Heterotypic piRNA Ping-Pong requires qin, a protein with both E3 ligase and Tudor domains

piRNAs guide PIWI proteins to silence transposons in animal germ cells. Reciprocal cycles of piRNA-directed RNA cleavage--catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila melanogaster--expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. We show that qin, a piRNA pathway gene whose protein product contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, a perinuclear structure implicated in transposon silencing. In qin mutants, less Ago3 binds Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, and DNA damage accumulates in nurse cells and oocytes. We propose that Qin enforces heterotypic Ping-Pong between Aub and Ago3, ensuring that transposons are silenced and maintaining the integrity of the germline genome.     

The interplay of transposon silencing genes in the Drosophila melanogaster germline

Complexes of Piwi proteins and Piwi-interacting RNAs (piRNAs) carry out the repression of transposable elements in animal gonads. The Piwi protein clade is represented in D. melanogaster by three members: Piwi, Aub and Ago3. Piwi protein functions in the nuclei of somatic and germinal ovarian cells, whereas Aub and Ago3 are cytoplasmic proteins of germinal cells. Aub and Ago3 interact with each other in the perinuclear nuage organelle to perform piRNA amplification via the ping-pong mechanism. Previously, derepression of several transposable elements as a result of mutations in the piRNA silencing system was shown. Here we quantify the increase in expression level of an enlarged number of retrotransposons due to the mutations in the piwi gene, nuage components coding aub, mael and spn-E genes and the RNA helicase armi gene mutation that impairs Piwi nuclear localization, but not the ping-pong cycle. We reveal that piwi, armi, aub, spn-E and mael genes participate together in the repression of several transposons (HMS-Beagle, Gate and HeT-A), whereas silencing of land G elements requires the same genes except piwi. We suggest that Armi has other functions besides the localizing of Piwi protein in the nuclei. Our data suggest also a role of cytoplasmic Aub, Spn-E and Mael nuage proteins in Piwi-mediated repression of Gate and HMS-Beagle transposons in the germline nuclei. As a whole, our results corroborate the idea that genome stabilization in the germline is realized by different silencing strategies specific for different transposable elements. At the same time, our data suggest the existence of yet unknown mechanisms of interplay between nuclear and cytoplasmic components of the piRNA machinery in the germline.     

Interplay of transposon-silencing genes in the germline of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Complexes of Piwi family proteins with short piRNAs (Piwi-interacting RNAs) are responsible for silencing transposable elements in animal reproductive organs. In Drosophila melanogaster, three proteins (Piwi, Aub, and Ago3) are members of the Piwi family. Piwi is the nuclear protein of somatic and germinal ovarian cells, whereas Aub and Ago3 are cytoplasmic proteins involved in piRNA amplification in perinuclear granules that constitute special organelles of germinal cells called nuage. Mutations in genes of the piRNA system are known to cause derepression of several transposable elements. In this study, we compared quantitatively changes in expression of a larger number of elements in the case of mutations in the piwi gene, genes aub, mael, and spn-E, which encode proteins of nuage granules, and armi gene coding an RNA helicase, the lack of which does not interfere with cytoplasmic piRNA amplification but disturbs nuclear localization of Piwi protein. We found that the genes piwi, armi, aub, spn-E, and mael interact to induce silencing of some retrotransposons (HMS-Beagle, Gate and HeT-A); the same genes, except piwi, are involved in repression of I and G elements. We propose that Armi is involved in control of not only nuclear Piwi localization. Our data suggest the relation of nuage proteins Aub, Spn-E, and Mael to Piwi-mediated silencing of retrotransposons Gate and HMS-Beagle in the nucleus. In general, our results corroborate the idea of genome stabilization by means of various silencing strategies specific to different transposable elements. At the same time, our data suggest the existence of yet unknown mechanisms of interplay between nuclear and cytoplasmic components of the piRNA machinery in germinal cells.     

Dynamic subcellular compartmentalization ensures fidelity of piRNA biogenesis in silkworms

PIWI‐interacting RNAs (piRNAs) guide PIWI proteins to silence transposable elements and safeguard fertility in germ cells. Many protein factors required for piRNA biogenesis localize to perinuclear ribonucleoprotein (RNP) condensates named nuage, where target silencing and piRNA amplification are thought to occur. In mice, some of the piRNA factors are found in discrete cytoplasmic foci called processing bodies (P‐bodies). However, the dynamics and biological significance of such compartmentalization of the piRNA pathway remain unclear. Here, by analyzing the subcellular localization of functional mutants of piRNA factors, we show that piRNA factors are actively compartmentalized into nuage and P‐bodies in silkworm cells. Proper demixing of nuage and P‐bodies requires target cleavage by the PIWI protein Siwi and ATP hydrolysis by the DEAD‐box helicase BmVasa, disruption of which leads to promiscuous overproduction of piRNAs deriving from non‐transposable elements. Our study highlights a role of dynamic subcellular compartmentalization in ensuring the fidelity of piRNA biogenesis.     

PAPI, a novel TUDOR-domain protein, complexes with AGO3, ME31B and TRAL in the nuage to silence transposition

The nuage is a germline-specific perinuclear structure that remains functionally elusive. Recently, the nuage in Drosophila was shown to contain two of the three PIWI proteins - Aubergine and Argonaute 3 (AGO3) - that are essential for germline development. The PIWI proteins bind to PIWI-interacting RNAs (piRNAs) and function in epigenetic regulation and transposon control. Here, we report a novel nuage component, PAPI (Partner of PIWIs), that contains a TUDOR domain and interacts with all three PIWI proteins via symmetrically dimethylated arginine residues in their N-terminal domain. In adult ovaries, PAPI is mainly cytoplasmic and enriched in the nuage, where it partially colocalizes with AGO3. The localization of PAPI to the nuage does not require the arginine methyltransferase dPRMT5 or AGO3. However, AGO3 is largely delocalized from the nuage and becomes destabilized in the absence of PAPI or dPRMT5, indicating that PAPI recruits PIWI proteins to the nuage to assemble piRNA pathway components. As expected, papi deficiency leads to transposon activation, phenocopying piRNA mutants. This further suggests that PAPI is involved in the piRNA pathway for transposon silencing. Moreover, AGO3 and PAPI associate with the P body component TRAL/ME31B complex in the nuage and transposon activation is observed in tral mutant ovaries. This suggests a physical and functional interaction in the nuage between the piRNA pathway components and the mRNA-degrading P-body components in transposon silencing. Overall, our study reveals a function of the nuage in safeguarding the germline genome against deleterious retrotransposition via the piRNA pathway.     

Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI‐interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl‐arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domain‐containing proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor‐like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon‐derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality‐controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud.     

Distinct sets of PIWI proteins produce arbovirus and transposon-derived piRNAs in Aedes aegypti mosquito cells

The PIWI-interacting RNA (piRNA) pathway is essential for transposon silencing in many model organisms. Its remarkable efficiency relies on a sophisticated amplification mechanism known as the ping-pong loop. In Alphavirus-infected Aedes mosquitoes, piRNAs with sequence features that suggest ping-pong-dependent biogenesis are produced from viral RNA. The PIWI family in Aedes mosquitoes is expanded when compared to other model organisms, raising the possibility that individual PIWI proteins have functionally diversified in these insects. Here, we show that Piwi5 and Ago3, but none of the other PIWI family members, are essential for piRNA biogenesis from Sindbis virus RNA in infected Aedes aegypti cells. In contrast, the production of piRNAs from transposons relies on a more versatile set of PIWI proteins, some of which do not contribute to viral piRNA biogenesis. These results indicate that functional specialization allows distinct mosquito PIWI proteins to process RNA from different endogenous and exogenous sources.     

An in vivo RNAi assay identifies major genetic and cellular requirements for primary piRNA biogenesis in Drosophila

In Drosophila, PIWI proteins and bound PIWI‐interacting RNAs (piRNAs) form the core of a small RNA‐mediated defense system against selfish genetic elements. Within germline cells, piRNAs are processed from piRNA clusters and transposons to be loaded into Piwi/Aubergine/AGO3 and a subset of piRNAs undergoes target‐dependent amplification. In contrast, gonadal somatic support cells express only Piwi, lack signs of piRNA amplification and exhibit primary piRNA biogenesis from piRNA clusters. Neither piRNA processing/loading nor Piwi‐mediated target silencing is understood at the genetic, cellular or molecular level. We developed an in vivo RNAi assay for the somatic piRNA pathway and identified the RNA helicase Armitage, the Tudor domain containing RNA helicase Yb and the putative nuclease Zucchini as essential factors for primary piRNA biogenesis. Lack of any of these proteins leads to transposon de‐silencing, to a collapse in piRNA levels and to a failure in Piwi‐nuclear accumulation. We show that Armitage and Yb interact physically and co‐localize in cytoplasmic Yb bodies, which flank P bodies. Loss of Zucchini leads to an accumulation of Piwi and Armitage in Yb bodies, indicating that Yb bodies are sites of primary piRNA biogenesis.     

The Bombyx ovary-derived cell line endogenously expresses PIWI/PIWI-interacting RNA complexes

Genetic studies and large-scale sequencing experiments have revealed that the PIWI subfamily proteins and PIWI-interacting RNAs (piRNAs) play an important role in germ line development and transposon control. Biochemical studies in vitro have greatly contributed to the understanding of small interfering RNA (siRNA) and microRNA (miRNA) pathways. However, in vitro analyses of the piRNA pathway have been thus far quite challenging, because their expression is largely restricted to the germ line. Here we report that Bombyx mori ovary-derived cultured cell line, BmN4, endogenously expresses two PIWI subfamily proteins, silkworm Piwi (Siwi) and Ago3 (BmAgo3), and piRNAs associated with them. Siwi-bound piRNAs have a strong bias for uridine at their 5′ end and BmAgo3-bound piRNAs are enriched for adenine at position 10. In addition, Siwi preferentially binds antisense piRNAs, whereas BmAgo3 binds sense piRNAs. Moreover, we identified many pairs in which Siwi-bound antisense and BmAgo3-bound sense piRNAs are overlapped by precisely 10 nt at their 5′ ends. These signatures are known to be important for secondary piRNA biogenesis in other organisms. Taken together, BmN4 is a unique cell line in which both primary and secondary steps of piRNA biogenesis pathways are active. This cell line would provide useful tools for analysis of piRNA biogenesis and function.     

Testosterone alters testis function through regulation of piRNA expression in rats

Piwi-interacting RNAs (piRNAs) play a role in gene silencing of retrotransposons, maintenance of spermatogenesis and maturation in germlines. The piRNA and PIWI protein are essential for fertility. To reveal piRNA function associated with testosterone, we investigated the expression of piRNA and piwi protein in normal male rats and testosterone-treated rats. Normal Sprague–Dawley (SD) rats were randomly selected and sacrificed at neonatal to late adolescence stage stages (2, 9, 16, 20, 24, 28, 35, and 42 days, n = 6 each). Additional SD rats were divided into four groups: group 1 received weekly injections of testosterone enanthate (8 mg/100 g) during 1–3 weeks; group 2 during 3–5 weeks; group 3 during 1–5 weeks; and group 4 was the control (n = 20 each). These animals were sacrificed at an age of 60 days. We investigated piRNA, PIWI, and Ago3 protein levels using real-time PCR, Western blot, and immunohistochemistry in each group. In normal rats, PIWI protein and piRNA were expressed at P24. The expression of PIWI and piRNA gradually increased from adolescence to adulthood on Western blot, real-time PCR and immunohistochemistry. In testosterone-treated rats, the expression of PIWI protein was analyzed by Western blot and shown to be significantly increased in group 1 (neonatal to juvenile injection). In real-time PCR, the expression of piRNA after testosterone treatment was increased in all groups (G1 166.8 ± 2.7; G2 113.3 ± 4.6; G3 70.2 ± 1.5 vs. control, 32.87 ± 2.0, all p < 0.001). The expression of testosterone in adolescence induces the development of male genitourinary organs and spermatogenesis. At the same time, the sexual hormones may activate the piRNA and PIWI protein. Our data demonstrate that patterns of piRNA and PIWI expression are similar to the secretion pattern of testosterone, and that piRNA expression was increased after testosterone treatment. Therefore, testosterone may affect testis function through the regulation of piRNA expression in rats.     

Mitochondrial protein BmPAPI modulates the length of mature piRNAs

PIWI proteins and their associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons in animal germlines. The molecular mechanisms and components responsible for piRNA biogenesis remain elusive. PIWI proteins contain conserved symmetrical dimethylarginines (sDMAs) that are specifically targeted by TUDOR domain-containing proteins. Here we report that the sDMAs of PIWI proteins play crucial roles in PIWI localization and piRNA biogenesis in Bombyx mori-derived BmN4 cells, which harbor fully functional piRNA biogenesis machinery. Moreover, RNAi screenings for Bombyx genes encoding TUDOR domain-containing proteins identified BmPAPI, a Bombyx homolog of Drosophila PAPI, as a factor modulating the length of mature piRNAs. BmPAPI specifically recognized sDMAs and interacted with PIWI proteins at the surface of the mitochondrial outer membrane. BmPAPI depletion resulted in 3′-terminal extensions of mature piRNAs without affecting the piRNA quantity. These results reveal the BmPAPI-involved piRNA precursor processing mechanism on mitochondrial outer membrane scaffolds.     

Spatio-temporal requirements for transposable element piRNA-mediated silencing during Drosophila oogenesis

During Drosophila oogenesis, transposable element (TE) repression involves the Piwi-interacting RNA (piRNA) pathway which ensures genome integrity for the next generation. We developed a transgenic model to study repression of the Idefix retrotransposon in the germline. Using a candidate gene KD-approach, we identified differences in the spatio-temporal requirements of the piRNA pathway components for piRNA-mediated silencing. Some of them (Aub, Vasa, Spn-E) are necessary in very early stages of oogenesis within the germarium and appear to be less important for efficient TE silencing thereafter. Others (Piwi, Ago3, Mael) are required at all stages of oogenesis. Moreover, during early oogenesis, in the dividing cysts within the germarium, Idefix anti-sense transgenes escape host control, and this is associated with very low piwi expression. Silencing of P-element-based transgenes is also strongly weakened in these cysts. This region, termed the ‘Piwiless pocket’ or Pilp, may ensure that new TE insertions occur and are transmitted to the next generation, thereby contributing to genome dynamics. In contrast, piRNA-mediated silencing is strong in germline stem cells in which TE mobilization is tightly repressed ensuring the continued production of viable germline cysts.     

Identification and characterization of Piwi subfamily in insects

As a subfamily of Argonaute proteins, Piwi is poorly understood compared with Ago subfamily until recent discovery of Piwi protein interacting with piRNA. We did a large scale screening of insect genomes to identify piwi-like genes. Full or partial cDNA sequences were obtained by EST elongation and GENSCAN. We found that the exon numbers were totally different between vertebrates and invertebrates, approximately 20 exons in mammals but only 6-9 exons in insects. This infers either intron insertion or loss occurred during evolution. Characterized PAZ, c-terminal PIWI domains exist in almost all predicted Piwi-like proteins. We found six conserved motifs, which contain active catalytic triad "Asp-Asp-His/Lys" required for slicer activity. The expression of siwi1 and siwi2 in Bombyx mori were verified with RT-PCR. Phylogenetic tree inferred by Bayesian algorithm indicates invertebrate Piwi-like proteins are classified into three clades, of which Ago3 clade is closer to mammalian Piwi proteins.     

Primary piRNA biogenesis: caught up in a Maelstrom

Precursors for most Piwi‐interacting RNAs (piRNAs) are indistinguishable from other RNA polymerase II‐transcribed long non‐coding RNAs. So, it is currently unclear how they are recognized as substrates by the piRNA processing machinery that resides in cytoplasmic granules called nuage. In this issue, Castaneda et al (2014) reveal a role for the nuage component and nucleo‐cytoplasmic shuttling protein Maelstrom in mouse piRNA biogenesis.