RESUMO
Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.
Assuntos
Gammaretrovirus/genética , Phascolarctidae/genética , RNA Interferente Pequeno/genética , Animais , Elementos de DNA Transponíveis , Gammaretrovirus/metabolismo , Gammaretrovirus/patogenicidade , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Genoma , Células Germinativas/metabolismo , Células Germinativas/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Phascolarctidae/virologia , Splicing de RNA , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, whereas the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 colocalizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts colocalization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.
Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Dano ao DNA , Elementos de DNA Transponíveis , Feminino , Células Germinativas/citologia , Masculino , Membrana Nuclear/metabolismoRESUMO
Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P elements and resident transposons and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P elements are silenced, and P element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles, and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.
Assuntos
Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Evolução Molecular , Animais , Drosophila melanogaster/metabolismo , Feminino , Inativação Gênica , Masculino , Ovário/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Piwi-interacting RNAs (piRNAs) silence transposons and maintain genome integrity during germline development. In Drosophila, transposon-rich heterochromatic clusters encode piRNAs either on both genomic strands (dual-strand clusters) or predominantly one genomic strand (uni-strand clusters). Primary piRNAs derived from these clusters are proposed to drive a ping-pong amplification cycle catalyzed by proteins that localize to the perinuclear nuage. We show that the HP1 homolog Rhino is required for nuage organization, transposon silencing, and ping-pong amplification of piRNAs. rhi mutations virtually eliminate piRNAs from the dual-strand clusters and block production of putative precursor RNAs from both strands of the major 42AB dual-strand cluster, but not of transcripts or piRNAs from the uni-strand clusters. Furthermore, Rhino protein associates with the 42AB dual-strand cluster,but does not bind to uni-strand cluster 2 or flamenco. Rhino thus appears to promote transcription of dual-strand clusters, leading to production of piRNAs that drive the ping-pong amplification cycle.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Inativação Gênica , Animais , Imunoprecipitação da Cromatina , Drosophila melanogaster/genética , Heterocromatina/metabolismo , RNA Interferente Pequeno/metabolismo , Transcrição GênicaRESUMO
piRNAs guide PIWI proteins to silence transposons in animal germ cells. Reciprocal cycles of piRNA-directed RNA cleavage--catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila melanogaster--expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. We show that qin, a piRNA pathway gene whose protein product contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, a perinuclear structure implicated in transposon silencing. In qin mutants, less Ago3 binds Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, and DNA damage accumulates in nurse cells and oocytes. We propose that Qin enforces heterotypic Ping-Pong between Aub and Ago3, ensuring that transposons are silenced and maintaining the integrity of the germline genome.
Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inativação Gênica , Genoma de Inseto , Oócitos/metabolismo , Ovário/metabolismo , RNA Interferente Pequeno/genética , Complexo de Inativação Induzido por RNA/genética , Ubiquitina-Proteína Ligases/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Biologia Computacional , Dano ao DNA , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Fertilidade , Inativação Gênica/fisiologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Mutação , Oócitos/citologia , Ovário/citologia , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Estrutura Terciária de Proteína/genética , Clivagem do RNA , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Small repeat-associated siRNAs (rasiRNAs) mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila rasiRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization as well as asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellate silencing. Furthermore, rasiRNA pathway mutations lead to germline-specific accumulation of gamma-H2Av foci characteristic of DNA damage. We conclude that rasiRNA-based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline.
Assuntos
Padronização Corporal/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Animais , Quinase do Ponto de Checagem 2 , RNA Helicases DEAD-box/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Mutação em Linhagem Germinativa , Microtúbulos/metabolismo , Modelos Biológicos , Ovário/citologia , Ovário/patologia , Fatores de Iniciação de Peptídeos/metabolismo , Fosforilação , Transporte Proteico , RNA Helicases/metabolismo , Supressão Genética , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Microtubules and the plus-end-directed microtubule motor Kinesin I are required for the selective accumulation of oskar mRNA at the posterior cortex of the Drosophila melanogaster oocyte, which is essential to posterior patterning and pole plasm assembly. We present evidence that microtubule minus ends associate with the entire cortex, and that Kinesin and microtubules are not required for oskar mRNA association with the posterior pole, but prevent ectopic localization of this transcript and the pole plasm proteins Oskar and Vasa to other cortical regions. Cortical binding of oskar mRNA seems to be dependent on the actin cytoskeleton. We conclude that most of the actin-rich oocyte cortex can support pole plasm assembly, and propose that Kinesin restricts pole plasm formation to the posterior by moving oskar mRNA away from microtubule-rich lateral and anterior cortical regions.
Assuntos
Cinesinas/metabolismo , Oócitos/metabolismo , Animais , Polaridade Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Hibridização in Situ Fluorescente , Cinesinas/química , Cinesinas/genética , Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Mutação , Oócitos/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Progression through the G(2)/M transition following DNA damage is linked to cytokinesis failure and mitotic death. In four different transformed cell lines and two human embryonic stem cell lines, we find that DNA damage triggers mitotic chromatin decondensation and global phosphorylation of histone H2AX, which has been associated with apoptosis. However, extended time-lapse studies in HCT116 colorectal cancer cells indicate that death does not take place during mitosis, but 72% of cells die within 3 days of mitotic exit. By contrast, only 11% of cells in the same cultures that remained in interphase died, suggesting that progression through mitosis enhances cell death following DNA damage. These time-lapse studies also confirmed that DNA damage leads to high rates of cytokinesis failure, but showed that cells that completed cytokinesis following damage died at higher rates than cells that failed to complete division. Therefore, post-mitotic cell death is not a response to cytokinesis failure or polyploidy. We also show that post-mitotic cell death is largely independent of p53 and is only partially suppressed by the apical caspase inhibitor Z-VAD-FMK. These findings suggest that progression through mitosis following DNA damage initiates a p53- and caspase-independent cell death response that prevents propagation of genetic lesions.
Assuntos
Morte Celular/genética , Dano ao DNA , Mitose , Caspases , Linhagem Celular , Linhagem Celular Tumoral , Citocinese , Células-Tronco Embrionárias , Células HCT116 , Humanos , Cinética , Proteína Supressora de Tumor p53RESUMO
The 13 syncytial cleavage divisions that initiate Drosophila embryogenesis are under maternal genetic control. The switch to zygotic regulation of development at the midblastula transition (MBT) follows mitosis 13, when the cleavage divisions terminate, transcription increases and the blastoderm cellularizes. Embryos mutant for grp, which encodes Checkpoint kinase 1 (Chk1), are DNA-replication-checkpoint defective and fail to cellularize, gastrulate or to initiate high-level zygotic transcription at the MBT. The mnk (also known as loki) gene encodes Checkpoint kinase 2 (Chk2), which functions in DNA-damage signal transduction. We show that mnk grp double-mutant embryos are replication-checkpoint defective but cellularize, gastrulate and activate high levels of zygotic gene expression. We also show that grp mutant embryos accumulate DNA double-strand breaks and that DNA-damaging agents induce a mnk-dependent block to cellularization and zygotic gene expression. We conclude that the DNA-replication checkpoint maintains genome integrity during the cleavage divisions, and that checkpoint mutations lead to DNA damage that induces a novel Chk2-dependent block at the MBT.
Assuntos
Dano ao DNA/genética , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Blástula/metabolismo , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Drosophila/metabolismo , Desenvolvimento Embrionário , Feminino , Masculino , Transdução de SinaisRESUMO
Polarization of the microtubule cytoskeleton during early oogenesis is required to specify the posterior of the Drosophila oocyte, which is essential for asymmetric mRNA localization during mid-oogenesis and for embryonic axis specification. The posterior determinant oskar mRNA is translationally silent until mid-oogenesis. We show that mutations in armitage and three components of the RNAi pathway disrupt oskar mRNA translational silencing, polarization of the microtubule cytoskeleton, and posterior localization of oskar mRNA. armitage encodes a homolog of SDE3, a presumptive RNA helicase involved in posttranscriptional gene silencing (RNAi) in Arabidopsis, and is required for RNAi in Drosophila ovaries. Armitage forms an asymmetric network associated with the polarized microtubule cytoskeleton and is concentrated with translationally silent oskar mRNA in the oocyte. We conclude that RNA silencing is essential for establishment of the cytoskeletal polarity that initiates embryonic axis specification and for translational control of oskar mRNA.
Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Embrião não Mamífero/embriologia , Biossíntese de Proteínas/genética , RNA Helicases/genética , Interferência de RNA/fisiologia , Sequência de Aminoácidos/genética , Animais , Proteínas de Arabidopsis/genética , Sequência de Bases/genética , Polaridade Celular/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , DNA Complementar/análise , DNA Complementar/genética , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Mutação , Oócitos/citologia , Oócitos/metabolismo , RNA Helicases/isolamento & purificaçãoRESUMO
The putative RNA helicase, Armitage (Armi), is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. Here, we show that armi is required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi. Our results suggest that armi is required for RISC maturation.