RESUMO
Small noncoding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies in Drosophila ovaries have demonstrated the function of the piRNA pathway in transposon silencing and therefore genome defense. However, the ability of the piRNA program to respond to different transposon landscapes and the role of piRNAs in regulating host gene expression remain poorly understood. Here, we comprehensively analyzed piRNA expression and defined the repertoire of their targets in Drosophila melanogaster testes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, we identified new piRNA clusters and established complex satellites as dual-strand piRNA clusters. While sharing most piRNA clusters, the two sexes employ them differentially to combat the sex-specific transposon landscape. We found two piRNA clusters that produce piRNAs antisense to four host genes in testis, including CG12717/pirate, a SUMO protease gene. piRNAs encoded on the Y chromosome silence pirate, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly, pirate is targeted by endogenous siRNAs in a sibling species, Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-coding gene.
Assuntos
Adaptação Fisiológica/genética , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Feminino , Masculino , Caracteres Sexuais , Fatores SexuaisRESUMO
The piRNA pathway is an adaptive mechanism that maintains genome stability by repression of selfish genomic elements. In the male germline of Drosophila melanogaster repression of Stellate genes by piRNAs generated from Supressor of Stellate (Su(Ste)) locus is required for male fertility, but both Su(Ste) piRNAs and their targets are absent in other Drosophila species. We found that D. melanogaster genome contains multiple X-linked non-coding genomic repeats that have sequence similarity to the protein-coding host gene vasa. In the male germline, these vasa-related AT-chX repeats produce abundant piRNAs that are antisense to vasa; however, vasa mRNA escapes silencing due to imperfect complementarity to AT-chX piRNAs. Unexpectedly, we discovered AT-chX piRNAs target vasa of Drosophila mauritiana in the testes of interspecies hybrids. In the majority of hybrid flies, the testes were strongly reduced in size and germline content. A minority of hybrids maintained wild-type array of premeiotic germ cells in the testes, but in them harmful Stellate genes were derepressed due to the absence of Su(Ste) piRNAs, and meiotic failures were observed. Thus, the piRNA pathway contributes to reproductive isolation between D. melanogaster and closely related species, causing hybrid male sterility via misregulation of two different host protein factors.
Assuntos
Quimera/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila/genética , Inativação Gênica , Genoma de Inseto , Proteínas Quinases/genética , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Quimera/metabolismo , Cruzamentos Genéticos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Fertilidade , Infertilidade , Masculino , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Isolamento Reprodutivo , Alinhamento de Sequência , Espermatozoides/metabolismo , Espermatozoides/patologia , Testículo/anormalidades , Testículo/metabolismoRESUMO
The tight interaction between somatic and germline cells is conserved in animal spermatogenesis. The testes of Drosophila melanogaster are the model of choice to identify processes responsible for mature gamete production. However, processes of differentiation and soma-germline interactions occurring in somatic cyst cells are currently understudied. Here we focused on the comparison of transcriptome expression patterns of early and mature somatic cyst cells to find out the developmental changes taking place in them. We employed a FACS-based approach for the isolation of early and mature somatic cyst cells from fly testes, subsequent preparation of RNA-Seq libraries, and analysis of gene differential expression in the sorted cells. We found increased expression of genes involved in cell cycle-related processes in early cyst cells, which is necessary for the proliferation and self-renewal of a crucial population of early cyst cells, cyst stem cells. Genes proposedly required for lamellipodium-like projection organization for proper cyst formation were also detected among the upregulated ones in early cyst cells. Gene Ontology and interactome analyses of upregulated genes in mature cyst cells revealed a striking over-representation of gene categories responsible for metabolic and catabolic cellular processes, as well as genes supporting the energetic state of the cells provided by oxidative phosphorylation that is carried out in mitochondria. Our comparative analyses of differentially expressed genes revealed major peculiarities in early and mature cyst cells and provide novel insight into their regulation, which is important for male fertility.
Assuntos
Cistos , Proteínas de Drosophila , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Masculino , Espermatogênese , Testículo/metabolismoRESUMO
DDX3 subfamily DEAD-box RNA helicases are essential developmental regulators of RNA metabolism in eukaryotes. belle, the single DDX3 ortholog in Drosophila, is required for fly viability, fertility, and germline stem cell maintenance. Belle is involved both in translational activation and repression of target mRNAs in different tissues; however, direct targets of Belle in the testes are essentially unknown. Here we showed that belle RNAi knockdown in testis cyst cells caused a disruption of adhesion between germ and cyst cells and generation of tumor-like clusters of stem-like germ cells. Ectopic expression of ß-integrin in cyst cells rescued early stages of spermatogenesis in belle knockdown testes, indicating that integrin adhesion complexes are required for the interaction between somatic and germ cells in a cyst. To address Belle functions in spermatogenesis in detail we performed cross-linking immunoprecipitation and sequencing (CLIP-seq) analysis and identified multiple mRNAs that interacted with Belle in the testes. The set of Belle targets includes transcripts of proteins that are essential for preventing the tumor-like clusters of germ cells and for sustaining spermatogenesis. By our hypothesis, failures in the translation of a number of mRNA targets additively contribute to developmental defects observed in the testes with belle knockdowns both in cyst cells and in the germline.
Assuntos
Carcinogênese/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Células Germinativas/metabolismo , RNA Helicases/metabolismo , Animais , Animais Geneticamente Modificados , Carcinogênese/patologia , Diferenciação Celular , Proliferação de Células , Drosophila melanogaster/citologia , Cadeias beta de Integrinas/metabolismo , Masculino , Modelos Biológicos , Fenótipo , Biossíntese de Proteínas , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogênese , Testículo/metabolismo , Testículo/ultraestrutura , Transcriptoma/genética , TransgenesRESUMO
Human DDX3 paralogs are housed on the X chromosome (DDX3X) as well as in the non- recombining region Yq11 of the Y-chromosome (DDX3Y or DBY). A gene encoding RNA helicase DDX3Y is located in the AZoospermia Factor a (AZFa) region of the Y-chromosome and expressed only in male germ cells. Deletions encompassing the DDX3Y gene lead to azoospermia and cause Sertoli Cell-Only Syndrome (SCOS) in humans. SCOS is characterized by a complete germ cell lack with preservation of somatic Sertoli cells. This review summarizes current advances in the study of DDX3Y functions in maintenance and development of early male germ cells. Data obtained from a mouse xenotransplantation model reveals that DDX3Y expression is enough to drive germ cell differentiation of AZFa-deleted human induced pluripotent stem cells (iPSCs) and for activation of the specific set of germline developmental genes. Results achieved using the testes of Drosophila demonstrate that DDX3Y homolog Belle is required cell-autonomously for mitotic progression and survival of germline stem cells and spermatogonia as the upstream regulator of mitotic cyclin expression.