The piRNA pathway in flies: highlights and future directions

Abstract

Piwi proteins, together with their bound Piwi-interacting RNAs, constitute an evolutionarily conserved, germline-specific innate immune system. The piRNA pathway is one of the key mechanisms for silencing transposable elements in the germline, thereby preserving genome integrity between generations. Recent work from several groups has significantly advanced our understanding of how piRNAs arise from discrete genomic loci, termed piRNA clusters, and how these Piwi-piRNA complexes enforce transposon silencing. Here, we discuss these recent findings, as well as highlight some aspects of piRNA biology that continue to escape our understanding.

Figure 1

A model for piRNA biogenesis in the Drosophila ovary. (a) Two distinct piRNA pathways are active in a stage 10 egg chamber of the Drosophila ovary. The nurse cells that provide nutrients to the oocyte and the oocyte itself make up the germ cells of the ovary, shown in blue. The monolayer of somatic follicle cells surrounding the oocyte is shown in green. Nuclei are indicated as circles within each cell. (b) In follicle cells, primary piRNAs arise from flamenco and are processed through a cascade of enzymatic cuts. Transcription by RNA polymerase II (Pol2) depends on deposition of Histone 3 Lysine 9 trimethyl marks (H3K9me3) by Eggless (Egg). Regulatory cis-acting elements, indicated as green boxes, upstream of the transcriptional start site could affect Pol2 recruitment and transcription. Additionally, clusters could carry cis elements within themselves, shown in red, that affect downstream processing. After processing of the primary cluster transcript by unknown activities, piRNA intermediates are cleaved by the nuclease, Zucchini (Zuc). After 5′ end formation, transcripts with a U at the first position are preferentially loaded into Piwi. Trimming activity, which could be carried out by redundant nucleases, shortens the transcript to its mature length. This process is coupled to 2′-O-methylation by Hen1. (c) The transcription of clusters in germ cells can occur bidirectionally. In addition to Egg, the HP1 homolog Rhino (Rhi) and Cutoff (Cuff) are essential for transcription. Subsequently, the helicase UAP56 binds the primary transcript and escorts it to the nuclear periphery. There, it is handed over to another RNA helicase Vasa (Vas) and arrives at its site of biogenesis, the nuage. After primary processing by similar machinery as in (a), primary piRNAs are loaded into Piwi and Aub, and potentially Ago3. These primary piRNAs can be used to kick-start the ping-pong amplification cycle, which silences transposons post-transcriptionally.

Figure 2

Transcriptional silencing of transposable elements by Piwi-piRNA complexes in the soma. The X chromosome of Drosophila melanogaster (chrX) is shown. A simplistic view of its chromatin state is indicated in shades of gray. The transcriptionally active euchromatin in white harbors a full-length copy of the retroelement, ZAM (indicated as a red box). An inactive remnant of the same element (in red) can be found within the flamenco piRNA cluster in pericentromeric heterochromatin. After transcription and processing of flamenco, this fragment gives rise to antisense piRNAs that are loaded into Piwi in the cytoplasm (indicated as red piRNA species). Upon reimport into the nucleus, these Piwi-piRNA complexes recognize active transcription of the full-length ZAM copy by RNA polymerase II (Pol2) based on sequence complementarity. This recognition leads to the recruitment of additional factors such as Maelstrom (Mael) and unknown chromatin remodelers. Ultimately, the deposition of H3K9me3 marks leads to loss of Pol2 occupancy and the transcriptional silencing of ZAM.