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Haifan Lin - One of the best experts on this subject based on the ideXlab platform.
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maternal Piwi regulates primordial germ cell development to ensure the fertility of female progeny in drosophila
bioRxiv, 2021Co-Authors: Lauren E Gonzalez, Xiongzhuo Tang, Haifan LinAbstract:ABSTRACT In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. Piwi proteins and their associated small noncoding Piwi-interacting RNAs (piRNAs), which guide Piwi to target RNAs by base-pairing, are among the maternal components deposited into the germline of the early embryo in Drosophila. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using Piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal Piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the Piwi/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal Piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the Piwi/piRNA pathway is involved in germline sex determination in Drosophila.
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heat shock protein dnaja1 stabilizes Piwi proteins to support regeneration and homeostasis of planarian schmidtea mediterranea
Journal of Biological Chemistry, 2019Co-Authors: Haifan Lin, Chen Wang, Zhenzhen Yang, Fanghao Guo, Shuo Shi, Xiaoshuai Han, An Zeng, Qing JingAbstract:Piwi proteins are key regulators of germline and somatic stem cells throughout different evolutionary lineages. However, how Piwi proteins themselves are regulated remains largely unknown. To identify candidate proteins that interact with Piwi proteins and regulate their stability, here we established a yeast two-hybrid (Y2H) assay in the planarian species Schmidtea mediterranea We show that DNAJA1, a heat shock protein 40 family member, interacts with the Piwi protein SMEDWI-2, as validated by the Y2H screen and co-immunoprecipitation assays. We found that DNAJA1 is enriched in planarian adult stem cells, the nervous system, and intestinal tissues. DNAJA1-knockdown abolished planarian regeneration and homeostasis, compromised stem cell maintenance and Piwi-interacting RNA (piRNA) biogenesis, and deregulated SMEDWI-1/2 target genes. Mechanistically, we observed that DNAJA1 is required for the stability of SMEDWI-1 and SMEDWI-2 proteins. Furthermore, we noted that human DNAJA1 binds to Piwi-like RNA-mediated gene silencing 1 (PiwiL1) and is required for PiwiL1 stability in human gastric cancer cells. In summary, our results reveal not only an evolutionarily conserved functional link between Piwi and DNAJA1 that is essential for Piwi protein stability and piRNA biogenesis, but also an important role of DNAJA1 in the control of proteins involved in stem cell regulation.
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a critical role for nucleoporin 358 nup358 in transposon silencing and pirna biogenesis in drosophila
Journal of Biological Chemistry, 2018Co-Authors: Rasesh Y Parikh, Haifan Lin, Vamsi K. GangarajuAbstract:Piwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)–dependent ping-pong cycle generates most germline piRNAs. Loading of antisense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. Here, we show that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. Our results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing.
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Posttranscriptional regulation of gene expression by Piwi proteins and piRNAs.
Molecular Cell, 2014Co-Authors: Toshiaki Watanabe, Haifan LinAbstract:Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for gametogenesis, embryogenesis, and stem cell maintenance in animals. Piwi proteins act on transposon RNAs by cleaving the RNAs and by interacting with factors involved in RNA regulation. Additionally, piRNAs generated from transposons and psuedogenes can be used by Piwi proteins to regulate mRNAs at the posttranscriptional level. Here we discuss piRNA biogenesis, recent findings on posttranscriptional regulation of mRNAs by the piRNA pathway, and the potential importance of this posttranscriptional regulation for a variety of biological processes such as gametogenesis, developmental transitions, and sex determination.
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Piwi proteins and Piwi-interacting RNAs function in Hydra somatic stem cells
Proceedings of the National Academy of Sciences, 2013Co-Authors: Celina E. Juliano, Na Liu, Adrian Reich, Jessica Götzfried, Mei Zhong, Selen Uman, Robert A. Reenan, Gary M. Wessel, Robert Steele, Haifan LinAbstract:Piwi proteins and their bound Piwi-interacting RNAs (piRNAs) are found in animal germlines and are essential for fertility, but their functions outside of the gonad are not well understood. The cnidarian Hydra is a simple metazoan with well-characterized stem/progenitor cells that provides a unique model for analysis of Piwi function. Here we report that Hydra has two Piwi proteins, Hydra Piwi (Hywi) and Hydra Piwi-like (Hyli), both of which are expressed in all Hydra stem/progenitor cells, but not in terminally differentiated cells. We identified ∼15 million piRNAs associated with Hywi and/or Hyli and found that they exhibit the ping-pong signature of piRNA biogenesis. Hydra Piwi proteins are strictly cytoplasmic and thus likely act as posttranscriptional regulators. To explore this function, we generated a Hydra transcriptome for piRNA mapping. piRNAs map to transposons with a 25- to 35-fold enrichment compared with the abundance of transposon transcripts. By sequencing the small RNAs specific to the interstitial, ectodermal, and endodermal lineages, we found that the targeting of transposons appears to be largely restricted to the interstitial lineage. We also identified putative nontransposon targets of the pathway unique to each lineage. Finally we demonstrate that hywi function is essential in the somatic epithelial lineages. This comprehensive analysis of the Piwi–piRNA pathway in the somatic stem/progenitor cells of a nonbilaterian animal suggests that this pathway originated with broader stem cell functionality.
Kuniaki Saito - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of Drosophila Piwi.
Nature Communications, 2020Co-Authors: Sonomi Yamaguchi, Naoki Matsumoto, Kazumichi M. Nishida, Ryuichiro Ishitani, Keitaro Yamashita, Asako Kajiya, Seiichi Hirano, Naoshi Dohmae, Kuniaki SaitoAbstract:Piwi-clade Argonaute proteins associate with Piwi-interacting RNAs (piRNAs), and silence transposons in animal gonads. Here, we report the crystal structure of the Drosophila Piwi-clade Argonaute Piwi in complex with endogenous piRNAs, at 2.9 A resolution. A structural comparison of Piwi with other Argonautes highlights the Piwi-specific structural features, such as the overall domain arrangement and metal-dependent piRNA recognition. Our structural and biochemical data reveal that, unlike other Argonautes including silkworm Siwi, Piwi has a non-canonical DVDK tetrad and lacks the RNA-guided RNA cleaving slicer activity. Furthermore, we find that the Piwi mutant with the canonical DEDH catalytic tetrad exhibits the slicer activity and readily dissociates from less complementary RNA targets after the slicer-mediated cleavage, suggesting that the slicer activity could compromise the Piwi-mediated co-transcriptional silencing. We thus propose that Piwi lost the slicer activity during evolution to serve as an RNA-guided RNA-binding platform, thereby ensuring faithful co-transcriptional silencing of transposons. Piwi-clade Argonautes bind Piwi-interacting RNAs (piRNAs) to silence transposons and maintain genome integrity in animal gonads. Here the authors present the crystal structure of a Drosophila Piwi–piRNA complex isolated from cultured fly ovarian somatic cells and find that it contains a non-canonical DVDK tetrad, and lacks slicer activity in vitro.
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nuclear rna export factor variant initiates pirna guided co transcriptional silencing
The EMBO Journal, 2019Co-Authors: Kensaku Murano, Yuka W Iwasaki, Hirotsugu Ishizu, Aoi Shibuya, Akane Mashiko, Shu Kondo, Shungo Adachi, Saori Suzuki, Kuniaki SaitoAbstract: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.
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nuclear rna export factor variant initiates pirna guided co transcriptional silencing
bioRxiv, 2019Co-Authors: Kensaku Murano, Yuka W Iwasaki, Hirotsugu Ishizu, Aoi Shibuya, Akane Mashiko, Shu Kondo, Shungo Adachi, Saori Suzuki, Kuniaki SaitoAbstract:Summary 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 Nxf2-Panx-p15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Highlights Nxf2 plays an essential role in the Piwi–piRNA pathway Formation of Piwi-Panx-Nxf2-p15 (PPNP) complexes stabilizes both Panx and Nxf2 The PPNP complex triggers transcriptional silencing before heterochromatin formation Nxf2 directly binds to target transcripts in a Piwi-dependent manner
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Piwi modulates chromatin accessibility by regulating multiple factors including histone h1 to repress transposons
Molecular Cell, 2016Co-Authors: Yuka W Iwasaki, Haruhiko Siomi, Mikiko C. Siomi, Hirotsugu Ishizu, Aoi Shibuya, Kensaku Murano, Yumiko Iyoda, Kuniaki SaitoAbstract: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.
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small rna profiling and characterization of pirna clusters in the adult testes of the common marmoset a model primate
RNA, 2014Co-Authors: Takamasa Hirano, Yuka W Iwasaki, Masanori Imamura, Naomi Seki, Erika Sasaki, Kuniaki Saito, Hideyuki OkanoAbstract:Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant Piwi in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.
Bernd Wullich - One of the best experts on this subject based on the ideXlab platform.
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Piwi like 1 protein expression is a prognostic factor for renal cell carcinoma patients
Scientific Reports, 2019Co-Authors: Christine Stohr, Rudolf Jung, Arndt Hartmann, Bernd Wullich, Sandra Steffens, Iris Polifka, Andreas Kahlmeyer, Philipp Ivanyi, Florian Weber, Sven WachAbstract:The Piwi-like genes belong to the Argonaute gene family and are conserved in plants, animals and humans. In addition to their essential role in the germ line and as stem cell-associated genes, Piwi-like proteins play a role in different cancer types but have yet to be studied in renal cell carcinoma (RCC). We investigated tissue micro arrays (TMAs) with tumor samples of two independent cohorts of RCC patients (N = 265 and N = 345); we used immunohistochemistry to assess the protein expression of Piwi-like 1. Applying an immunoreactive score (IRS), we found Piwi-like 1 positivity (IRS > 0) in 28.3% and 14.8% of the tumors in cohorts 1 and 2, respectively. Piwi-like 1 positivity was correlated with Fuhrman grade, tumor stage and the presence of distant metastasis (P < 0.005). Moreover, in univariate and multivariate analyses (adjusted to Fuhrman grade and tumor stage), Piwi-like 1 positivity was associated with a shorter cancer-specific survival in the patients in the second cohort. In addition, Piwi-like 1 expression allowed to further distinguish the RCC patients with high Fuhrman grade, high tumor stage, distant metastasis or high pre-operative levels of C-reactive protein, as Piwi-like 1 positivity was associated with a shorter cancer-specific survival in both cohorts. Our data encourage further investigations to enlighten the role of Piwi-like 1 and its function as a marker of poor prognosis in RCC patients.
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Piwi like 1 and 2 protein expression levels are prognostic factors for muscle invasive urothelial bladder cancer patients
Scientific Reports, 2018Co-Authors: Markus Eckstein, Rudolf Jung, Katrin Weigelt, Danijel Sikic, Robert Stohr, Carol Geppert, Abbas Agaimy, Verena Lieb, Arndt Hartmann, Bernd WullichAbstract:Piwi-like proteins are essential for stem-cell maintenance and self-renewal in multicellular organisms. We analyzed the expression of Piwi-like 1 and Piwi-like 2 by immunohistochemistry (IHC) in 95 muscle invasive bladder cancer (MIBC) samples using tissue microarray. Application of an immunoreactive score (IRS) revealed 37 and 45 patients who were Piwi-like 1 and -2 positive (IRS > 2). IHC results were correlated with clinico-pathological and survival data. The expression of both proteins was positively correlated with each other, lymph node metastasis and expression of CK20 and GATA 3. A negative correlation for both proteins was detected for disease-specific survival (DSS), recurrence, Ki67/MIB1 proliferation index, and CK5 expression. Detection of Piwi-like 1 protein positivity was associated with poor DSS (P = 0.019; log rank test, Kaplan-Meier analysis), and in multivariate Cox’s analysis (adjusted to tumor stage and tumor grade), it was an independent prognostic factor for DSS (RR = 2.16; P = 0.011). Piwi-like 2 positivity was associated with DSS (P = 0.008) and recurrence-free survival (RFS; P = 0.040), and in multivariate Cox’s analysis, Piwi-like 2 positivity was an independent prognostic factor for DSS (RR = 2.46; P = 0.004) and RFS (RR = 3.0; P = 0.003). Most interestingly, in the basal type patient subgroup (CK5+/GATA3−), Piwi-like 2 positivity was associated with poorer DSS, OS and RFS (P < 0.001, P = 0.004 and P = 0.05; log rank test). In multivariate analysis, Piwi-like 2 positivity was an independent prognostic factor for DSS (RR = 12.70; P = 0.001), OS (RR = 6.62; = 0.008) and RFS (RR=13.0; P = 0.040). In summary, Piwi-like 1 and -2 positivity are associated with clinico-pathological factors and survival. Both Piwi-like proteins are suggested as biomarkers for MIBC patients.
Alexei A Aravin - One of the best experts on this subject based on the ideXlab platform.
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Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state
Genes & Development, 2013Co-Authors: Adrien Le Thomas, Alexei A Aravin, Alicia K. Rogers, Alexandre Webster, Georgi K. Marinov, Susan E. Liao, Edward M. Perkins, Junho K. Hur, Katalin Fejes TóthAbstract:In the metazoan germline, Piwi proteins and associated Piwi-interacting RNAs (piRNAs) provide a defense system against the expression of transposable elements. In the cytoplasm, piRNA sequences guide Piwi complexes to destroy complementary transposon transcripts by endonucleolytic cleavage. However, some Piwi family members are nuclear, raising the possibility of alternative pathways for piRNA-mediated regulation of gene expression. We found that Drosophila Piwi is recruited to chromatin, colocalizing with RNA polymerase II (Pol II) on polytene chromosomes. Knockdown of Piwi in the germline increases expression of transposable elements that are targeted by piRNAs, whereas protein-coding genes remain largely unaffected. Derepression of transposons upon Piwi depletion correlates with increased occupancy of Pol II on their promoters. Expression of piRNAs that target a reporter construct results in a decrease in Pol II occupancy and an increase in repressive H3K9me3 marks and heterochromatin protein 1 (HP1) on the reporter locus. Our results indicate that Piwi identifies targets complementary to the associated piRNA and induces transcriptional repression by establishing a repressive chromatin state when correct targets are found.
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proteomic analysis of murine Piwi proteins reveals a role for arginine methylation in specifying interaction with tudor family members
Genes & Development, 2009Co-Authors: Vasily V Vagin, James A Wohlschlegel, Zophonias O Jonsson, Xinhua Huang, Shinichiro Chuma, Angelique Girard, Alexei A AravinAbstract: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.
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Developmentally Regulated piRNA Clusters Implicate MILI in Transposon Control
Science (New York N.Y.), 2007Co-Authors: Alexei A Aravin, Angelique Girard, Ravi Sachidanandam, Katalin Fejes-toth, Gregory J HannonAbstract:Nearly half of the mammalian genome is composed of repeated sequences. In Drosophila, Piwi proteins exert control over transposons. However, mammalian Piwi proteins, MIWI and MILI, partner with Piwi-interacting RNAs (piRNAs) that are depleted of repeat sequences, which raises questions about a role for mammalian Piwi's in transposon control. A search for murine small RNAs that might program Piwi proteins for transposon suppression revealed developmentally regulated piRNA loci, some of which resemble transposon master control loci of Drosophila. We also find evidence of an adaptive amplification loop in which MILI catalyzes the formation of piRNA 5' ends. Mili mutants derepress LINE-1 (L1) and intracisternal A particle and lose DNA methylation of L1 elements, demonstrating an evolutionarily conserved role for Piwi proteins in transposon suppression.
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a novel class of small rnas bind to mili protein in mouse testes
Nature, 2006Co-Authors: Alexei A Aravin, Dimos Gaidatzis, Sebastien Pfeffer, Mariana Lagosquintana, Pablo Landgraf, Nicola IovinoAbstract:Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes. A subgroup of the Argonaute proteins--known as the 'Piwi family'--is required for germ- and stem-cell development in invertebrates, and two Piwi members--MILI and MIWI--are essential for spermatogenesis in mouse. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5' uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26-31 nucleotides (nt) in length--clearly distinct from the 21-23 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)--and we refer to them as 'Piwi-interacting RNAs' or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis.
Yuka W Iwasaki - One of the best experts on this subject based on the ideXlab platform.
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nuclear rna export factor variant initiates pirna guided co transcriptional silencing
The EMBO Journal, 2019Co-Authors: Kensaku Murano, Yuka W Iwasaki, Hirotsugu Ishizu, Aoi Shibuya, Akane Mashiko, Shu Kondo, Shungo Adachi, Saori Suzuki, Kuniaki SaitoAbstract: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.
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nuclear rna export factor variant initiates pirna guided co transcriptional silencing
bioRxiv, 2019Co-Authors: Kensaku Murano, Yuka W Iwasaki, Hirotsugu Ishizu, Aoi Shibuya, Akane Mashiko, Shu Kondo, Shungo Adachi, Saori Suzuki, Kuniaki SaitoAbstract:Summary 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 Nxf2-Panx-p15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Highlights Nxf2 plays an essential role in the Piwi–piRNA pathway Formation of Piwi-Panx-Nxf2-p15 (PPNP) complexes stabilizes both Panx and Nxf2 The PPNP complex triggers transcriptional silencing before heterochromatin formation Nxf2 directly binds to target transcripts in a Piwi-dependent manner
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Piwi modulates chromatin accessibility by regulating multiple factors including histone h1 to repress transposons
Molecular Cell, 2016Co-Authors: Yuka W Iwasaki, Haruhiko Siomi, Mikiko C. Siomi, Hirotsugu Ishizu, Aoi Shibuya, Kensaku Murano, Yumiko Iyoda, Kuniaki SaitoAbstract: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.
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small rna profiling and characterization of pirna clusters in the adult testes of the common marmoset a model primate
RNA, 2014Co-Authors: Takamasa Hirano, Yuka W Iwasaki, Masanori Imamura, Naomi Seki, Erika Sasaki, Kuniaki Saito, Hideyuki OkanoAbstract:Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant Piwi in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.
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dmgtsf1 is necessary for Piwi pirisc mediated transcriptional transposon silencing in the drosophila ovary
Genes & Development, 2013Co-Authors: Hitoshi Ohtani, Haruhiko Siomi, Mikiko C. Siomi, Yuka W Iwasaki, Aoi Shibuya, Kuniaki SaitoAbstract:The Piwi-piRNA (Piwi-interacting RNA) complex (Piwi-piRISC) in Drosophila ovarian somatic cells represses transposons transcriptionally to maintain genome integrity; however, the underlying mechanisms remain obscure. Here, we reveal that DmGTSF1, a Drosophila homolog of gametocyte-specific factor 1 (GTSF1) (which is required for transposon silencing in mouse testes), is necessary for Piwi-piRISC to repress target transposons and neighboring genes. DmGTSF1 depletion affected neither piRNA biogenesis nor nuclear import of Piwi-piRISC. DmGTSF1 mutations caused derepression of transposons and loss of ovary follicle layers, resulting in female infertility. We suggest that DmGTSF1, a nuclear Piwi interactor, is an integral factor in Piwi-piRISC-mediated transcriptional silencing.