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-boxRESUMO
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 , BiopolímerosRESUMO
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/genéticaRESUMO
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/metabolismoRESUMO
In Drosophila ovaries, distinct Piwi-interacting RNA (piRNA) pathways defend against transposons in somatic and germline cells. Germline piRNAs predominantly arise from bidirectional clusters and are amplified by the ping-pong cycle. In this study, we characterize a novel Drosophila gene, kumo and show that it encodes a conserved germline piRNA pathway component. Kumo contains five tudor domains and localizes to nuage, a unique structure present in animal germline cells, which is considered to be the processing site for germline piRNAs. Transposons targeted by the germline piRNA pathway are derepressed in kumo mutant females. Moreover, germline piRNA production is significantly reduced in mutant ovaries, thereby indicating that kumo is required to generate germline piRNAs. Kumo localizes to the nuage as well as to nucleus early female germ cells, where it is required to maintain cluster transcript levels. Our data suggest that kumo facilitates germline piRNA production by promoting piRNA cluster transcription in the nucleus and piRNA processing at the nuage.
Assuntos
Proteínas de Drosophila/fisiologia , Células Germinativas , RNA Interferente Pequeno/metabolismo , Animais , Núcleo Celular/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , MutaçãoRESUMO
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/citologiaRESUMO
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/fisiologiaRESUMO
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/químicaRESUMO
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/metabolismoRESUMO
During mitosis, faithful inheritance of genetic material is achieved by chromosome segregation, as mediated by the condensin I and II complexes. Failed chromosome segregation can result in neoplasm formation, infertility, and birth defects. Recently, the germ-line-specific DEAD-box RNA helicase Vasa was demonstrated to promote mitotic chromosome segregation in Drosophila by facilitating robust chromosomal localization of Barren (Barr), a condensin I component. This mitotic function of Vasa is mediated by Aubergine and Spindle-E, which are two germ-line components of the Piwi-interacting RNA pathway. Faithful segregation of chromosomes should be executed both in germ-line and somatic cells. However, whether a similar mechanism also functions in promoting chromosome segregation in somatic cells has not been elucidated. Here, we present evidence that belle (vasa paralog) and the RNA interference pathway regulate chromosome segregation in Drosophila somatic cells. During mitosis, belle promotes robust Barr chromosomal localization and chromosome segregation. Belle's localization to condensing chromosomes depends on dicer-2 and argonaute2. Coimmunoprecipitation experiments indicated that Belle interacts with Barr and Argonaute2 and is enriched at endogenous siRNA (endo-siRNA)-generating loci. Our results suggest that Belle functions in promoting chromosome segregation in Drosophila somatic cells via the endo-siRNA pathway. DDX3 (human homolog of belle) and DICER function in promoting chromosome segregation and hCAP-H (human homolog of Barr) localization in HeLa cells, indicating a conserved function for those proteins in human cells. Our results suggest that the RNA helicase Belle/DDX3 and the RNA interference pathway perform a common role in regulating chromosome segregation in Drosophila and human somatic cells.
Assuntos
Segregação de Cromossomos/fisiologia , RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/metabolismo , Mitose/fisiologia , RNA Helicases/metabolismo , Interferência de RNA/fisiologia , Animais , Proteínas Argonautas , Western Blotting , Proteínas de Ciclo Celular , Imunoprecipitação da Cromatina , Segregação de Cromossomos/genética , Drosophila , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Imunoprecipitação , Mitose/genética , RNA Interferente Pequeno/genética , Complexo de Inativação Induzido por RNARESUMO
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/genéticaRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
The accurate transfer of genetic material in germline cells during the formation of gametes is important for the continuity of the species. However, animal germline cells face challenges from transposons, which seek to spread themselves in the genome. This review focuses on studies in Drosophila melanogaster on how the genome protects itself from such a mutational burden via a class of gonad-specific small interfering RNAs, known as piRNAs (Piwi-interacting RNAs). In addition to silencing transposons, piRNAs also regulate other processes, such as chromosome segregation, mRNA degradation and germline differentiation. Recent studies revealed two modes of piRNA processing primary processing and secondary processing (also known as ping-pong amplification). The primary processing pathway functions in both germline and somatic cells in the Drosophila ovaries by processing precursor piRNAs into 2329 nt piRNAs. In contrast, the secondary processing pathway functions only in the germline cells where piRNAs are amplified in a feed-forward loop and require the Piwi-family proteins Aubergine and Argonaute3. Aubergine and Argonaute3 localize to a unique structure found in animal germline cells, the nuage, which has been proposed to function as a compartmentalized site for the ping-pong cycle. The nuage and the localized proteins are well-conserved, implying the importance of the piRNA amplification loop in animal germline cells. Nuage components include various types of proteins that are known to interact both physically and genetically, and therefore appear to be assembled in a sequential order to exert their function, resulting in a macromolecular RNA-protein complex dedicated to the silencing of transposons.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , RNA Interferente Pequeno/genética , Regiões 3' não Traduzidas , Animais , Proteínas Argonautas/genética , Padronização Corporal , Elementos de DNA Transponíveis , Proteínas de Drosophila/genética , Feminino , Inativação Gênica , Masculino , Mutação , Ovário/metabolismo , Fatores de Iniciação de Peptídeos/genética , RNA Mensageiro Estocado/metabolismoRESUMO
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.
RESUMO
Stem cells in animal tissues are often located and controlled by special tissue microenvironments known as niches. Studies of stem cell niches in model systems such as Drosophila have revealed adhesive interactions, cell cycle modifications and intercellular signals that operate to control stem cell behavior. Candidate niches and regulatory molecules have also been identified in many mammalian tissues, including bone marrow, skin, gut and brain. While niches are an ancient evolutionary device with conserved features across diverse organisms, we suggest that certain niches display important differences in their organization and function.
Assuntos
Células-Tronco/citologia , Animais , Adesão Celular , Divisão Celular , Linhagem da Célula , Proliferação de Células , Drosophila , Humanos , Modelos Animais , Modelos Biológicos , Transdução de Sinais , Distribuição TecidualRESUMO
Many tissues including blood, skin, gut and germ cells are continuously maintained by tissue stem cells. Under certain conditions, however, other organs can undergo repair using stem-cell-like progenitors generated by cell de-differentiation. Cell fates have been broadened experimentally, but mechanisms allowing de-differentiation to a stem cell state are poorly known. Germline stem cells begin to differentiate by forming interconnected germ cell cysts (cystocytes), and under certain conditions male mouse cystocytes have been postulated to revert into functional progenitors. Here we report that four- and eight-cell Drosophila germline cystocytes generated either in second instar larval ovaries or in adults over-producing the BMP4-like stem cell signal Decapentaplegic efficiently convert into single stem-like cells. These de-differentiated cells can develop into functional germline stem cells and support normal fertility. Our results show that cystocytes represent a relatively abundant source of regenerative precursors that might help replenish germ cells after depletion by genotoxic chemicals, radiation or normal ageing. More generally, Drosophila cystocytes now provide a system for studying de-differentiation and its potential as a source of functional stem cells.
Assuntos
Diferenciação Celular , Linhagem da Célula , Drosophila melanogaster/citologia , Ovário/citologia , Óvulo/citologia , Células-Tronco/citologia , Animais , Apoptose , Feminino , Resposta ao Choque Térmico , Larva/citologiaRESUMO
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 GênicaRESUMO
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.