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Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci

ABSTRACT: 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.

SUBMITTER: Dr. Yuka, W Iwasaki

PROVIDER: S-SCDT-EMBOJ-2021-108345 | biostudies-other |

REPOSITORIES: biostudies-other

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Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci.

Project description: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.

| S-EPMC8441340 | biostudies-literature

Piwi–piRNA complexes induce stepwise changes in nuclear architecture at target loci

Project description:PIWI-interacting RNAs (piRNAs) are germline-specific small RNAs that form effector complexes with PIWI proteins (Piwi–piRNA complexes), and plays a critical role for preserving genomic integrity by repressing transposable elements (TEs). Drosophila Piwi transcriptionally silences specific targets through heterochromatin formation, and increases H3K9me3 and histone H1. Additionally, Nxf2, a nuclear RNA export factor variant, has been identified as a co-factor. Using ChEP and DamID-seq, we uncover 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 interaction 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.

2021-07-01 | GSE158082 | GEO

Piwi–piRNA complexes induce stepwise changes in nuclear architecture at target loci

Project description:Piwi–piRNA complexes induce stepwise changes in nuclear architecture at target loci

| PRJNA663907 | ENA

Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity.

Project description:To test the directness of factors in initiating PIWI-directed gene silencing, we employed a Piwi-interacting RNA (piRNA)-targeted reporter assay in Drosophila ovary somatic sheet (OSS) cells [1]. This assay confirmed direct silencing roles for piRNA biogenesis factors and PIWI-associated factors [2-12] but suggested that chromatin-modifying proteins may act downstream of the initial silencing event. Our data also revealed that RNA-polymerase-II-associated proteins like PAF1 and RTF1 antagonize PIWI-directed silencing. PAF1 knockdown enhances PIWI silencing of reporters when piRNAs target the transcript region proximal to the promoter. Loss of PAF1 suppresses endogenous transposable element (TE) transcript maturation, whereas a subset of gene transcripts and long-non-coding RNAs adjacent to TE insertions are affected by PAF1 knockdown in a similar fashion to piRNA-targeted reporters. Additionally, transcription activation at specific TEs and TE-adjacent loci during PIWI knockdown is suppressed when PIWI and PAF1 levels are both reduced. Our study suggests a mechanistic conservation between fission yeast PAF1 repressing AGO1/small interfering RNA (siRNA)-directed silencing [13, 14] and Drosophila PAF1 opposing PIWI/piRNA-directed silencing.

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Exosomes/microvesicles target SARS-CoV-2 via innate and RNA-induced immunity with PIWI-piRNA system.

Project description:Murine neural stem cells (NSCs) were recently shown to release piRNA-containing exosomes/microvesicles (Ex/Mv) for exerting antiviral immunity, but it remains unknown if these Ex/Mv could target SARS-CoV-2 and whether the PIWI-piRNA system is important for these antiviral actions. Here, using in vitro infection models, we show that hypothalamic NSCs (htNSCs) Ex/Mv provided an innate immunity protection against SARS-CoV-2. Importantly, enhanced antiviral actions were achieved by using induced Ex/Mv that were derived from induced htNSCs through twice being exposed to several RNA fragments of SARS-CoV-2 genome, a process that was designed not to involve protein translation of these RNA fragments. The increased antiviral effects of these induced Ex/Mv were associated with increased expression of piRNA species some of which could predictably target SARS-CoV-2 genome. Knockout of piRNA-interacting protein PIWIL2 in htNSCs led to reductions in both innate and induced antiviral effects of Ex/Mv in targeting SARS-CoV-2. Taken together, this study demonstrates a case suggesting Ex/Mv from certain cell types have innate and adaptive immunity against SARS-CoV-2, and the PIWI-piRNA system is important for these antiviral actions.

| S-EPMC8645330 | biostudies-literature

PIWI Slicing and EXD1 Drive Biogenesis of Nuclear piRNAs from Cytosolic Targets of the Mouse piRNA Pathway.

Project description:PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing in the two compartments is coordinated is not known. Here we demonstrate that endonucleolytic slicing of a transcript by the cytosolic mouse PIWI protein MILI acts as a trigger to initiate its further 5'?3' processing into non-overlapping fragments. These fragments accumulate as new piRNAs within both cytosolic MILI and the nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) as a partner of the MIWI2 piRNA biogenesis factor TDRD12. EXD1 homodimers are inactive as a nuclease but function as an RNA adaptor within a PET (PIWI-EXD1-Tdrd12) complex. Loss of Exd1 reduces sequences generated by MILI slicing, impacts biogenesis of MIWI2 piRNAs, and de-represses LINE1 retrotransposons. Thus, piRNA biogenesis triggered by PIWI slicing, and promoted by EXD1, ensures that the same guides instruct PIWI proteins in the nucleus and cytoplasm.

| S-EPMC4712191 | biostudies-literature

Altered expression of porcine Piwi genes and piRNA during development.

Project description:Three Sus scrofa Piwi genes (Piwil1, Piwil2 and Piwil4) encoding proteins of 861, 985 and 853 aminoacids, respectively, were cloned and sequenced. Alignment of the Piwi proteins showed the high identity between Sus scrofa and Homo sapiens. Relative transcript abundance of porcine Piwil1, Piwil2 and Piwil4 genes in testes, ovaries and oocytes derived from sexually immature and mature animals was examined using Real-Time PCR. Expression of the three Piwi mRNAs was proved to be tissue specific and restricted exclusively to the gonads. In testes of adult pigs the highest relative transcript abundance was observed for the Sus scrofa Piwil1 gene. On the other hand, in testes of neonatal pigs the Piwil1 transcript level was over 2-fold reduced while the level of Piwil2 transcript was higher. As regards the expression of the Piwil4 transcript, its level was 34-fold elevated in testes of neonatal piglet when compared to adult male. In ovaries of prepubertal and pubertal female pigs transcript abundance of the three Piwi genes was significantly reduced in comparison with testes. However, similarly to testes, in ovaries of neonatal pigs the Piwil2 gene was characterized by the highest relative transcript abundance among the three Piwi genes analysed. In prepubertal and pubertal oocytes Piwil1 transcript was the most abundant whereas the expression of Piwil4 was undetectable. We also demonstrated that expression of piRNA occurs preferentially in the gonads of adult male and female pigs. Moreover, a piRNA subset isolated from ovaries was 2-3 nucleotides longer than the piRNA from testes.

| S-EPMC3431407 | biostudies-literature

The emerging role of the piRNA/piwi complex in cancer.

Project description:Piwi interacting RNAs (piRNAs) constitute novel small non-coding RNA molecules of approximately 24-31 nucleotides in length that often bind to members of the piwi protein family to play regulatory roles. Recently, emerging evidence suggests that in addition to the mammalian germline, piRNAs are also expressed in a tissue-specific manner in a variety of human tissues and modulate key signaling pathways at the transcriptional or post-transcriptional level. In addition, a growing number of studies have shown that piRNA and PIWI proteins, which are abnormally expressed in various cancers, may serve as novel biomarkers and therapeutic targets for tumor diagnostics and treatment. However, the functions of piRNAs in cancer and their underlying mechanisms remain incompletely understood. In this review, we discuss current findings regarding piRNA biogenetic processes, functions, and emerging roles in cancer, providing new insights regarding the potential applications of piRNAs and piwi proteins in cancer diagnosis and clinical treatment.

| S-EPMC6688334 | biostudies-literature

Structural basis for piRNA 2'-O-methylated 3'-end recognition by Piwi PAZ (Piwi/Argonaute/Zwille) domains.

Project description:Argonaute and Piwi proteins are key players in the RNA silencing pathway, with the former interacting with micro-RNAs (miRNAs) and siRNAs, whereas the latter targets piwi-interacting RNAs (piRNAs) that are 2'-O-methylated (2(')-OCH(3)) at their 3' ends. Germline-specific piRNAs and Piwi proteins play a critical role in genome defense against transposable elements, thereby protecting the genome against transposon-induced defects in gametogenesis and fertility. Humans contain four Piwi family proteins designated Hiwi1, Hiwi2, Hiwi3, and Hili. We report on the structures of Hili-PAZ (Piwi/Argonaute/Zwille) domain in the free state and Hiwi1 PAZ domain bound to self-complementary 14-mer RNAs (12-bp + 2-nt overhang) containing 2(')-OCH(3) and 2'-OH at their 3' ends. These structures explain the molecular basis underlying accommodation of the 2(')-OCH(3) group within a preformed Hiwi1 PAZ domain binding pocket, whose hydrophobic characteristics account for the preferential binding of 2(')-OCH(3) over 2'-OH 3' ends. These results contrast with the more restricted binding pocket for the human Ago1 PAZ domain, which exhibits a reverse order, with preferential binding of 2'-OH over 2(')-OCH(3) 3' ends.

| S-EPMC3024652 | biostudies-literature

Single Cell Imaging of Nuclear Architecture Changes.

Project description:The dynamic architecture of chromatin, the macromolecular complex comprised primarily of DNA and histones, is vital for eukaryotic cell growth. Chemical and conformational changes to chromatin are important markers of functional and developmental processes in cells. However, chromatin architecture regulation has not yet been fully elucidated. Therefore, novel approaches to assessing chromatin changes at the single-cell level are required. Here we report the use of FTIR imaging and microfluidic cell-stretcher chips to assess changes to chromatin architecture and its effect on the mechanical properties of the nucleus in immune cells. FTIR imaging enables label-free chemical imaging with subcellular resolution. By optimizing the FTIR methodology and coupling it with cell segmentation analysis approach, we have identified key spectral changes corresponding to changes in DNA levels and chromatin conformation at the single cell level. By further manipulating live single cells using pressure-driven microfluidics, we found that chromatin decondensation - either during general transcriptional activation or during specific immune cell maturation - can ultimately lead to nuclear auxeticity which is a new biological phenomenon recently identified. Taken together our findings demonstrate the tight and, potentially bilateral, link between extra-cellular mechanotransduction and intra-cellular nuclear architecture.

| S-EPMC6668442 | biostudies-literature

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