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 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.     

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals.     

piRNA：一类新的非编码小RNA

piRNA（Piwi-interacting RNA）是从哺乳动物生殖细胞中分离得到的一类长度约为30nt的小RNA,并且这种小RNA与PIWI蛋白家族成员相结合才能发挥它的调控作用。目前,越来越多的文献表明piRNA在生殖细胞的生长发育中的调控是由于Piwi-piRNA复合物引起的基因沉默导致的,但由于对piRNA的研究尚处于初级阶段,它的一些具体的功能和生源论尚在研究当中。本文主要综述了piRNA的最新研究进展。     

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.     

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.     

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.     

Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization

Piwi proteins such as Drosophila Aubergine (Aub) and mouse Miwi are essential for germline development and for primordial germ cell (PGC) specification. They bind piRNAs and contain symmetrically dimethylated arginines (sDMAs), catalyzed by dPRMT5. PGC specification in Drosophila requires maternal inheritance of cytoplasmic factors, including Aub, dPRMT5, and Tudor (Tud), that are concentrated in the germ plasm at the posterior end of the oocyte. Here we show that Miwi binds to Tdrd6 and Aub binds to Tudor, in an sDMA-dependent manner, demonstrating that binding of sDMA-modified Piwi proteins with Tudor-domain proteins is an evolutionarily conserved interaction in germ cells. We report that in Drosophila tud¹ mutants, the piRNA pathway is intact and most transposons are not de-repressed. However, the localization of Aub in the germ plasm is severely reduced. These findings indicate that germ plasm assembly requires sDMA modification of Aub by dPRMT5, which, in turn, is required for binding to Tudor. Our study also suggests that the function of the piRNA pathway in PGC specification may be independent of its role in transposon control.     

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.