RESUMO
PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, we uncover an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Argonautas/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Helicases DEAD-box/metabolismo , Elementos de DNA Transponíveis , Inativação Gênica , Células Germinativas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Gênica , Proteína Exportina 1RESUMO
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
Although animals have evolved multiple mechanisms to suppress transposons, "leaky" mobilizations that cause mutations and diseases still occur. This suggests that transposons employ specific tactics to accomplish robust propagation. By directly tracking mobilization, we show that, during a short and specific time window of oogenesis, retrotransposons achieve massive amplification via a cell-type-specific targeting strategy. Retrotransposons rarely mobilize in undifferentiated germline stem cells. However, as oogenesis proceeds, they utilize supporting nurse cells-which are highly polyploid and eventually undergo apoptosis-as factories to massively manufacture invading products. Moreover, retrotransposons rarely integrate into nurse cells themselves but, instead, via microtubule-mediated transport, they preferentially target the DNA of the interconnected oocytes. Blocking microtubule-dependent intercellular transport from nurse cells significantly alleviates damage to the oocyte genome. Our data reveal that parasitic genomic elements can efficiently hijack a host developmental process to propagate robustly, thereby driving evolutionary change and causing disease.
Assuntos
Drosophila melanogaster/genética , Elementos Nucleotídeos Longos e Dispersos , Oogênese , RNA Interferente Pequeno , Retroelementos , Retroviridae/genética , Animais , Proteínas de Drosophila , Feminino , Biblioteca Gênica , Inativação Gênica , Células Germinativas , Proteínas de Fluorescência Verde/metabolismo , Hibridização in Situ Fluorescente , Masculino , Oócitos/metabolismo , Células-Tronco/metabolismoRESUMO
In the male mouse germ line, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germ line remain unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of these pathogenic alleles, we discovered that the uncharacterized protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in fetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterized protein essential for orchestrating piRNA-directed DNA methylation.
Assuntos
Metilação de DNA , RNA de Interação com Piwi , Masculino , Humanos , Animais , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas/metabolismo , Células Germinativas/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Elementos de DNA Transponíveis/genética , Mamíferos/metabolismoRESUMO
PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs) and silence transposable elements in animal gonads. Here, we report the crystal structure of a silkworm PIWI-clade Argonaute, Siwi, bound to the endogenous piRNA, at 2.4 Å resolution. Siwi adopts a bilobed architecture consisting of N-PAZ and MID-PIWI lobes, in which the 5' and 3' ends of the bound piRNA are anchored by the MID-PIWI and PAZ domains, respectively. A structural comparison of Siwi with AGO-clade Argonautes reveals notable differences in their nucleic-acid-binding channels, likely reflecting the distinct lengths of their guide RNAs and their mechanistic differences in guide RNA loading and cleavage product release. In addition, the structure reveals that Siwi and prokaryotic, but not eukaryotic, AGO-clade Argonautes share unexpected similarities, such as metal-dependent 5'-phosphate recognition and a potential structural transition during the catalytic-tetrad formation. Overall, this study provides a critical starting point toward a mechanistic understanding of piRNA-mediated transposon silencing.
Assuntos
Proteínas Argonautas/química , Bombyx/metabolismo , Proteínas de Insetos/química , RNA Interferente Pequeno/química , Animais , Proteínas Argonautas/isolamento & purificação , Bombyx/química , Bombyx/genética , Linhagem Celular , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Inativação Gênica , Humanos , Proteínas de Insetos/isolamento & purificação , Conformação de Ácido Nucleico , RNA Interferente Pequeno/isolamento & purificaçãoRESUMO
PIWI-interacting RNAs (piRNAs) guide transposable element repression in animal germ lines. In Drosophila, piRNAs are produced from heterochromatic loci, called piRNA clusters, which act as information repositories about genome invaders. piRNA generation by dual-strand clusters depends on the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex, which is deposited on clusters guided by piRNAs, forming a positive feedback loop in which piRNAs promote their own biogenesis. However, how piRNA clusters are formed before cognate piRNAs are present remains unknown. Here, we report spontaneous de novo piRNA cluster formation from repetitive transgenic sequences. Cluster formation occurs over several generations and requires continuous trans-generational maternal transmission of small RNAs. We discovered that maternally supplied small interfering RNAs (siRNAs) trigger de novo cluster activation in progeny. In contrast, siRNAs are dispensable for cluster function after its establishment. These results reveal an unexpected interplay between the siRNA and piRNA pathways and suggest a mechanism for de novo piRNA cluster formation triggered by siRNAs.
Assuntos
Proteínas de Drosophila , RNA de Interação com Piwi , Animais , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Herança Materna , Drosophila/genética , Cromatina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMO
PIWI-interacting RNAs (piRNAs) are a class of small RNAs that are 24-31 nucleotides in length. They associate with PIWI proteins, which constitute a germline-specific subclade of the Argonaute family, to form effector complexes known as piRNA-induced silencing complexes, which repress transposons via transcriptional or posttranscriptional mechanisms and maintain germline genome integrity. In addition to having a role in transposon silencing, piRNAs in diverse organisms function in the regulation of cellular genes. In some cases, piRNAs have shown transgenerational inheritance to pass on the memory of "self" and "nonself," suggesting a contribution to various cellular processes over generations. Many piRNA factors have been identified; however, both the molecular mechanisms leading to the production of mature piRNAs and the effector phases of gene silencing are still enigmatic. Here, we summarize the current state of our knowledge on the biogenesis of piRNA, its biological functions, and the underlying mechanisms.
Assuntos
RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas/metabolismo , Elementos de DNA Transponíveis , Inativação Gênica , HumanosRESUMO
In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal trimming and 2'-O-methylation. Both trimming and methylation influence piRNA stability. Our biochemical data show that multiple mechanisms destabilize unmethylated mouse piRNAs, depending on whether the piRNA 5' or 3' sequence is complementary to a trigger RNA. Unlike target-directed degradation of microRNAs, complementarity-dependent destabilization of piRNAs in mice and flies is blocked by 3' terminal 2'-O-methylation and does not require base pairing to both the piRNA seed and the 3' sequence. In flies, 2'-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent destruction. By contrast, pre-piRNA trimming protects mouse piRNAs from a degradation pathway unaffected by trigger complementarity. In testis lysate and in vivo, internal or 3' terminal uridine- or guanine-rich tracts accelerate pre-piRNA decay. Loss of both trimming and 2'-O-methylation causes the mouse piRNA pathway to collapse, demonstrating that these modifications collaborate to stabilize piRNAs.
Assuntos
Proteínas Argonautas/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Separação Celular , Drosophila melanogaster , Feminino , Citometria de Fluxo , Expressão Gênica , Inativação Gênica , Técnicas Genéticas , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Processamento de Proteína Pós-Traducional , RNA de Cadeia Dupla , Espermatócitos/metabolismo , Espermatogônias/metabolismo , Testículo/metabolismoRESUMO
Histone H3.3 lysine-to-methionine substitutions K27M and K36M impair the deposition of opposing chromatin marks, H3K27me3/me2 and H3K36me3/me2. We show that these mutations induce hypotrophic and disorganized eyes in Drosophila eye primordia. Restriction of H3K27me3 spread in H3.3K27M and its redistribution in H3.3K36M result in transcriptional deregulation of PRC2-targeted eye development and of piRNA biogenesis genes, including krimp. Notably, both mutants promote redistribution of H3K36me2 away from repetitive regions into active genes, which associate with retrotransposon de-repression in eye discs. Aberrant expression of krimp represses LINE retrotransposons but does not contribute to the eye phenotype. Depletion of H3K36me2 methyltransferase ash1 in H3.3K27M, and of PRC2 component E(z) in H3.3K36M, restores the expression of eye developmental genes and normal eye growth, showing that redistribution of antagonistic marks contributes to K-to-M pathogenesis. Our results implicate a novel function for H3K36me2 and showcase convergent downstream effects of oncohistones that target opposing epigenetic marks.
Assuntos
Cromatina/química , Elementos de DNA Transponíveis , Histonas/química , Histonas/genética , Discos Imaginais/metabolismo , Mutação , Animais , Animais Geneticamente Modificados , Centrômero/ultraestrutura , Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Metilação de DNA , Drosophila melanogaster , Epigênese Genética , Humanos , Lisina/química , Metionina/química , Camundongos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Fenótipo , RNA-SeqRESUMO
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
Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila, facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Inativação Gênica/fisiologia , Complexos Multiproteicos/metabolismo , Animais , Dimerização , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Piwi-interacting RNAs (piRNAs) constitute a class of small RNAs that bind PIWI proteins and are essential to repress transposable elements in the animal germline, thereby promoting genome stability and maintaining fertility. C. elegans piRNAs (21U RNAs) are transcribed individually from minigenes as precursors that require 5' and 3' processing. This process depends on the PETISCO complex, consisting of four proteins: IFE-3, TOFU-6, PID-3, and ERH-2. We used biochemical and structural biology approaches to characterize the PETISCO architecture and its interaction with RNA, together with its effector proteins TOST-1 and PID-1. These two proteins define different PETISCO functions: PID-1 governs 21U processing, whereas TOST-1 links PETISCO to an unknown process essential for early embryogenesis. Here, we show that PETISCO forms an octameric assembly with each subunit present in two copies. Determination of structures of the TOFU-6/PID-3 and PID-3/ERH-2 subcomplexes, supported by in vivo studies of subunit interaction mutants, allows us to propose a model for the formation of the TOFU-6/PID-3/ERH-2 core complex and its functionality in germ cells and early embryos. Using NMR spectroscopy, we demonstrate that TOST-1 and PID-1 bind to a common surface on ERH-2, located opposite its PID-3 binding site, explaining how PETISCO can mediate different cellular roles.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Elementos de DNA Transponíveis , Células Germinativas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , RNA Interferente Pequeno/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Proteínas Argonautas/metabolismo , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica/genética , Inativação Gênica/fisiologia , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/genética , RNA Interferente Pequeno/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Transcrição Gênica/genéticaRESUMO
Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Inativação Gênica/fisiologia , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Células Germinativas/metabolismo , Proteínas Nucleares/metabolismo , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/metabolismo , RNA Nuclear/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Piwi-interacting RNAs (piRNAs) direct PIWI proteins to transposons to silence them, thereby preserving genome integrity and fertility. The piRNA population can be expanded in the ping-pong amplification loop. Within this process, piRNA-associated PIWI proteins (piRISC) enter a membraneless organelle called nuage to cleave their target RNA, which is stimulated by Gtsf proteins. The resulting cleavage product gets loaded into an empty PIWI protein to form a new piRISC complex. However, for piRNA amplification to occur, the new RNA substrates, Gtsf-piRISC, and empty PIWI proteins have to be in physical proximity. In this study, we show that in silkworm cells, the Gtsf1 homolog BmGtsf1L binds to piRNA-loaded BmAgo3 and localizes to granules positive for BmAgo3 and BmVreteno. Biochemical assays further revealed that conserved residues within the unstructured tail of BmGtsf1L directly interact with BmVreteno. Using a combination of AlphaFold modeling, atomistic molecular dynamics simulations, and in vitro assays, we identified a novel binding interface on the BmVreteno-eTudor domain, which is required for BmGtsf1L binding. Our study reveals that a single eTudor domain within BmVreteno provides two binding interfaces and thereby interconnects piRNA-loaded BmAgo3 and BmGtsf1L.
Assuntos
Bombyx , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Bombyx/genética , Bombyx/metabolismo , RNA de Interação com Piwi , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Domínio TudorRESUMO
In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. We propose that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins.
Assuntos
Elementos de DNA Transponíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , RNA Polimerase II/metabolismo , RNA Guia de Cinetoplastídeos/biossíntese , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/biossíntese , Transcrição Gênica , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Ligação Proteica , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Polimerase II/genética , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltransferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified.
Assuntos
Heterocromatina , RNA de Interação com Piwi , Animais , Masculino , Camundongos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Metilação de DNA , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
The Piwi-interacting RNA (piRNA) pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialized machinery that converts long single-stranded precursors into small RNAs of â¼25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and the correct localization of the indispensable piRNA biogenesis factor Armitage (Armi). We found that Gasz and Daed interact with each other and likely provide a mitochondrial "anchoring platform" to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb bodies in a manner that depends on Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing; namely, how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , RNA Helicases/metabolismo , RNA Interferente Pequeno/biossíntese , Animais , Linhagem Celular , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnicas de Silenciamento de Genes , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/metabolismoRESUMO
The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.