Abstract
Epigenetic information can be inherited for multiple generations (termed transgenerational epigenetic inheritance or TEI)1,2. Non-coding RNAs have emerged as important mediators of TEI, although the mechanism(s) by which non-coding RNAs mediate TEI remains poorly understood. dsRNA-mediated gene silencing (RNAi) in C. elegans is a robust example of RNA-directed TEI3–5. To further our understanding of RNA-directed TEI, we conducted a genetic screen in C. elegans to identify genes required for RNAi inheritance. Our screen identified the conserved RNA helicase/Zn finger protein ZNFX-1 and the Argonaute protein WAGO-4. We find that WAGO-4 and ZNFX-1 act cooperatively in inheriting generations to mark mRNAs of genes undergoing heritable silencing to maintain small interfering (si)RNA expression across generations. Additionally, we find that ZNFX-1/WAGO-4 localize to a liquid droplet organelle termed the P granule in early germline blastomeres. Later in development, ZNFX-1/WAGO-4 demix from P granule components to form an independent liquid droplet organelle that we term the Z granule. In the adult germline, Z granules assemble into ordered tri-droplet assemblages with P granules and another germline droplet-like foci termed the Mutator foci. This work identifies conserved RNA-binding proteins that contribute to RNA-directed TEI in C. elegans, defines a liquid droplet organelle termed the Z granule, and shows that liquid droplet organelles can undergo developmentally regulated cycles of mixing/demixing and assemble into spatially ordered multi-droplet structures. We speculate that temporal and spatial ordering of liquid droplets helps cells organize and coordinate the complex RNA processing pathways underlying gene regulatory systems, such as RNA-directed TEI.