piRNA Biogenesis in Drosophila melanogaster

Abstract

The PIWI-interacting RNA (piRNA) pathway is a conserved defense system that protects the genome integrity of the animal germline from deleterious transposable elements. Targets of silencing are recognized by small noncoding piRNAs that are processed from long precursor molecules. Although piRNAs and other classes of small noncoding RNAs, such as miRNAs and small interfering (si)RNAs, interact with members of the same family of Argonaute (Ago) proteins and their function in target repression is similar, the biogenesis of piRNAs differs from those of the other two small RNAs. Recently, many aspects of piRNA biogenesis have been revealed in Drosophila melanogaster. In this review, we elaborate on piRNA biogenesis in Drosophila somatic and germline cells. We focus on the mechanisms by which piRNA precursor transcription is regulated and highlight recent work that has advanced our understanding of piRNA precursor processing to mature piRNAs. We finish by discussing current models to the still unresolved question of how piRNA precursors are selected and channeled into the processing machinery.

Figure 1. Transcription of piRNA clusters

Figure 2. Processing of the 5'- and the 3'-end of piRNA

(A) The 5’-end of piRNAs can be formed either through the endonuclease (slicer) activity of the cytoplasmic Piwi proteins, Aub and Ago3, or through cleavage by the endonuclease Zucchini. Slicer cleavage of piRNA precursors is guided by complementary piRNA. The 5’-end of a slicer product is shifted by exactly 10 nt relative to the 5’ end of the guide piRNA. Cleavage mediated by Zuc is independent of guide piRNA. piRNAs formed through slicer-dependent mechanism are loaded into Aub and Ago3, while piRNAs formed by Zuc are loaded into Piwi and Aub. The 3’-end of piRNAs can be formed by three mechanisms: through endonucleolytic 25 cleavage by Zuc or slicer or by 3’ to 5’ trimming of longer precursors by the exonuclease Nibbler. (B) piRNA processing in somatic follicular and germline cells of Drosophila ovary. Only Piwi, but not Aub and Ago3, is expressed in follicular cells. Therefore, in these cells both ends of mature piRNAs are formed exclusively through Zuc-mediated processing with possible contribution of a 3’-end trimming activity. The single Zuc cleavage can simultaneously generate the 5’-end of a downstream and the 3’-end of an upstream RNA resulting in a characteristic phased pattern of piRNAs. Slicer-dependent and Zuc-dependent processing co-exists and the two pathways cooperate in germline nurse cells. When a new piRNA is formed by the slicer-dependent mechanism and loaded into Aub or Ago3, it can guide formation of the next piRNA giving rise to the so-called ping-pong cycle. Products of Aub-guided cleavage are predominantly loaded into Ago3, while products of Ago3-guided cleavage are loaded onto Aub. Slicer-dependent cleavage by Aub or Ago3 also directs Zuc-dependent substrate RNA processing.

Figure 3. Selection of piRNA precursors for processing

The selection mechanisms of piRNA precursors seems to differ between somatic (follicular) and germline cells. In follicular cells, specific sequences in piRNA precursors are recognized in the cytoplasm through binding by Yb or other yet-to-be-identified RNA-binding proteins (X). It was proposed that Yb, which forms cytoplasmic granules called Yb-bodies, recruit other factors necessary for piRNA processing such as Zuc, Vret and Armi. Two models were proposed for selection of piRNA precursors in germline cells. According to the ‘persistent nuclear mark’ model piRNA precursors are marked and licensed for processing in the nucleus. The mark – probably an RNA-binding protein – was proposed to shuttle with the piRNA precursor into the cytoplasm and activate the processing machinery. The identity of the mark is not known, although its deposition was proposed to depend on the RDC complex, which is present on chromatin of piRNA-generating loci. Normal mRNAs – that are destined for translation – are bound by the exon-junction complex (EJC) loaded on mRNA as a result of productive splicing. It was proposed that stalled splicing of piRNA precursors might induce marking of piRNA precursors and trigger their processing in the cytoplasm. The TREX complex, which is loaded on piRNA precursors in an RDC-dependent fashion is a candidate for such nuclear mark. According to the second model, selection of RNA for processing only happens in the cytoplasm and is governed by complementary piRNAs that recognize potential precursors and trigger their processing through slicer- and zuc-dependent mechanisms.