FBS Colloquia No.371Germline Biology Group

Analysis of the Molecular Function of Squash, a Crucial piRNA Component for Germline Genome Integrity

PIWI-interacting RNAs (piRNAs) are essential for repressing the expression of mobile transposable elements (TEs), thereby safeguarding the genomic integrity in animal gonads. In Drosophila, piRNAs originate from TE RNAs and piRNA precursor transcripts derived from genomic loci known as piRNA clusters. These precursors undergo processing into mature piRNA within a specialized, membrane-less organelle termed nuage, facilitated by a feed-forward amplification mechanism referred to as ping-pong cycle. Nuage encompasses a repertoire of proteins, each possessing distinct domains and motifs crucial for piRNA biogenesis. This includes PIWI-family proteins (Aubergine and Argonaute3), RNA helicases (Vasa and Spindle-E), and Tudor domain-containing proteins such as Krimper and Tejas.

Squash (squ), another Drosophila protein localized to nuage, is unique in that it lacks identifiable domains or motifs. The loss of squ function results in female sterility, upregulation of some TEs, and a reduction of piRNAs in the ovaries. However, the molecular mechanisms underlying these phenotypes remain poorly understood. Our comprehensive analysis elucidates the dysfunctional interplay among piRNA pathway components in the absence of squ, characterized by the severe mislocalization of Krimper from nuage, decreased expression of Ago3, and reduced Aub-bound piRNAs. Additionally, Squ was found to co-immunoprecipitate with the RNA helicase Spindle-E in both ovarian lysate and S2 cells, suggesting a direct interaction. Moreover, the accumulation of piRNA cluster transcripts from loci 42AB and 38C near nuage in the absence of squ function suggests an interruption in their processing pathway. This presentation will explore the potential molecular roles of Squ in piRNA-mediated genome defense mechanisms.

The female germ cell-specific apoptosis regulation in Drosophila

Apoptosis is a form of programmed cell death that is essential for development and tissue homeostasis in multicellular organisms, and functions to remove unwanted or damaged cells. While the basis of the apoptotic machinery in Drosophila is well known, its regulation in germline cells remains elusive. Here, we identify stand still (stil) as a crucial regulator specific to germline survival through the repression of the proapoptotic gene reaper (rpr). stil encodes a 35.8 kDa BED-type zinc finger protein, which is localized onto chromosomes. The loss of stil function almost completely abrogated ovarian germline cells by the time of eclosion and resulted in the upregulation of proapoptotic genes, including rpr and hid. By a TUNEL labeling assay, we found that stil-deficient female germline cells underwent apoptotic cell death. Reporter assays using Drosophila S2 cells showed that Stil repressed the transcription from the rpr promoter region via the N-terminal BED-type zinc finger domain, suggesting that Stil interacts with the regulatory region to repress its expression. Our study highlights the crucial role of Stil in safeguarding female germline cells from apoptosis, providing new insights into the molecular mechanisms underlying germline cell survival.