Expression and Localization of Piwi-interacting RNAs in Drosophila During Oogenesis

Abstract

The activity of transposable elements (TEs), repetitive mobile DNA sequences found in eukaryotic genomes, has been linked to DNA damage and mutation. Thus, it is necessary for organisms to develop cellular mechanisms for maintaining genomic integrity. One type of small non-coding RNA, Piwi-interacting RNAs (piRNAs), has been associated with transposon silencing during development in various organisms. They are distinguishable from other small non-coding RNAs by their interactions with Piwi family proteins, as well as a unique biogenesis mechanism. We use a Drosophila model to characterize piRNA biogenesis and potential pathway factors. In general, piRNAs are thought to be produced as a long transcript from "clusters", regions within the genome enriched with transposons. The long piRNA transcripts are subsequently processed into shorter piRNA strands in the perinuclear nuage organelle. Two distinct pathways have been proposed: the "Ping-Pong pathway" and "primary processing pathway". In order to characterize these pathways, we use a novel small molecule fluorescent in situ hybridization (smFISH) method to detect the presence of piRNAs with confocal microscopy. Our results indicate that piRNA transcripts from the same cluster accumulate in distinct nuage regions within germline cells. Additionally, not all piRNAs transcripts may reach the processing stage of piRNA biogenesis. Most significantly, we detected transcription of the uni-strand cluster flamenco¸which was previously believed to be specific to the somatic cells. Our findings indicate that the hypothesized Ping-Pong pathway and primary processing pathway may not be exclusively applicable to piRNA biogenesis in the germline or soma. Further studies should be performed to localize piRNA biogenesis mechanisms and identify potential biogenesis factors.