Essential functions of RNA helicase Vasa in maintaining germline stem cells and piRNA-guided Stellate silencing in Drosophila spermatogenesis
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ABSTRACT:
SUBMITTER: Adashev V
PROVIDER: S-EPMC11341464 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:PIWI-interacting RNAs (piRNAs) play critical and conserved roles in transposon silencing and gene regulation in the animal germline. Two distinct piRNA populations are present during mouse spermatogenesis: pre-pachytene piRNAs in fetal/neonatal testes and pachytene piRNAs in adult testes. PNLDC1 is required for both pre-pachytene piRNA and pachytene piRNA 3' end maturation in multiple species. However, whether PNLDC1 is the bona fide piRNA trimmer and the physiological role of 3' trimming of two distinct piRNA populations in spermatogenesis remain unclear. Here, by inactivating Pnldc1 exonuclease activity in vitro and in mice, we reveal that PNLDC1 trimmer activity is required for both pre-pachytene piRNA and pachytene piRNA 3' end trimming and male fertility. Furthermore, conditional inactivation of Pnldc1 in postnatal germ cells causes LINE1 transposon de-repression and spermatogenic arrest in mice. This indicates that pachytene piRNA trimming, but not pre-pachytene piRNA trimming, is essential for mouse germ cell development and transposon silencing. Our findings highlight the potential of inhibiting germline piRNA trimmer activity as a potential means for male contraception.
| S-EPMC10793440 | biostudies-literature
Project description:The PIWI-interacting RNA (piRNA) pathway is a small RNA-based immune system that controls the expression of transposons and maintains genome integrity in animal gonads. In Drosophila, piRNA-guided silencing is achieved, in part, via co-transcriptional repression of transposons by Piwi. This depends on Panoramix (Panx); however, precisely how an RNA binding event silences transcription remains to be determined. Here we show that Nuclear Export Factor 2 (Nxf2) and its co-factor, Nxt1, form a complex with Panx and are required for co-transcriptional silencing of transposons in somatic and germline cells of the ovary. Tethering of Nxf2 or Nxt1 to RNA results in silencing of target loci and the concomitant accumulation of repressive chromatin marks. Nxf2 and Panx proteins are mutually required for proper localization and stability. We mapped the protein domains crucial for the Nxf2/Panx complex formation and show that the amino-terminal portion of Panx is sufficient to induce transcriptional silencing.
| S-EPMC6677536 | biostudies-literature
Project description:The mammalian Phospholipase D MitoPLD facilitates mitochondrial fusion by generating the signaling lipid phosphatidic acid (PA). The Drosophila MitoPLD homolog Zucchini (Zuc), a proposed cytoplasmic nuclease, is required for piRNA generation, a critical event in germline development. We show that Zuc localizes to mitochondria and has MitoPLD-like activity. Conversely, MitoPLD(-/-) mice exhibit the meiotic arrest, DNA damage, and male sterility characteristic of mice lacking piRNAs. The primary function of MitoPLD seems to be the generation of mitochondrial-surface PA. This PA in turn recruits the phosphatase Lipin 1, which converts PA to diacylglycerol and promotes mitochondrial fission, suggesting a mechanism for mitochondrial morphology homeostasis. MitoPLD and Lipin 1 have opposing effects on mitochondria length and on intermitochondrial cement (nuage), a structure found between aggregated mitochondria that is implicated in piRNA generation. We propose that mitochondrial-surface PA generated by MitoPLD/Zuc recruits or activates nuage components critical for piRNA production.
| S-EPMC3061402 | biostudies-literature
Project description:Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposition, regulate translation, and guide epigenetic programming in the germline. Here, we show that an evolutionarily conserved Tudor and KH domain-containing protein, Tdrkh (a.k.a. Tdrd2), is required for spermatogenesis and involved in piRNA biogenesis. Tdrkh partners with Miwi and Miwi2 via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is a mitochondrial protein often juxtaposed to pi-bodies and piP-bodies and is required for Tdrd1 cytoplasmic localization and Miwi2 nuclear localization. Tdrkh mutants display meiotic arrest at the zygotene stage, attenuate methylation of Line1 DNA, and upregulate Line1 RNA and protein, without inducing apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs but accumulate a distinct population of 1'U-containing, 2'O-methylated 31-37?nt RNAs that largely complement the missing mature piRNAs. Our results demonstrate that the primary piRNA biogenesis pathway involves 3'?5' processing of 31-37?nt intermediates and that Tdrkh promotes this final step of piRNA biogenesis but not the ping-pong cycle. These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent.
| S-EPMC3981179 | biostudies-literature
Project description:DEAD-box RNA helicase Vasa is required for gonad development and fertility in multiple animals. In Drosophila, Vasa performs essential functions in oogenesis, including the maintenance of germline stem cells (GSCs), piRNA silencing of mobile elements, translation regulation, and primordial germ cell specification. Despite its evident significance, the mechanistic basis of Vasa action and its precise role in spermatogenesis become incomprehensible. Several papers affirm the fertility of males carrying vasa mutations. However, it is also shown that Vasa is essential for piRNA-mediated repression of Stellate genes needed for the maintenance of male fertility.Here we found that loss-of-function vasa mutations led to a rapid decline in GSC maintenance in the testes, a severe loss of total germ cell content, and a strong decrease in male fertility over time. With the aid of analysis of small RNA libraries, we revealed that collapse of piRNA biogenesis in the absence of vasa expression. Despite that, we did not reveal increasing cell death events in the early germ cells of vasa mutant testes. The introduction of the transgene rhino copy, encoding a nuclear component of the piRNA pathway, in vasa mutant background allowed us to rescue premeiotic stages of spermatogenesis, including GSC maintenance and the development of spermatogonia and spermatocytes. However, the progression of spermatocytes through meiosis and the fertility of the rhino transgene-rescued males were disrupted by strong Stellate gene derepression owing to the absence of corresponding piRNAs. We have shown that Vasa functions in spermatogenesis are essential at two separate developmental stages: in GSCs for their maintenance and in spermatocytes for the repression of Stellate genes.
2024-08-02 | GSE269988 | GEO
Project description:DEAD-box RNA helicase Vasa is required for gonad development and fertility in multiple animals. In Drosophila, Vasa performs essential functions in oogenesis, including the maintenance of germline stem cells (GSCs), piRNA silencing of mobile elements, translation regulation, and primordial germ cell specification. Despite its evident significance, the mechanistic basis of Vasa action and its precise role in spermatogenesis become incomprehensible. Several papers affirm the fertility of males carrying vasa mutations. However, it is also shown that Vasa is essential for piRNA-mediated repression of Stellate genes needed for the maintenance of male fertility.Here we found that loss-of-function vasa mutations led to a rapid decline in GSC maintenance in the testes, a severe loss of total germ cell content, and a strong decrease in male fertility over time. With the aid of analysis of small RNA libraries, we revealed that collapse of piRNA biogenesis in the absence of vasa expression. Despite that, we did not reveal increasing cell death events in the early germ cells of vasa mutant testes. The introduction of the transgene rhino copy, encoding a nuclear component of the piRNA pathway, in vasa mutant background allowed us to rescue premeiotic stages of spermatogenesis, including GSC maintenance and the development of spermatogonia and spermatocytes. However, the progression of spermatocytes through meiosis and the fertility of the rhino transgene-rescued males were disrupted by strong Stellate gene derepression owing to the absence of corresponding piRNAs. We have shown that Vasa functions in spermatogenesis are essential at two separate developmental stages: in GSCs for their maintenance and in spermatocytes for the repression of Stellate genes.
2024-08-02 | GSE269987 | GEO
Project description:PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ∼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways.
| S-EPMC4561483 | biostudies-literature
Project description:Vasa is a conserved RNA-helicase found in the germ lines of all metazoans tested. Whereas Vasa presence is often indicated as a metric for germline determination in animals, it is also expressed in stem cells of diverse origin. Recent research suggests, however, that Vasa has a much broader function, including a significant role in cell cycle regulation. Results herein indicate that Vasa is utilized widely, and often induced transiently, during development in diverse somatic cells and adult precursor tissues. We identified that Vasa in the sea urchin is essential for: (1) general mRNA translation during embryogenesis, (2) developmental re-programming upon manipulations to the embryo and (3) larval wound healing. We also learned that Vasa interacted with mRNAs in the perinuclear area and at the spindle in an Importin-dependent manner during cell cycle progression. These results suggest that, when present, Vasa functions are essential to contributing to developmental regulation.
| S-EPMC4460737 | biostudies-literature
Project description: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.
| S-EPMC6717896 | biostudies-literature
Project description:Piwi-interacting RNAs are small regulatory RNAs with key roles in transposon silencing and regulation of gametogenesis. The production of mature piwi-interacting RNAs requires a critical step of trimming piwi-interacting RNA intermediates to achieve optimally sized piwi-interacting RNAs. The poly(A)-specific ribonuclease family deadenylase PNLDC1 is implicated in piwi-interacting RNA trimming in silkworms. The physiological function of PNLDC1 in mammals remains unknown. Using Pnldc1-deficient mice, here we show that PNLDC1 is required for piwi-interacting RNA biogenesis, transposon silencing, and spermatogenesis. Pnldc1 mutation in mice inhibits piwi-interacting RNA trimming and causes accumulation of untrimmed piwi-interacting RNA intermediates with 3' end extension, leading to severe reduction of mature piwi-interacting RNAs in the testis. Pnldc1 mutant mice exhibit disrupted LINE1 retrotransposon silencing and defect in spermiogenesis. Together, these results define PNLDC1 as a mammalian piwi-interacting RNA biogenesis factor that protects the germline genome and ensures normal sperm production in mice.piRNAs are regulatory RNAs that play a critical role in transposon silencing and gametogenesis. Here, the authors provide evidence that mammalian PNLDC1 is a regulator of piRNA biogenesis, transposon silencing and spermatogenesis, protecting the germline genome in mice.
| S-EPMC5635004 | biostudies-literature