RESUMO
Piwi-interacting RNAs (piRNAs) silence transposons in animal germ cells. piRNAs are thought to derive from long transcripts spanning transposon-rich genomic loci and to direct an autoamplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, triggers production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to produce a second antisense piRNA. Here, we describe strong loss-of-function mutations in ago3, allowing a direct genetic test of this model. We find that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. We also detect a second, Ago3-independent piRNA pathway centered on Piwi. Transposons targeted by this second pathway often reside in the flamenco locus, which is expressed in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germline.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Folículo Ovariano/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Retroelementos , Animais , Proteínas Argonautas , Feminino , Inativação Gênica , Mutação , RNA Interferente Pequeno/metabolismoRESUMO
PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAsprimary and secondaryare defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction.
Assuntos
Proteínas Argonautas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Testículo/fisiopatologia , Fatores de Transcrição/metabolismo , Animais , Proteínas Argonautas/genética , Elementos de DNA Transponíveis/genética , Técnicas de Inativação de Genes , Masculino , Meiose/genética , Camundongos , Mutação , RNA Interferente Pequeno/metabolismo , Testículo/metabolismo , Fatores de Transcrição/genéticaRESUMO
The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Conectoma/métodos , Hipocampo/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Neurônios/metabolismo , RNA/genética , Animais , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular Neuronais/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Hipocampo/citologia , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/citologia , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase/métodos , Cultura Primária de Células , RNA/metabolismo , Sindbis virus/genética , Sindbis virus/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Transfecção , Proteína Vermelha FluorescenteRESUMO
In germ cells, Piwi proteins interact with a specific class of small noncoding RNAs, piwi-interacting RNAs (piRNAs). Together, these form a pathway that represses transposable elements, thus safeguarding germ cell genomes. Basic models describe the overall operation of piRNA pathways. However, the protein compositions of Piwi complexes, the critical protein-protein interactions that drive small RNA production and target recognition, and the precise molecular consequences of conserved localization to germline structures, call nuage, remains poorly understood. We purified the three murine Piwi family proteins, MILI, MIWI, and MIWI2, from mouse germ cells and characterized their interacting protein partners. Piwi proteins were found in complex with PRMT5/WDR77, an enzyme that dimethylates arginine residues. By immunoprecipitation with specific antibodies and by mass spectrometry, we found that Piwi proteins are arginine methylated at conserved positions in their N termini. These modifications are essential to direct complex formation with specific members of the Tudor protein family. Recognition of methylarginine marks by Tudor proteins can drive the localization of Piwi proteins to cytoplasmic foci in an artificial setting, supporting a role for this interaction in Piwi localization to nuage, a characteristic that correlates with proper operation of the piRNA pathway and transposon silencing in multiple organisms.
Assuntos
Arginina/metabolismo , Proteínas/metabolismo , Ribonucleoproteínas/metabolismo , Testículo/metabolismo , Animais , Proteínas Argonautas , Proteínas de Ciclo Celular , Linhagem Celular , Elementos de DNA Transponíveis/fisiologia , Humanos , Masculino , Metilação , Camundongos , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases , Proteínas/isolamento & purificação , Proteômica , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells.
Assuntos
Grânulos Citoplasmáticos/fisiologia , Células Germinativas/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermátides/metabolismo , Espermatogênese/fisiologia , Animais , Biomarcadores/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Imunofluorescência , Perfilação da Expressão Gênica , Células Germinativas/ultraestrutura , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermátides/ultraestruturaRESUMO
Piwi proteins and their associated small RNAs are essential for fertility in animals. In part, this is due to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and, as such, form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , RNA Interferente Pequeno/biossíntese , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Domínio Catalítico , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Genes de Insetos , Glicerol-3-Fosfato O-Aciltransferase/genética , Masculino , Dados de Sequência Molecular , Mutação , Fosfolipídeos/metabolismo , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
In the male germline in mammals, chromatoid bodies, a specialized assembly of cytoplasmic ribonucleoprotein (RNP), are structurally evident during meiosis and haploidgenesis, but their developmental origin and regulation remain elusive. The tudor domain containing proteins constitute a conserved class of chromatoid body components. We show that tudor domain containing 7 (Tdrd7), the deficiency of which causes male sterility and age-related cataract (as well as glaucoma), is essential for haploid spermatid development and defines, in concert with Tdrd6, key biogenesis processes of chromatoid bodies. Single and double knockouts of Tdrd7 and Tdrd6 demonstrated that these spermiogenic tudor genes orchestrate developmental programs for ordered remodeling of chromatoid bodies, including the initial establishment, subsequent RNP fusion with ubiquitous processing bodies/GW bodies and later structural maintenance. Tdrd7 suppresses LINE1 retrotransposons independently of piwi-interacting RNA (piRNA) biogenesis wherein Tdrd1 and Tdrd9 operate, indicating that distinct Tdrd pathways act against retrotransposons in the male germline. Tdrd6, in contrast, does not affect retrotransposons but functions at a later stage of spermiogenesis when chromatoid bodies exhibit aggresome-like properties. Our results delineate that chromatoid bodies assemble as an integrated compartment incorporating both germline and ubiquitous features as spermatogenesis proceeds and that the conserved tudor family genes act as master regulators of this unique RNP remodeling, which is genetically linked to the male germline integrity in mammals.
Assuntos
Cromatina/metabolismo , Ribonucleoproteínas/metabolismo , Espermatogênese , Animais , Cromossomos Artificiais Bacterianos , Masculino , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiologiaRESUMO
Derepression of transposable elements (TEs) in the course of epigenetic reprogramming of the mouse embryonic germline necessitates the existence of a robust defense that is comprised of PIWI/piRNA pathway and de novo DNA methylation machinery. To gain further insight into biogenesis and function of piRNAs, we studied the intracellular localization of piRNA pathway components and used the combination of genetic, molecular, and cell biological approaches to examine the performance of the piRNA pathway in germ cells of mice lacking Maelstrom (MAEL), an evolutionarily conserved protein implicated in transposon silencing in fruit flies and mice. Here we show that principal components of the fetal piRNA pathway, MILI and MIWI2 proteins, localize to two distinct types of germinal cytoplasmic granules and exhibit differential association with components of the mRNA degradation/translational repression machinery. The first type of granules, pi-bodies, contains the MILI-TDRD1 module of the piRNA pathway and is likely equivalent to the enigmatic "cementing material" first described in electron micrographs of rat gonocytes over 35 years ago. The second type of granules, piP-bodies, harbors the MIWI2-TDRD9-MAEL module of the piRNA pathway and signature components of P-bodies, GW182, DCP1a, DDX6/p54, and XRN1 proteins. piP-bodies are found predominantly in the proximity of pi-bodies and the two frequently share mouse VASA homolog (MVH) protein, an RNA helicase. In Mael-mutant gonocytes, MIWI2, TDRD9, and MVH are lost from piP-bodies, whereas no effects on pi-body composition are observed. Further analysis revealed that MAEL appears to specifically facilitate MIWI2-dependent aspects of the piRNA pathway including biogenesis of secondary piRNAs, de novo DNA methylation, and efficient downregulation of TEs. Cumulatively, our data reveal elaborate cytoplasmic compartmentalization of the fetal piRNA pathway that relies on MAEL function.
Assuntos
Compartimento Celular , Citoplasma/metabolismo , Feto/metabolismo , RNA/metabolismo , Animais , Ciclo Celular , Grânulos Citoplasmáticos/metabolismo , Metilação de DNA , Elementos de DNA Transponíveis , Células Germinativas/metabolismo , Camundongos , Processamento Pós-Transcricional do RNARESUMO
Here we show that RNA interference (RNAi) machinery operates in Drosophila melanogaster 1.688 satellite transcription. Mutation in the spn-E gene, known to be involved in RNAi in the oocytes, causes an increase of satellite transcript abundance. Transcripts of both strands of 1.688 satellite repeats in germinal tissues were detected. The strength of the effects of the spn-E mutation differs for 1.688 satellite DNA subfamilies and is more pronounced for autosomal pericentromeric satellites compared to the X-linked centromeric ones. The spn-E(1) mutation causes an increase of the H3-AcK9 mark and TAF1 (a component of the polymerase II transcriptional complex) occupancy in the chromatin of autosomal pericentromeric repeats. Thus, we revealed that RNAi operates in ovaries to maintain the silenced state of centromeric and pericentromeric 1.688 repeats.
Assuntos
DNA Satélite/genética , Drosophila melanogaster/genética , Ovário/fisiologia , Interferência de RNA/fisiologia , Transcrição Gênica , Animais , Cromatina/genética , Cromatina/ultraestrutura , Clonagem Molecular , Primers do DNA , Feminino , Mutação , Oócitos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
microRNAs (miRNAs) are single-stranded, 21- to 23-nucleotide cellular RNAs that control the expression of cognate target genes. Primary miRNA (pri-miRNA) transcripts are transformed to mature miRNA by the successive actions of two RNase III endonucleases. Drosha converts pri-miRNA transcripts to precursor miRNA (pre-miRNA); Dicer, in turn, converts pre-miRNA to mature miRNA. Here, we show that normal processing of Drosophila pre-miRNAs by Dicer-1 requires the double-stranded RNA-binding domain (dsRBD) protein Loquacious (Loqs), a homolog of human TRBP, a protein first identified as binding the HIV trans-activator RNA (TAR). Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs(f00791) mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis.
Assuntos
Drosophila melanogaster/fisiologia , Células Germinativas/fisiologia , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células-Tronco/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Feminino , Masculino , Dados de Sequência Molecular , RNA Helicases/metabolismo , Interferência de RNA/fisiologia , Proteínas de Ligação a RNA/genética , Ribonuclease III/metabolismoRESUMO
RNA interference (RNAi) is considered as a defense against expansion of transposable elements. The proteins related to RNA helicase and Argonaute families are involved in RNAi process in different organisms. It was shown that Argonaute AUBERGINE and putative RNA helicase SPINDLE-E proteins were essential for RNAi in Drosophila. Here, we describe the role of aubergine (aub) and spindle-E (spn-E) genes in the control of LTR retrotransposon copia and nonLTR telomeric Het-A and I retrotransposons in ovaries. spn-E mutation causes a drastically increased lacZ expression driven by copia LTR. For the first time we show the involvement of AUBERGINE protein and VASA RNA helicase, essential for oocyte patterning, in the retrotransposon silencing. spn-E, vasa and aub mutations cause similar accumulation of both I element and Het-A transcripts in the developing oocyte. VASA and AUBERGINE proteins are known as components of perinuclear ribonucleoprotein particles in germ cells, and spn-E mutation disturbs protein content of the particles. We suggest participation of these proteins in the same silencing pathway.
Assuntos
RNA Helicases DEAD-box/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Inativação Gênica , Fatores de Iniciação de Peptídeos/fisiologia , Interferência de RNA , Retroelementos/genética , Animais , RNA Helicases DEAD-box/fisiologia , Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Drosophila melanogaster , Feminino , Óperon Lac , Mutação , Ovário/metabolismo , RNA Helicases/química , Sequências Repetidas TerminaisRESUMO
In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. Our data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways.