A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors

PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) form the core of a gonad-specific small RNA silencing pathway that protects the animal genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology as TUDOR domains of various proteins bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analysed the Drosophila TUDOR protein family and identified four previously not characterized TUDOR domain-containing proteins (CG4771, CG14303, CG11133 and CG31755) as essential piRNA pathway factors. We characterized CG4771 (Vreteno) in detail and demonstrate a critical role for this protein in primary piRNA biogenesis. Vreteno physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Vreteno also interacts with the Tdrd12 orthologues CG11133 (Brother of Yb) and CG31755 (Sister of Yb), which are essential for the primary piRNA pathway in the germline and probably replace the function of the related but soma-specific factor Yb.     

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.     

The role of piRNA and Piwi proteins in regulation of germline development

A new group of small noncoding RNAs of 24-30 nucleotides in length, piRNAs, are mainly expressed in germline cells. They form complexes with Piwi proteins, members of the Argonaute family and unlike other small RNAs they are created without RNase Dicer participation. They are present in male and female germinal cells of numerous animals, from flies to humans. The piRNA biogenesis mechanism is unknown, however, it is postulated that they are formed from long single-stranded RNA precursors coded by repetitive sequences occurring in the genome. A large part of piRNA corresponds to retrotranspozon sequences, which may indicate their participation in silencing the mobile elements and maintaining genome integrity of germinal cells. However, disruption of the piRNA biosynthesis pathway and mutations genes encoding Piwi proteins cause the activation of transpozons and a number of defects in the course of gametogenesis, resulting in reproduction disturbance. In this review, the current state of knowledge on the structure, biogenesis and function of piRNA and their interactions with Piwi proteins is presented.     

Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline

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.     

The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs

Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present in a single organism. Despite this complexity, specific Piwi proteins incorporate piRNAs with distinct nucleotide- and transposon strand-biases (antisense or sense) of unknown origin. Here, we examined the contribution of structural domains in Piwi proteins toward defining these biases. We report the first crystal structure of the MID domain from a Piwi Argonaute and use docking experiments to show its ability to specify recognition of 5′ uridine (1U-bias) of piRNAs. Mutational analyses reveal the importance of 5′ end-recognition within the MID domain for piRNA biogenesis in vivo. Finally, domain-swapping experiments uncover an unexpected role for the MID-PIWI module of a Piwi protein in dictating the transposon strand-orientation of its bound piRNAs. Our work identifies structural features that allow distinguishing individual Piwi members during piRNA biogenesis.     

A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice

piRNAs and Piwi proteins have been implicated in transposon control and are linked to transposon methylation in mammals. Here we examined the construction of the piRNA system in the restricted developmental window in which methylation patterns are set during mammalian embryogenesis. We find robust expression of two Piwi family proteins, MIWI2 and MILI. Their associated piRNA profiles reveal differences from Drosophila wherein large piRNA clusters act as master regulators of silencing. Instead, in mammals, dispersed transposon copies initiate the pathway, producing primary piRNAs, which predominantly join MILI in the cytoplasm. MIWI2, whose nuclear localization and association with piRNAs depend upon MILI, is enriched for secondary piRNAs antisense to the elements that it controls. The Piwi pathway lies upstream of known mediators of DNA methylation, since piRNAs are still produced in dnmt3L mutants, which fail to methylate transposons. This implicates piRNAs as specificity determinants of DNA methylation in germ cells.     

shutdown is a component of the Drosophila piRNA biogenesis machinery

In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors, which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown (shu), a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown.     

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.     

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.