes. Among these factors, microbes facilitate vital functions for host animals such as nutritional intake, metabolic regulation, and enhancing fertility under poor nutrition conditions. However, detailed molecular mechanisms by which microbes control germline maturation, leading to reproduction, remain largely unknown. In this study, we show that environmental microbes exert a beneficial effect on Drosophila oogenesis by promoting germline stem cell (GSC) proliferation and subsequent egg maturation via acceleration of ovarian cell division and suppression of apoptosis. Moreover, insulin-related signaling is not required; rather, the ecdysone pathway is necessary for microbe-induced increase of GSCs and promotion of egg maturation, while juvenile hormone contributes only to increasing GSC numbers, suggesting that hormonal pathways are activated at different stages of oogenesis. Our findings reveal that environmental microbes can enhance host reproductivity by modulating host hormone release and promoting oogenesis.


Assuntos
Drosophila , Oogênese , Animais , Feminino , Drosophila/metabolismo , Oogênese/fisiologia , Ovário , Células-Tronco/metabolismo , Células Germinativas/metabolismo
3.
J Cell Biol ; 222(10)2023 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-37555815

RESUMO

PIWI-interacting RNAs (piRNAs), which protect genome from the attack by transposons, are produced and amplified in membraneless granules called nuage. In Drosophila, PIWI family proteins, Tudor-domain-containing (Tdrd) proteins, and RNA helicases are assembled and form nuage to ensure piRNA production. However, the molecular functions of the Tdrd protein Tejas (Tej) in piRNA biogenesis remain unknown. Here, we conduct a detailed analysis of the subcellular localization of fluorescently tagged nuage proteins and behavior of piRNA precursors. Our results demonstrate that Tej functions as a core component that recruits Vasa (Vas) and Spindle-E (Spn-E) into nuage granules through distinct motifs, thereby assembling nuage and engaging precursors for further processing. Our study also reveals that the low-complexity region of Tej regulates the mobility of Vas. Based on these results, we propose that Tej plays a pivotal role in piRNA precursor processing by assembling Vas and Spn-E into nuage and modulating the mobility of nuage components.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , RNA de Interação com Piwi , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , RNA de Interação com Piwi/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo
4.
Sci Adv ; 9(29): eadh0397, 2023 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-37467338

RESUMO

PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. Here, we show that endogenous protein coding sequences (CDSs), which are normally used for translation, serve as origins of noncoding piRNA biogenesis in Drosophila melanogaster testes. The product, namely, CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show that CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute 2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering RNAs (siRNAs) and microRNAs (miRNAs) specify precursors to be processed into piRNAs. We further demonstrate that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, our data illustrate a unique strategy used by male germ line, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis.


Assuntos
Proteínas de Drosophila , MicroRNAs , Animais , Masculino , Drosophila/genética , RNA Interferente Pequeno/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA de Interação com Piwi , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Espermatogênese/genética
5.
Genes Cells ; 28(8): 539-552, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37249032

RESUMO

A long-standing assumption in molecular biology posits that the conservation of protein and nucleic acid sequences emphasizes the functional significance of biomolecules. These conserved sequences fold into distinct secondary and tertiary structures, enable highly specific molecular interactions, and regulate complex yet organized molecular processes within living cells. However, recent evidence suggests that biomolecules can also function through primary sequence regions that lack conservation across species or gene families. These regions typically do not form rigid structures, and their inherent flexibility is critical for their functional roles. This review examines the emerging roles and molecular mechanisms of "nondomain biomolecules," whose functions are not easily predicted due to the absence of conserved functional domains. We propose the hypothesis that both domain- and nondomain-type molecules work together to enable flexible and efficient molecular processes within the highly crowded intracellular environment.


Assuntos
Proteínas , Proteínas/genética , Sequência Conservada , Biopolímeros
6.
Front Mol Biosci ; 9: 818302, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35425810

RESUMO

Silencing of transposable elements (TEs) by Piwi-interacting RNAs (piRNAs) is crucial for maintaining germline genome integrity and fertility in animals. To repress TEs, PIWI clade Argonaute proteins cooperate with several Tudor domain-containing (Tdrd) proteins at membraneless perinuclear organelles, called nuage, to produce piRNAs to repress transposons. Here, we identify and characterize Kotsubu (Kots), one of the Drosophila Tudor domain-containing protein-1 (Tdrd1) orthologs, encoded by the CG9925 gene, that localizes to the nuage in gonads. We further show the dynamic localization of Kots in the male germline, where it shows perinuclear signals in spermatogonia but forms large cytoplasmic condensates in the spermatocytes that overlap with components of piNG-body, a nuage-associated organelle. The loss of kots results in a notable upregulation of stellate and a corresponding reduction in the suppressor of stellate piRNAs in the mutants. Furthermore, a moderate yet significant reduction of other piRNAs was observed in kots mutant testes. Taken together, we propose that Kots functions in the piRNA pathway, predominantly in the male germline by forming discrete cytoplasmic granules.

7.
Cell Rep ; 33(6): 108380, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33176138

RESUMO

In gene silencing, Hsp90 chaperone machinery assists Argonaute (Ago) binding and unwinding of silencing small RNA (sRNA) duplexes. This enables the formation of effector RNA-induced silencing complex (RISC) that often displays cargo preferences. Hence, in Drosophila, microRNAs (miRNAs) and small-interfering RNAs (siRNAs) are differentially sorted into Ago1-RISC and Ago2-RISC, respectively. Here, we identify fly Cyclophilin 40 (Cyp40) as a testis-specialized Hsp90 co-chaperone essential for spermatogenesis and for modulating Ago2-RISC formation. We show that testis-distinctive Ago-sorting and strand-selection mechanisms accumulate a unique set of miRNAs on Ago2. Cyp40 interacts with duplex-incorporating Ago2 through Hsp90 in vitro and selectively promotes the build-up of Ago2-bound miRNAs, but not endogenous siRNAs, in vivo. Moreover, one of Cyp40-dependent Ago2-sorted miRNAs is required for late spermatogenesis, unraveling the physiological relevance of the unconventional yet conserved Drosophila miRNA-Ago2 sorting pathway. Collectively, these results identify RISC-regulatory roles for Hsp90 machinery and, more generally, highlight the tissue-specific adaptation of sRNA pathways through chaperone diversification.


Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , MicroRNAs/metabolismo , Peptidil-Prolil Isomerase F/metabolismo , Maturação do Esperma/fisiologia , Animais , Drosophila , Inativação Gênica
8.
PLoS One ; 15(4): e0231114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32243476

RESUMO

Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. In this study, we examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. Our results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. We propose that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Fusão Celular , Sequência Conservada , Ciclina B , Regulação para Baixo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Regulação da Expressão Gênica , Meiose , Proteínas Mutantes/metabolismo , Mutação/genética , Oogênese , Ovário/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Tiorredoxinas/metabolismo
10.
Nat Commun ; 9(1): 1735, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29728561

RESUMO

In metazoan germline, Piwi-interacting RNAs (piRNAs) provide defence against transposons. Piwi-piRNA complex mediates transcriptional silencing of transposons in nucleus. Heterochromatin protein 1a (HP1a) has been proposed to function downstream of Piwi-piRNA complex in Drosophila. Here we show that HP1a germline knockdown (HP1a-GLKD) leads to a reduction in the total and Piwi-bound piRNAs mapping to clusters and transposons insertions, predominantly in the regions close to telomeres and centromeres, resulting in derepression of a limited number of transposons from these regions. In addition, HP1a-GLKD increases the splicing of transcripts arising from clusters in above regions, suggesting HP1a also functions upstream to piRNA processing. Evolutionarily old transposons enriched in the pericentric regions exhibit significant loss in piRNAs targeting these transposons upon HP1a-GLKD. Our study suggests that HP1a functions to repress transposons in a chromosomal compartmentalised manner.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Heterocromatina/genética , RNA Interferente Pequeno/genética , Telômero/genética , Animais , Animais Geneticamente Modificados , Centrômero/genética , Centrômero/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Germinativas/metabolismo , Heterocromatina/metabolismo , Mutagênese Insercional , RNA Interferente Pequeno/biossíntese , Telômero/metabolismo
11.
Genetics ; 208(2): 435-471, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29378808

RESUMO

Gametogenesis represents the most dramatic cellular differentiation pathways in both female and male flies. At the genome level, meiosis ensures that diploid germ cells become haploid gametes. At the epigenome level, extensive changes are required to turn on and shut off gene expression in a precise spatiotemporally controlled manner. Research applying conventional molecular genetics and cell biology, in combination with rapidly advancing genomic tools have helped us to investigate (1) how germ cells maintain lineage specificity throughout their adult reproductive lifetime; (2) what molecular mechanisms ensure proper oogenesis and spermatogenesis, as well as protect genome integrity of the germline; (3) how signaling pathways contribute to germline-soma communication; and (4) if such communication is important. In this chapter, we highlight recent discoveries that have improved our understanding of these questions. On the other hand, restarting a new life cycle upon fertilization is a unique challenge faced by gametes, raising questions that involve intergenerational and transgenerational epigenetic inheritance. Therefore, we also discuss new developments that link changes during gametogenesis to early embryonic development-a rapidly growing field that promises to bring more understanding to some fundamental questions regarding metazoan development.


Assuntos
Gametogênese , Células Germinativas/metabolismo , Animais , Diferenciação Celular , Autorrenovação Celular , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Humanos , Meiose , Mitose , RNA Interferente Pequeno , Transdução de Sinais
12.
RNA ; 22(7): 1044-54, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27208314

RESUMO

PIWI-interacting RNAs (piRNAs) are predominantly produced in animal gonads to suppress transposons during germline development. Our understanding about the piRNA biogenesis and function is predominantly from studies of the Drosophila female germline. piRNA pathway function in the male germline, however, remains poorly understood. To study overall and stage-specific features of piRNAs during spermatogenesis, we analyzed small RNAs extracted from entire wild-type testes and stage-specific arrest mutant testes enriched with spermatogonia or primary spermatocytes. We show that most active piRNA clusters in the female germline do not majorly contribute to piRNAs in testes, and abundance patterns of piRNAs mapping to different transposon families also differ between male and female germlines. piRNA production is regulated in a stage-specific manner during spermatogenesis. The piRNAs in spermatogonia-enriched testes are predominantly transposon-mapping piRNAs, and almost half of those exhibit a ping-pong signature. In contrast, the primary spermatocyte-enriched testes have a dramatically high amount of piRNAs targeting repeats like suppressor of stellate and AT-chX The transposon-mapping piRNAs in the primary spermatocyte stages lacking Argonaute3 expression also show a ping-pong signature, albeit to a lesser extent. Consistently, argonaute3 mutant testes also retain ping-pong signature-bearing piRNAs, suggesting that a noncanonical ping-pong cycle might act during spermatogenesis. Our study shows stage-specific regulation of piRNA biogenesis during spermatogenesis: An active ping-pong cycle produces abundant transposon-mapping piRNAs in spermatogonia, while in primary spermatocytes, piRNAs act to suppress the repeats and transposons.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Interferente Pequeno/metabolismo , Espermatogênese/genética , Animais , Drosophila , Proteínas de Drosophila/genética , Inativação Gênica , Masculino , RNA Interferente Pequeno/genética
13.
Semin Cell Dev Biol ; 47-48: 17-31, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26582251

RESUMO

Small non-coding RNAs are indispensable to many biological processes. A class of endogenous small RNAs, termed PIWI-interacting RNAs (piRNAs) because of their association with PIWI proteins, has known roles in safeguarding the genome against inordinate transposon mobilization, embryonic development, and stem cell regulation, among others. This review discusses the biogenesis of animal piRNAs and their diverse functions together with their PIWI protein partners, both in the germline and in somatic cells, and highlights the evolutionarily conserved aspects of these molecular players in animal biology.


Assuntos
Proteínas Argonautas/genética , Desenvolvimento Embrionário/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Animais , Proteínas Argonautas/metabolismo , Elementos de DNA Transponíveis/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Filogenia , Ligação Proteica , RNA Interferente Pequeno/classificação , RNA Interferente Pequeno/metabolismo
14.
Curr Protoc Stem Cell Biol ; 34: 2E.3.1-2E.3.15, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26237568

RESUMO

This unit describes a method for isolating undifferentiated, stem cell-like germline cells from adult Drosophila ovaries. Here, we demonstrate that this population of cells can be effectively purified from hand-dissected ovaries in considerably large quantities. Tumor ovaries with expanded populations of undifferentiated germline cells are first removed from fly abdomens and dissociated into a cell suspension with the aid of protease treatment. The target cells, which express Vasa-green fluorescent protein (GFP) fusion protein under the control of the germline-specific vasa promoter, are specifically selected from the suspension via fluorescence-activated cell sorting (FACS). These protocols can be adapted to isolate other cell types from fly ovaries, such as somatic follicle cells or escort cells, by driving GFP expression in the respective target cells.


Assuntos
Células-Tronco Adultas , Separação Celular/métodos , Ovário , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Feminino , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Ovário/citologia , Ovário/metabolismo
15.
BMC Biol ; 12: 61, 2014 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-25287931

RESUMO

BACKGROUND: Piwi-interacting RNAs (piRNAs) are a special class of small RNAs that provide defense against transposable elements in animal germline cells. In Drosophila, germline piRNAs are thought to be processed at a unique perinuclear structure, the nuage, that houses piRNA pathway proteins including the Piwi clade of Argonaute family proteins, along with several Tudor domain proteins, RNA helicases and nucleases. We previously demonstrated that Tudor domain protein Tejas (Tej), an ortholog of vertebrate Tdrd5, is an important component of the piRNA pathway. RESULTS: In the current study, we identified the paralog of the Drosophila tej gene, tapas (tap), which is an ortholog of vertebrate Tdrd7. Like Tej, Tap is localized at the nuage. Alone, tap loss leads to a mild increase in transposon expression and decrease in piRNAs targeting transposons expressed in the germline. The tap gene genetically interacts with other piRNA pathway genes and we also show that Tap physically interacts with piRNA pathway components, such as Piwi family proteins Aubergine and Argonaute3 and the RNA helicases Spindle-E and Vasa. Together with tej, tap is required for survival of germline cells during early stages and for polarity formation. We further observed that loss of tej and tap together results in more severe defects in the piRNA pathway in germline cells compared to single mutants: the double-mutant ovaries exhibit mis-localization of piRNA pathway components and significantly greater reduction of piRNAs against transposons predominantly expressed in germline compared to single mutants. The single or double mutants did not have any reduction in piRNAs mapping to transposons predominantly expressed in gonadal somatic cells or those derived from unidirectional clusters such as flamenco. Consistently, the loss of both tej and tap function resulted in mis-localization of Piwi in germline cells, whereas Piwi remained localized to the nucleus in somatic cells. CONCLUSIONS: Our observations suggest that tej and tap work together for germline maintenance. tej and tap also function in a synergistic manner to maintain examined piRNA components at the perinuclear nuage and for piRNA production in Drosophila germline cells.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Germinativas/citologia , Neuropeptídeos/genética , RNA Interferente Pequeno/genética , Retroelementos , Fatores de Transcrição/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/citologia , Neuropeptídeos/metabolismo , Ovário/citologia , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Interferente Pequeno/isolamento & purificação , Fatores de Transcrição/metabolismo
17.
Dev Biol ; 386(1): 237-51, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24355748

RESUMO

To preserve genome integrity, an evolutionarily conserved small RNA-based silencing mechanism involving PIWI proteins and PIWI-interacting RNAs (piRNAs) represses potentially deleterious transposons in animals. Although there has been extensive research into PIWI proteins in bilaterians, these proteins remain to be examined in ancient phyla. Here, we investigated the PIWI proteins Hywi and Hyli in the cnidarian Hydra, and found that both PIWI proteins are enriched in multipotent stem cells, germline stem cells, and in the female germline. Hywi and Hyli localize to the nuage, a perinuclear organelle that has been implicated in piRNA-mediated transposon silencing, together with other conserved nuage and piRNA pathway components. Our findings provide the first report of nuage protein localization patterns in a non-bilaterian. Hydra PIWI proteins possess symmetrical dimethylarginines: modified residues that are known to aid in PIWI protein localization to the nuage and proper piRNA loading. piRNA profiling suggests that transposons are the major targets of the piRNA pathway in Hydra. Our data suggest that piRNA biogenesis through the ping-pong amplification cycle occurs in Hydra and that Hywi and Hyli are likely to preferentially bind primary and secondary piRNAs, respectively. Presumptive piRNA clusters are unidirectionally transcribed and primarily give rise to piRNAs that are antisense to transposons. These results indicate that various conserved features of PIWI proteins, the piRNA pathway, and their associations with the nuage were likely established before the evolution of bilaterians.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hydra/genética , RNA Interferente Pequeno/metabolismo , Animais , Evolução Biológica , Mapeamento de Sequências Contíguas , Citoplasma/metabolismo , Elementos de DNA Transponíveis , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Inativação Gênica , Células Germinativas/citologia , Proteínas de Fluorescência Verde/metabolismo , Hydra/fisiologia , Camundongos , Oligonucleotídeos Antissenso/metabolismo , Filogenia , Interferência de RNA , Células-Tronco/citologia
18.
Development ; 139(24): 4505-13, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23136393

RESUMO

In Drosophila, Maelstrom is a conserved component of the perinuclear nuage, a germline-unique structure that appears to serve as a site for Piwi-interacting RNA (piRNA) production to repress deleterious transposons. Maelstrom also functions in the nucleus as a transcriptional regulator to repress the expression of microRNA-7, a process that is essential for the proper differentiation of germline stem cells. In this paper, we report another function of Maelstrom in regulating oocyte determination independently of its transposon silencing and germline stem cell differentiation activities. In Drosophila, the conserved serine 138 residue in Maelstrom is required for its phosphorylation, an event that promotes oocyte determination. Phosphorylation of Maelstrom is required for the repression of the pachytene checkpoint protein Sir2, but not for transposon silencing or for germline stem cell differentiation. We identify Polo as a kinase that mediates the phosphorylation of Maelstrom. Our results suggest that the Polo-mediated phosphorylation of Maelstrom may be a mechanism that controls oocyte determination by inactivating the pachytene checkpoint via the repression of Sir2 in Drosophila ovaries.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila , Oócitos/metabolismo , Oogênese , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Elementos de DNA Transponíveis/genética , Drosophila/genética , Drosophila/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Feminino , Inativação Gênica , Células Germinativas/citologia , Células Germinativas/metabolismo , Células Germinativas/fisiologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histona Desacetilases/fisiologia , Oócitos/fisiologia , Oogênese/genética , Oogênese/fisiologia , Ovário/metabolismo , Estágio Paquíteno/genética , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Sirtuínas/genética , Sirtuínas/metabolismo , Sirtuínas/fisiologia
19.
Curr Protoc Stem Cell Biol ; Chapter 2: Unit2E.3, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22872426

RESUMO

This unit describes a method for isolating undifferentiated, stem cell-like germline cells from adult Drosophila ovaries. Here, we demonstrate that this population of cells can be effectively purified from hand-dissected ovaries in considerably large quantities. Tumor ovaries with expanded populations of undifferentiated germline cells are first removed from fly abdomens and dissociated into a cell suspension with the aid of protease treatment. The target cells, which express Vasa-green fluorescent protein (GFP) fusion protein under the control of the germline-specific vasa promoter, are specifically selected from the suspension via fluorescence-activated cell sorting (FACS). These protocols can be adapted to isolate other cell types from fly ovaries, such as somatic follicle cells or escort cells, by driving GFP expression in the respective target cells.


Assuntos
Envelhecimento/fisiologia , Diferenciação Celular , Separação Celular/métodos , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Células Germinativas/citologia , Ovário/citologia , Animais , Dissecação , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Peptídeo Hidrolases/metabolismo , Suspensões , Fixação de Tecidos
20.
Development ; 139(13): 2255-66, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22669818

RESUMO

Tudor domain proteins function as molecular adaptors, binding methylated arginine or lysine residues on their substrates to promote physical interactions and the assembly of macromolecular complexes. Here, we discuss the emerging roles of Tudor domain proteins during development, most notably in the Piwi-interacting RNA pathway, but also in other aspects of RNA metabolism, the DNA damage response and chromatin modification.


Assuntos
RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Arginina/química , Arginina/metabolismo , Cromatina/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Desenvolvimento Embrionário , Feminino , Humanos , Lisina/química , Lisina/metabolismo , Masculino , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/química
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