PIWI-interacting RNAs (piRNAs) are small RNAs that defend genomes against transposons and regulate endogenous gene expression in animal reproductive systems[1]. Many of the piRNA pathway factors are enriched in various perinuclear foci, including P/Z granules and Mutator granules[2-4]. How these liquid condensates interact and coordinate for piRNA mediated gene silencing is not well understood. Using a sensitized piRNA reporter strain, we performed a candidate RNAi screen and identified CGH-1 as a novel factor required for piRNA silencing. CGH-1, a conserved DEAD box helicase, is a key component of P bodies. CGH-1 localizes to cytoplasmic storage bodies (germline P bodies) and perinuclear P granules in the C. elegans germline. Our imaging analyses suggest that CGH-1 regulates the localization of piRNA pathway factors. In both cgh-1 RNAi and knockout mutants, PIWI Argonaute PRG-1 is delocalized from perinuclear P granules; CSR-1, an Argonaute that counteracts piRNA silencing, is also not properly localized to P granules, while accumulation of two constitutive P granule components, PGL-1 and GLH-1, are largely normal. Perinuclear foci of Mutator protein MUT-16 and Z granule protein WAGO-4 are also partially dispersed in cgh-1 mutants. Interestingly, CGH-1 foci accumulate at the cytoplasmic side of P granules, suggesting CGH-1-containing bodies represent a separate but closely related granule to P granules, or that these bodies represent a subdomain of P granules. In addition, crosslinking and IP-MS identified P granule factors, including PRG-1, GLH-1 and CSR-1, together with P body components, as CGH-1 interactors, suggesting a role of CGH-1 in mediating the interactions between these two granules. As cgh-1 is expressed broadly and cgh-1 null mutants are lethal, we developed an auxin-inducible degron (AID) tagged CGH-1::degron::GFP strain to perform the germline specific depletion of GGH-1. Small RNA-sequencing suggests that piRNA levels are reduced slightly, while CSR-1, but not WAGO, bound 22G-RNAs have a 2-fold increase in cgh-1 depleted animals. In summary, our results support a model that the RNA helicase CGH-1 promotes piRNA silencing through regulating the liquid condensation of piRNA pathway factors at perinuclear foci. Our research further highlights the critical role of an RNA helicase in mediating the interaction between distinct phase-separated bodies. 1. Ozata et al., Nat Rev Genet (2019); 2. Lee et al., eLife (2020); 3. Placentino et al., EMBO J (2020); 4. Spichal et al., Nat Commun (2021)

Authors: DU, ZhenZhen, Zhang, Ying, He, Tao, Brown, Jordan, Hu, Yabing, He, Chenxia, Shi, Kun, Wu, Wei-Sheng, Lee, Heng-Chi, Zhang, Donglei

Affiliations:
- Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan
- Department of Electrical Engineering, National Cheng Kung University, Tainan