One Loop to Rule Them All: The Ping-Pong Cycle and piRNA-Guided Silencing

Abstract

The PIWI-interacting RNA (piRNA) pathway is a conserved defense mechanism that protects the genetic information of animal germ cells from the deleterious effects of molecular parasites, such as transposons. Discovered nearly a decade ago, this small RNA silencing system comprises PIWI-clade Argonaute proteins and their associated RNA-binding partners, the piRNAs. In this review, we highlight recent work that has advanced our understanding of how piRNAs preserve genome integrity across generations. We discuss the mechanism of piRNA biogenesis, give an overview of common themes as well as differences in piRNA-mediated silencing between species, and end by highlighting known and emerging functions of piRNAs.

Keywords: PIWI proteins; piRNA biogenesis; ping-pong amplification loop; post-transcriptional gene silencing (PTGS); transcriptional gene silencing (TGS); transposon control.

Figure 1

PIWI-interacting RNA (piRNA) Clusters and Transcription of piRNA Sources. The body of uni-strand piRNA clusters (left, top) carries repressive histone 3 lysine 9 trimethylation (H3K9me3) marks. Yet, clusters resemble coding genes in that their promoters contain histone 3 lysine 4 dimethylation (H3K4me2) marks. piRNA clusters also produce transcripts via RNA Polymerase II (Pol II) that have 5′ caps, undergo (alternative) splicing and are terminated by canonical poly(A) signals. Meiotic piRNA clusters show similar characteristics, yet often produce piRNAs from two nonoverlapping genomic strands (left, bottom). These clusters are transcribed from bidirectional promoters that are activated by the A-MYB transcription factor. By contrast, dual-strand clusters in Drosophila germ cells (right, top) carry H3K9me3 modifications, but show no H3K4me2 signatures typical for active promoters. Instead, these piRNA sources utilize Pol II-dependent noncanonical read-through transcription from neighboring coding genes. It is hypothesized that 3′ end processing of the nascent RNAs results in the release of uncapped piRNA precursors. Cutoff (Cuff), a component of the Rhino-Deadlock-Cutoff (Rhi-Del-Cuff) complex that is anchored on piRNA cluster bodies via the H3K9me3-binding capacity of Rhi, associates with these uncapped transcripts and protects them from degradation, splicing, and transcription termination. Finally, recruitment of UAP56 allows the delivery to processing sites located across the nuclear envelope (right, bottom). Abbreviations: CBC, cap-binding complex; Pld, phospholipase D; TSS, transcription start site; UAP56, U2AF65–associated protein.

Figure 2

De Novo Biogenesis of PIWI-interacting RNAs (piRNAs) in Somatic Cells. Following export from the nucleus, transcripts carrying signals of unknown nature (red question mark) are recognized as piRNA precursors and transported to the processing sites Yb bodies. There, Zucchini (Zuc) and its co-factors produce piRNA intermediates with a 5′ uracil. This processing step requires Vreteno (Vret), Minotaur (Mino), and Gasz, although the precise molecular mechanisms are not fully understood. Armi seems to be involved in resolving secondary structures of these piRNA intermediates before they are loaded into Piwi and undergo further processing. The 3′ end formation is carried out either by another cleavage event by Zuc, which results in the formation of phased Piwi-associated piRNAs, or, alternatively, piRNA intermediates are resected to their mature size by a putative exonuclease named ‘trimmer’ and its co-factor Papi. Following methylation by Hen1, mature piRNA-Piwi complexes enter the nucleus to exert silencing. Most factors required for de novo piRNA biogenesis are anchored in the outer mitochondrial membrane, suggesting a central role of mitochondria in this process.

Figure 3

The Ping-Pong Amplification Loop and Phased PIWI-interacting RNA (piRNA) Production. Argonaute3 (Ago3) associated with a sense piRNA recognizes and cleaves piRNA cluster transcripts to produce piRNA intermediates with a 5′ uracil (U), which are loaded into Aubergine (Aub), likely assisted by Spindle-E (Spn-E) (A). Maturation of piRNAs can follow two nonexclusive roads: cleavage by Zuc results in 3′ end formation of Aub-bound piRNAs, as well as production of phased primary piRNAs that associate with Piwi. Alternatively, 3′ end formation can occur via Papi-dependent trimming (B). The combination of both mechanisms is likely. Following Hen1-mediated methylation (C), mature Aub complexes receive symmetric dimethyl-arginine (sDMA) modifications at their amino-termini (D). sDMA-Aub is recruited by Krimper (Krimp), which also interacts with unloaded Ago3, thus bringing both factors in close proximity (E). Subsequent to piRNA-Aub-dependent detection and slicing of transposon RNAs, the 3′ cleavage product is loaded into Ago3 aided by Vasa (F). Maturation of Ago3-loaded intermediates via Zuc or trimming (G), followed by methylation by Hen1 (H) results in mature Ago3 complexes that in turn cleave cluster transcripts to start yet another cycle (I). In Drosophila ovaries, the ping-pong amplification loop is the predominant determinant that specifies piRNA precursors for Piwi (J). Capsuleen (Csul) and its cofactor Valois (Vls) add symmetric dimethyl-arginines (sDMA) modifications to Aub and Ago3. Qin inhibits homotypic Aub:Aub ping-pong by preventing Aub-sliced transcripts from becoming substrates for Aub (inset in center).

Figure 4

PIWI-interacting RNA (piRNA)-Mediated Transcriptional Silencing. In the Drosophila ovary, piRNA-Piwi/Asterix (Arx) complexes scan for, and detect, nascent transposon transcription. Upon target engagement, Piwi likely undergoes conformational changes that lead to the recruitment of Panoramix (Panx). This piRNA-protein (comprising Piwi, Arx, and Panx,) complex induces co-transcriptional repression through recruitment of general silencing machinery components. As a consequence, transposon bodies receive repressive histone 3 lysine 9 trimethylation (H3K9me3) marks, a modification produced by Eggless (Egg) and its cofactor Windei (Wde). Subsequent recruitment of HP1 to H3K9me3 leads to heterochromatin formation. In addition, Lysine-specific demethylase 1 (Lsd1) likely removes active histone 3 lysine 4 dimethylation (H3K4me2) marks from transposon promoter regions, leading to efficient suppression of transposons at the transcriptional level. Maelstrom (Mael), a putative single-stranded RNA-binding protein, is required for transcriptional silencing and blocks H3K9me3 spread.