(52) Establishing a Degron System in Drosophila Ovarian Somatic Sheath Cells (OSCs) to Better Understand the PIWI-Interacting RNA (piRNA) Pathway

Abstract Authors: Establishing a Degron System in Drosophila Ovarian Somatic Sheath Cells (OSCs) to Better Understand the PIWI-Interacting RNA (piRNA) Pathway

Hibo Hassan¹; Angel Jaimes2; Qingcai Meng²; Elizabeth Giordano³; Astrid Haase¹ 

Laboratory of Cellular & Developmental Biology, NIDDK, NIH, Bethesda, United States

Abstract Text:

PIWI-interacting RNA (piRNA) pathway function in animals and humans to protect genome integrity and regulate gene expression. Specifically found in the germline cells, non-coding small RNAs called piRNAs silence harmful mobile genetic elements, also known as transposons. Understanding how this pathway operates is important, as a dysfunctional piRNA pathway leads to the uncontrolled transposon activity and ultimately causes germ cell loss and infertility. Drosophila ovarian somatic sheath cells serve as an excellent model system, providing an ex vivo platform that preserves the highly conserved molecular mechanisms of a functional piRNA pathway. piRNA biogenesis starts with long single-stranded RNA precursors, which are cleaved by the endonuclease Zucchini (Zuc). This cleavage generates RNA fragments that are loaded into PIWI proteins to become piRNAs. Mature piRNAs guide their PIWI protein partners to recognize target transcripts through base-pair complementarity. Another key component of the pathway is the RNA helicase Armitage (Armi), which is involved in piRNA processing. However, the precise function of Armi and the nature of its interactions with other pathway components remain unclear. My project aims to probe the precise function of Armitage (Armi) in piRNA biogenesis. I aim to test the possible function of Armi at two distinct molecular steps: the recognition of piRNA precursors by Zuc and loading of piRNA fragments into Piwi proteins to form functional Piwi-piRNA complexes. To address this question, I am establishing an Armi-Degron allele in OSC. Adding a degron tag to Armi’s open reading frame will enable us to regulate Armi protein stability. Once a functional degron system is established, we can further explore how piRNA precursors and mature piRNAs behave upon timed degradation of Armitage. If Armitage is required for Zuc-mediated cleavage of piRNA precursors, we expect to observe an accumulation of long, uncut precursor transcripts in RNA-sequencing experiments and a reduction of mature piRNAs. If Armi functions in loading of piRNAs into Piwi proteins, we expect to observe a loss of Piwi-bound piRNAs without changes in piRNA-precursor levels. Results from my project will provide mechanistic insight into piRNA biogenesis and thus further our understanding of how the integrity of germline genomes is protected.