Drosophila Ovarian Somatic Cells Research Articles

A Genome-wide RNAi Screen Draws a Genetic Framework for Transposon Control and Primary piRNA Biogenesis in Drosophila

A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and components of the piRNA pathway, wecarried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels.

Gatekeepers for Piwi–piRNA complexes to enter the nucleus

RNA silencing pathways are now recognized to participate in essential cellular functions ranging from the regulation of mRNA turnover to the suppression of the activity of potentially deleterious transposable elements (TEs). Piwi-interacting RNAs (piRNAs) are germline-specific, small silencing RNAs that suppress TE activity and maintain genome integrity during germline development. In Drosophila ovarian somatic cells, piRNAs are processed from long single-stranded RNAs by a Dicer-independent pathway and are loaded onto Piwi in the cytoplasm. The Piwi-piRNA complexes are then transported into the nucleus to exert TE silencing. This mechanism involves gatekeepers for a functional Piwi-piRNA complex to be imported, which parallels with the Tetrahymena Twi1p-scan RNA pathway used to carry out the programmed DNA elimination.

P41. Primary piRNA biogenesis occurs through Yb bodies in the Drosophila ovarian somatic cells

PIWI proteins, a subset of Argonaute proteins that are the key factors in small RNA-mediated gene silencing, are expressed predominantly in the germline and are associated specifically with PIWI-interacting RNAs (piRNAs), small RNAs expressed predominantly in the germline. The PIWI-piRNA complex silences transposable elements and other genes that show complementarities to the piRNA sequences. Without functions of PIWI proteins and piRNAs, transposable elements become active, which has high potential to injure the genome. Thus, both PIWI proteins and piRNAs are essential for maintenance of genomic integrity and germline development. piRNA in Drosophila ovarian somatic cells (OSCs) are generated only through the primary processing pathway and are loaded onto Piwi, a member of PIWI proteins. However, factors required for the primary piRNA processing remain elusive. We focused on elucidating the mechanism of the primary processing pathway using the OSC line, which we have established from the female germline stem cell/ovarian somatic sheet (fGS/OSS) line, and found that primary piRNA biogenesis occur through Yb bodies, cytoplasmic structures that have been previously implicated in RNA processing. armitage (armi), a gene encoding an ATP-dependent RNA helicase, is necessary for primary piRNA biogenesis in OSCs. Armi localizes Piwi to Yb bodies by associating with Piwi and Yb, the main component of Yb bodies. piRNA intermediates are loaded onto the Armi-Piwi complex and processed into mature piRNAs. Loss of Yb caused depletion of Yb bodies and resulted in a reduction in overall piRNA levels. Yb bodies also impacted piRNA intermediates production, suggesting that they play important roles in primary piRNA biogenesis. zucchini (zuc), a gene encoding a putative cytoplasmic nuclease, is involved in processing piRNA intermediates into mature piRNAs. Without Armi, Yb or Zuc, Piwi is not loaded with piRNAs and does not localize to nucleus. A Piwi mutant that lacks the nuclear localization signal and therefore is not localized in the nucleus failed to silence transposons. These data suggest that Armi, Yb and Zuc are required for primary piRNA biogenesis and Piwi function. We further identified components of Yb bodies other than Armi and Yb and cloned piRNA intermediates. We will discuss in detail primary piRNA biogenesis based on our analyses.