RESUMEN
The nuclear lamina (NL) plays various roles and participates in nuclear integrity, chromatin organization, and transcriptional regulation. Lamin proteins, the main components of the NL, form a homogeneous meshwork structure under the nuclear envelope. Lamins are essential, but it is unknown whether their homogeneous distribution is important for nuclear function. Here, we found that PIGB, an enzyme involved in glycosylphosphatidylinositol (GPI) synthesis, is responsible for the homogeneous lamin meshwork in Drosophila. Loss of PIGB resulted in heterogeneous distributions of B-type lamin and lamin-binding proteins in larval muscles. These phenotypes were rescued by expression of PIGB lacking GPI synthesis activity. The PIGB mutant exhibited changes in lamina-associated domains that are large heterochromatic genomic regions in the NL, reduction of nuclear stiffness, and deformation of muscle fibers. These results suggest that PIGB maintains the homogeneous meshwork of the NL, which may be essential for chromatin distribution and nuclear mechanical properties.
Asunto(s)
Proteínas de Drosophila , Drosophila , Músculo Esquelético , Lámina Nuclear , Animales , Lamina Tipo B/genética , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Lámina Nuclear/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Glicosilfosfatidilinositoles/metabolismoRESUMEN
Telomeres in Drosophila are composed of sequential non-LTR retrotransposons HeT-A, TART and TAHRE. Although they are repressed by the PIWI-piRNA pathway or heterochromatin in the germline, the regulation of these retrotransposons in somatic cells is poorly understood. In this study, we demonstrated that specific splice variants of Mod(mdg4) repress HeT-A by blocking subtelomeric enhancers in ovarian somatic cells. Among the variants, we found that the Mod(mdg4)-N variant represses HeT-A expression the most efficiently. Subtelomeric sequences bound by Mod(mdg4)-N block enhancer activity within subtelomeric TAS-R repeats. This enhancer-blocking activity is increased by the tandem association of Mod(mdg4)-N to repetitive subtelomeric sequences. In addition, the association of Mod(mdg4)-N couples with the recruitment of RNA polymerase II to the subtelomeres, which reinforces its enhancer-blocking function. Our findings provide novel insights into how telomeric retrotransposons are regulated by the specific variants of insulator proteins associated with subtelomeric sequences.
Asunto(s)
Drosophila , Retroelementos , Telómero , Animales , Drosophila/genética , Drosophila/metabolismo , Heterocromatina , Retroelementos/genética , Telómero/genética , Telómero/metabolismo , Elementos de Facilitación GenéticosRESUMEN
Transposable elements (TEs) constitute a large proportion of the genome in multiple organisms. Therefore, anti-transposable element machineries are essential to maintain genomic integrity. PIWI-interacting RNAs (piRNAs) are a major force to repress TEs in Drosophila ovaries. Ovarian somatic cells (OSC), in which nuclear piRNA regulation is functional, have been used for research on piRNA pathway as a cell culture system to elucidate the molecular mechanisms underlying the piRNA pathway. Analysis of piRNA pathway using a reporter system to monitor the gene regulation or overexpression of specific genes would be a powerful approach. Here, we present the technical protocol to establish stable cell lines using the piggyBac system, adopted for OSCs. This easy, consistent, and timesaving protocol may accelerate research on the piRNA pathway.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Línea Celular , Elementos Transponibles de ADN/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Femenino , Ovario/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Bombyx Papi acts as a scaffold for Siwi-piRISC biogenesis on the mitochondrial surface. Papi binds first to Siwi via the Tudor domain and subsequently to piRNA precursors loaded onto Siwi via the K-homology (KH) domains. This second action depends on phosphorylation of Papi. However, the underlying mechanism remains unknown. Here, we show that Siwi targets Par-1 kinase to Papi to phosphorylate Ser547 in the auxiliary domain. This modification enhances the ability of Papi to bind Siwi-bound piRNA precursors via the KH domains. The Papi S547A mutant bound to Siwi, but evaded phosphorylation by Par-1, abrogating Siwi-piRISC biogenesis. A Papi mutant that lacked the Tudor and auxiliary domains escaped coordinated regulation by Siwi and Par-1 and bound RNAs autonomously. Another Papi mutant that lacked the auxiliary domain bound Siwi but did not bind piRNA precursors. A sophisticated mechanism by which Siwi cooperates with Par-1 kinase to promote Siwi-piRISC biogenesis was uncovered.
Asunto(s)
Bombyx , Animales , Bombyx/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Dominio TudorRESUMEN
PIWI-interacting RNAs (piRNAs) bind to PIWI proteins to assemble the piRISC, which represses germline transposons. Maelstrom (Mael) is necessary for piRISC biogenesis in germ cells, but its function remains unclear. Here, we show that Mael interconnects Spindle-E (Spn-E), a key piRISC biogenesis factor, with unloaded Siwi, one of two silkworm PIWI members. Mael also assembles a subset of nuage, a non-membranous organelle involved in piRISC biogenesis. Loss of Mael abrogated the Spn-E-Siwi interaction and Ago3-piRISC biogenesis, but Siwi-piRISC was produced. Bioinformatic analysis showed that Siwi-bound piRNAs in Mael-lacking cells were rich in transposon-targeting piRNAs as in normal cells but were biased toward transposons that are marginally controlled by Siwi-piRISC. This explains the impairment in Ago3-piRISC production because transposon mRNAs cleaved by Siwi are the origin of Ago3-loaded piRNAs. We argue that Mael plays a role in the production of primary Siwi-piRISC capable of regulating transposon expression in germ cells.
RESUMEN
Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CLpro) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CLpro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CLpro revealed that the Kcat/Km of the 3CLpro enzyme containing Ser108 was 58% lower than that of Pro108 3CLpro. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CLpro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CLpro inhibitor.
Asunto(s)
COVID-19 , Proteasas 3C de Coronavirus , Mutación Missense , Gravedad del Paciente , Adulto , Anciano , Sustitución de Aminoácidos , COVID-19/enzimología , COVID-19/genética , Proteasas 3C de Coronavirus/genética , Proteasas 3C de Coronavirus/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Many animals have a conserved adaptive genome defence system known as the Piwi-interacting RNA (piRNA) pathway, which is essential for germ cell development and function. Disruption of individual mouse Piwi genes results in male but not female sterility, leading to the assumption that PIWI genes play little or no role in mammalian oocytes. Here, we report the generation of PIWI-defective golden hamsters, which have defects in the production of functional oocytes. The mechanisms involved vary among the hamster PIWI genes, whereby the lack of PIWIL1 has a major impact on gene expression, including hamster-specific young transposon de-silencing, whereas PIWIL3 deficiency has little impact on gene expression in oocytes, although DNA methylation was reduced to some extent in PIWIL3-deficient oocytes. Our findings serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes, including humans.
Asunto(s)
Mesocricetus/metabolismo , Oocitos/metabolismo , ARN Interferente Pequeño/genética , Testículo/metabolismo , Animales , Proteínas Argonautas/genética , Cricetinae , Metilación de ADN/fisiología , Expresión Génica/fisiología , Células Germinativas/metabolismo , MasculinoRESUMEN
PIWI-interacting RNAs (piRNAs) are germline-specific small RNAs that form effector complexes with PIWI proteins (Piwi-piRNA complexes) and play critical roles for preserving genomic integrity by repressing transposable elements (TEs). Drosophila Piwi transcriptionally silences specific targets through heterochromatin formation and increases histone H3K9 methylation (H3K9me3) and histone H1 deposition at these loci, with nuclear RNA export factor variant Nxf2 serving as a co-factor. Using ChEP and DamID-seq, we now uncover a Piwi/Nxf2-dependent target association with nuclear lamins. Hi-C analysis of Piwi or Nxf2-depleted cells reveals decreased intra-TAD and increased inter-TAD interactions in regions harboring Piwi-piRNA target TEs. Using a forced tethering system, we analyze the functional effects of Piwi-piRNA/Nxf2-mediated recruitment of piRNA target regions to the nuclear periphery. Removal of active histone marks is followed by transcriptional silencing, chromatin conformational changes, and H3K9me3 and H1 association. Our data show that the Piwi-piRNA pathway can induce stepwise changes in nuclear architecture and chromatin state at target loci for transcriptional silencing.
Asunto(s)
Proteínas Argonautas/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica , Sitios Genéticos , ARN Interferente Pequeño/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Drosophila melanogaster , Heterocromatina/genética , Heterocromatina/metabolismo , Unión Proteica , ARN Interferente Pequeño/genéticaRESUMEN
Transposable elements form a major fraction of the genome in various eukaryotic species. Although deleterious effects of transpositions within the genome have been reported, recent findings suggest that transposable elements can function as novel regulatory elements to fine-tune gene expression. Transposable elements can impact the chromatin state through processes such as heterochromatin formation, enhancer-promoter interactions, and chromatin boundary formation, mainly because of the functions of chromatin-based pathways that regulate the expression of transposable elements via DNA methylation and repressive histone modification. Therefore, transposable elements can rewire the chromatin state and gene expression depending on their insertions. Here, we review the findings that reveal the role of transposable elements as modifiers of the chromatin state and gene expression as well as the molecular mechanisms capable of inducing these changes.
Asunto(s)
Cromatina , Elementos Transponibles de ADN , Cromatina/genética , Metilación de ADN , Elementos Transponibles de ADN/genética , Eucariontes , Expresión GénicaRESUMEN
SARS-CoV-2 whole-genome sequencing of samples from COVID-19 patients is useful for informing infection control. Datasets of these genomes assembled from multiple hospitals can give critical clues to regional or national trends in infection. Herein, we report a lineage summary based on data collected from hospitals located in the Tokyo metropolitan area. We performed SARS-CoV-2 whole-genome sequencing of specimens from 198 patients with COVID-19 at 13 collaborating hospitals located in the Kanto region. Phylogenetic analysis and fingerprinting of the nucleotide substitutions were performed to differentiate and classify the viral lineages. More than 90% of the identified strains belonged to Clade 20B, which has been prevalent in European countries since March 2020. Only two lineages (B.1.1.284 and B.1.1.214) were found to be predominant in Japan. However, one sample from a COVID-19 patient admitted to a hospital in the Kanto region in November 2020 belonged to the B.1.346 lineage of Clade 20C, which has been prevalent in the western United States since November 2020. The patient had no history of overseas travel or any known contact with anyone who had travelled abroad. Consequently, the Clade 20C strain belonging to the B.1.346 lineage appeared likely to have been imported from the western United States to Japan across the strict quarantine barrier. B.1.1.284 and B.1.1.214 lineages were found to be predominant in the Kanto region, but a single case of the B.1.346 lineage of clade 20C, probably imported from the western United States, was also identified. These results illustrate that a decentralized network of hospitals offers significant advantages as a highly responsive system for monitoring regional molecular epidemiologic trends.
Asunto(s)
COVID-19/virología , Genoma Viral , SARS-CoV-2/genética , Secuenciación Completa del Genoma/métodos , Humanos , FilogeniaRESUMEN
In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5'-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.
Asunto(s)
Proteínas Argonautas/metabolismo , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/genética , Femenino , Genómica , Masculino , Mesocricetus , Metafase , Fosforilación , ARN Interferente Pequeño/genética , Testículo/metabolismoRESUMEN
COVID-19 caused by SARS-CoV-2 is a worldwide problem. From the standpoint of hospital infection control, determining the source of infection is critical. We conducted the present study to evaluate the efficacy of using whole genome sequencing to determine the source of infection in hospitalized patients who do not have a clear infectious contact history. Recently, we encountered two seemingly separate COVID-19 clusters in a tertiary hospital. Whole viral genome sequencing distinguished the two clusters according to the viral haplotype. However, the source of infection was unclear in 14 patients with COVID-19 who were clinically unlinked to clusters #1 or #2. These patients, who had no clear history of infectious contact within the hospital ("undetermined source of infection"), had haplotypes similar to those in cluster #2 but did not have two of the mutations used to characterize cluster #2, suggesting that these 14 cases of "undetermined source of infection" were not derived from cluster #2. Whole viral genome sequencing can be useful for confirming that sporadic COVID-19 cases with an undetermined source of infection are indeed not part of clusters at the institutional level.
RESUMEN
The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Our results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Silenciador del Gen , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/metabolismo , Femenino , Regulación de la Expresión Génica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/metabolismo , Transcripción GenéticaRESUMEN
The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.
Asunto(s)
Sistema Cardiovascular/metabolismo , Linaje de la Célula , Células Madre Pluripotentes/metabolismo , Somitos/citología , Somitos/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Humanos , Masculino , Mesodermo , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Proteínas de Dominio T Box , Factores de Transcripción/genéticaRESUMEN
PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that bind to PIWI proteins to control transposons and maintain genome integrity in animal germ lines. piRNA 3' end formation in the silkworm Bombyx mori has been shown to be mediated by the 3'-to-5' exonuclease Trimmer (Trim; known as PNLDC1 in mammals), and piRNA intermediates are bound with PIWI anchored onto mitochondrial Tudor domain protein Papi. However, it remains unclear whether the Zucchini (Zuc) endonuclease and Nibbler (Nbr) 3'-to-5' exonuclease, both of which have pivotal roles in piRNA biogenesis in Drosophila, are required for piRNA processing in other species. Here we show that the loss of Zuc in Bombyx had no effect on the levels of Trim and Nbr, but resulted in the aberrant accumulation of piRNA intermediates within the Papi complex, and that these were processed to form mature piRNAs by recombinant Zuc. Papi exerted its RNA-binding activity only when bound with PIWI and phosphorylated, suggesting that complex assembly involves a hierarchical process. Both the 5' and 3' ends of piRNA intermediates within the Papi complex showed hallmarks of PIWI 'slicer' activity, yet no phasing pattern was observed in mature piRNAs. The loss of Zuc did not affect the 5'- and 3'-end formation of the intermediates, strongly supporting the idea that the 5' end of Bombyx piRNA is formed by PIWI slicer activity, but independently of Zuc, whereas the 3' end is formed by the Zuc endonuclease. The Bombyx piRNA biogenesis machinery is simpler than that of Drosophila, because Bombyx has no transcriptional silencing machinery that relies on phased piRNAs.
Asunto(s)
Bombyx/citología , Bombyx/genética , Endorribonucleasas/metabolismo , Células Germinativas/metabolismo , Proteínas Mitocondriales/metabolismo , ARN Interferente Pequeño/biosíntesis , Animales , Proteínas Argonautas/metabolismo , Drosophila , ARN Interferente Pequeño/genéticaRESUMEN
The assay for transposase-accessible chromatin using sequencing (ATAC-seq) was recently established as a method to profile open chromatin, which overcomes the sample size limitations of the alternative methods DNase/MNase-seq. To investigate the role of Piwi in heterochromatin formation around transposable element loci, we have used ATAC-seq to examine chromatin accessibility at target transposable elements in a Drosophila cultured cell line, ovarian somatic cells (OSCs). In this chapter, we describe our method to profile open chromatin structure in OSCs using ATAC-seq.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Ovario/citología , Análisis de Secuencia de ADN , Animales , Biología Computacional/métodos , Elementos Transponibles de ADN , Drosophila , Femenino , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , ARN Pequeño no Traducido , Análisis de Secuencia de ADN/métodosRESUMEN
Small RNAs are now known to be major regulatory factors of gene expression. Emerging methods based on deep-sequencing have enabled the analysis of small RNA expression in a high-throughput manner, leading to the identification of large numbers of small RNAs in various species. Moreover, profiling small RNA data together with transcriptome data enables transcriptional and post-transcriptional regulation mediated by small RNAs to be hypothesized. Here, we isolated PIWIL1 (MIWI)-associated small RNAs from mouse testes, and performed small RNA-seq analysis. In addition, directional RNA-seq was performed using Piwil1 mutant mouse testes. Using these data, we describe protocols for analyzing small RNA-seq reads to obtain profiles of small RNAs associated with PIWI proteins. We also present bioinformatic protocols for analyzing RNA-seq reads that aim to annotate expression of piRNA clusters and identify genes regulated by piRNAs.
Asunto(s)
Proteínas Argonautas/análisis , Proteínas Argonautas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Interferente Pequeño/análisis , ARN Interferente Pequeño/genética , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia de ARN/métodosRESUMEN
PIWI-interacting RNAs (piRNAs) mediate transcriptional and post-transcriptional silencing of transposable element (TE) in animal gonads. In Drosophila ovaries, Piwi-piRNA complexes (Piwi-piRISCs) repress TE transcription by modifying the chromatin state, such as by H3K9 trimethylation. Here, we demonstrate that Piwi physically interacts with linker histone H1. Depletion of Piwi decreases H1 density at a subset of TEs, leading to their derepression. Silencing at these loci separately requires H1 and H3K9me3 and heterochromatin protein 1a (HP1a). Loss of H1 increases target loci chromatin accessibility without affecting H3K9me3 density at these loci, while loss of HP1a does not impact H1 density. Thus, Piwi-piRISCs require both H1 and HP1a to repress TEs, and the silencing is correlated with the chromatin state rather than H3K9me3 marks. These findings suggest that Piwi-piRISCs regulate the interaction of chromatin components with target loci to maintain silencing of TEs through the modulation of chromatin accessibility.
Asunto(s)
Proteínas Argonautas/metabolismo , Ensamble y Desensamble de Cromatina , Elementos Transponibles de ADN , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Represión Epigenética , Histonas/metabolismo , Ovario/metabolismo , Animales , Proteínas Argonautas/genética , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Histonas/genética , Ovario/citología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , TransfecciónRESUMEN
In Drosophila germ cells, PIWI-interacting RNAs (piRNAs) are amplified through a PIWI slicer-dependent feed-forward loop termed the ping-pong cycle, yielding secondary piRNAs. However, the detailed mechanism remains poorly understood, largely because an ex vivo model system amenable to biochemical analyses has not been available. Here, we show that CRISPR-mediated loss of function of lethal (3) malignant brain tumor [l(3)mbt] leads to ectopic activation of the germ-specific ping-pong cycle in ovarian somatic cells. Perinuclear foci resembling nuage, the ping-pong center, appeared following l(3)mbt mutation. This activation of the ping-pong machinery in cultured cells will greatly facilitate elucidation of the mechanism underlying secondary piRNA biogenesis in Drosophila.