piRNA-Guided Genome Defense: From Biogenesis to Silencing

Abstract

PIWI-interacting RNAs (piRNAs) and their associated PIWI clade Argonaute proteins constitute the core of the piRNA pathway. In gonadal cells, this conserved pathway is crucial for genome defense, and its main function is to silence transposable elements. This is achieved through posttranscriptional and transcriptional gene silencing. Precursors that give rise to piRNAs require specialized transcription and transport machineries because piRNA biogenesis is a cytoplasmic process. The ping-pong cycle, a posttranscriptional silencing mechanism, combines the cleavage-dependent silencing of transposon RNAs with piRNA production. PIWI proteins also function in the nucleus, where they scan for nascent target transcripts with sequence complementarity, instructing transcriptional silencing and deposition of repressive chromatin marks at transposon loci. Although studies have revealed numerous factors that participate in each branch of the piRNA pathway, the precise molecular roles of these factors often remain unclear. In this review, we summarize our current understanding of the mechanisms involved in piRNA biogenesis and function.

Keywords: PIWI proteins; piRNA biogenesis; piRNA clusters; ping-pong loop; posttranscriptional gene silencing; transcriptional gene silencing; transposon control.

Figure 1

piRNA clusters are major piRNA source loci. (a) The flam locus is the major source of piRNAs in the follicle cells of the Drosophila ovary and a prime example for unistrand clusters. Like coding genes, flam features a promoter that is decorated with H3K4me3 marks and is controlled by the transcription factor CI. RNA pol II transcribes the flam locus; the resulting RNA is capped and shows splicing signatures. Unlike the promoter region, the body of flam is marked with H3K9me3. (b) Transcription from dual-strand clusters is noncanonical. Dual-strand clusters are decorated with H3K9me3 marks and, for unknown reasons, they are specifically bound by the HP1 homolog Rhi. Together with Del, Rhi serves as a binding platform for Moon, which recruits a specialized transcription initiation machinery and leads to RNA production by pol II. The accumulation of cluster-derived transcripts also depends on Cuff, which interacts with Rhi through Del. Cuff is thought to suppress transcription termination and to protect nascent RNAs from splicing and degradation. UAP56 and Thoc5 (the TREX complex) were proposed to transport cluster transcripts from their site of synthesis to their processing sites. Abbreviations: 42AB, the most prominent dual-strand piRNA cluster named for its cytological position; CI, Cubitus interruptus; Cuff, Cutoff; Del, Deadlock; DIP1, DISCO interacting protein 1; flam, flamenco; HP1, heterochromatin protein 1; Moon, Moonshiner; Nuage, perinuclear structures marked by the presence of PIWI proteins participating in the ping-pong loop; pol II, polymerase II; Rhi, Rhino; TFIIA, transcription factor II A; Thoc5, THO complex subunit 5; TRF2, TATA-box binding protein-related factor 2; UAP56, the conserved DEAD-box helicase U2AF65-associated protein also known as Hel25E; YR, dinucleotide signature of transcription initiation.

Figure 2

Biogenesis of piRNAs follows several routes. (a) Following export of precursor RNAs from the nucleus, piRNA biogenesis is initiated in nuage through ping-pong looping. There, precursor transcripts are recognized and cleaved by Ago3 in complex with a trigger piRNA. This slicing event gives rise to a responder piRNA, which is loaded into Aub. The remaining 3′ portion of the cleaved transcript (red) serves as substrate for further piRNA production. Subsequently, piRNA biogenesis proceeds in a 3′-oriented manner. The mitochondrial endonuclease Zuc generates waves of trail piRNAs approximately every 25 nt, guided by an as-yet-unidentified cofactor or by the footprint of Piwi itself (top inset). Several other factors participate in this process, including Vret, Mino, and Gasz, but their precise molecular functions remain to be clarified. The helicase activity of Armi is likely to unwind precursor RNAs before feeding them into Zuc. The loading of piRNAs relies on Shu and Hsp90 and causes a conformational change in Piwi, thereby allowing its nuclear translocation. As a final step, Hen1 methylates the 3′ end of mature, Piwi-loaded piRNAs prior to import to the nucleus. (b) The main steps of the ping-pong cycle. Aub bound to an antisense piRNA recognizes and cleaves a transposon mRNA. The resulting 3′ cleavage product is converted into a new sense piRNA that associates with Ago3; its 3′ end is generated through trimming by Nbr. Ago3 associated with a sense piRNA can in turn recognize and cleave cluster transcripts. The product of this slicing event reinitiates the cycle, becoming an Aub-bound piRNA, and the remaining 3′ slicing product (red) is processed into Piwi-loaded piRNAs via Zuc-mediated biogenesis. Specialized loading complexes ensure that products of slicing are loaded into the correct PIWI protein: Vasa and Krimp assist Ago3 loading, whereas Qin and Spn-E act on Aub. Furthermore, Qin prevents Aub cleavage products from being loaded into Aub, thus ensuring the directionality of the ping-pong cycle. This regulatory framework is based on posttranslational modifications of PIWI proteins in the form of sDMA residues, which are added by Csul and Vls. Abbreviations: Ago3, Argonaute3; Armi, Armitage; Aub, Aubergine; CH3, depicts a 2-O-methyl group; Csul, Capsuleen; Gasz, germ cell protein with ankyrin repeats/sterile-alpha motif/leucine zipper; Hen1, Hua enhancer 1; Hsp90, Heat shock protein 90; Krimp, Krimper; Mino, Minotaur; Nbr, Nibbler; nt, nucleotide; sDMA, symmetrical dimethylation of arginine; Shu, Shutdown; Spn-E, Spindle-E; Vls, Valois; Vret, Vreteno; Zuc, Zucchini.

Figure 3

A stepwise model of piRNA-guided transcriptional silencing. (a) During target recognition, Piwi–piRNA complexes scan the nucleus and recognize nascent transcripts of TEs through sequence complementarity. This process is likely assisted by the cofactors Arx and Panx and is regulated by the PAF1 complex—the latter which is associated with elongating RNA pol II. (b) Silencing is established through interactions with several elements of the heterochromatin silencing machinery, yet the molecular details of these interactions remain to be resolved. The histone methyltransferase Egg and its cofactor Wde deposit H3K9me2/3 marks on transposon bodies. Mael appears to function independently of this histone mark, yet is crucial for proper silencing. Furthermore, silencing of some TEs requires Lsd1, which likely removes activating H3K4me2 modifications prior to Egg’s function. (c) Upon establishment of silencing, HP1a and histone H1 cover the entire locus and cooperate to maintain its repressed status. Across all three steps, histone modifications generally associated with transcriptional activity, namely H3K4me3 and H3K4me2, are represented in green, whereas the repressive H3K9me2/3 mark is shown in red. Abbreviations: Arx, Asterix; Egg, Eggless/dSETDB1; HP1a, heterochromatin protein 1a; Lsd1, lysine-specific demethylase 1 [also known as su(var)3–3]; Mael, Maelstrom; Panx, Panoramix; pol II, polymerase II; TEs, transposable elements; Wde, Windei.

Figure 4

Intergenerationally inherited piRNAs function as epigenetic information carriers. (a) In addition to the maternal deposition of proteins and mRNAs, Drosophila embryos also inherit piRNAs associated with Aubergine (Aub) and Piwi. While Piwi is distributed in the nuclei of somatic and future germline cells throughout the entire embryo, Aub is specifically localized to germ cell progenitors known as pole cells. Upon activation of zygotic transcription, Piwi–piRNA complexes may engage with target transposon RNAs, resulting in recruitment of factors of the transcriptional silencing machinery and deposition of H3K9me2/3. Since Piwi expression in somatic cells is limited to the early stages of development, silencing of transposons must be maintained independently, likely through mechanisms involving heterochromatin protein 1a (HP1a). In developing germ cells, however, Piwi–piRNA complexes target nascent transcripts derived from transposon loci and piRNA clusters, and it is this mechanism that is thought to result in H3K9me2/3 establishment. Instead of recruiting HP1a, these loci are specifically bound by its homolog, Rhino (Rhi), through unknown mechanisms. Binding of Rhi and its cofactors Deadlock (Del), Moonshiner (Moon), and Cutoff (Cuff) ensures the production of piRNA cluster transcripts. Aub–piRNA complexes might serve to reinitiate the ping-pong cycle by generating new sense piRNAs for Ago3 loading, which can, in turn, produce a reshaped pool of trail piRNAs that associate with Piwi. This process might allow resetting of the piRNA pool and target diversification in each generation. (b) Maternally deposited piRNAs protect against hybrid dysgenesis. PIWI–piRNA complexes capable of targeting active transposon copies are maternally deposited. Males do not contribute piRNAs to their offspring. During embryonic development of the next generation, piRNAs scan for nascent transcripts with sequence complementarity. Targets are silenced through chromatin modifications such as H3K9me2/3. However, in cases where females lack piRNAs to protect against transposon insertions in the parental genome, these transposons can escape silencing and result in DNA damage and infertility.