Boermel, Mandy, Butter, Falk, Dietz, Sabrina, Schreier, Jan, Ketting, Rene, Seistrup, Ann-Sophie, Domingues, Antonio, Bronkhorst, Alfred, Oorschot, Viola, L'Hernault, Steven, Phillips, Carolyn, Nguyen, Dieu
[
International Worm Meeting,
2021]
Germ cells possess specialized perinuclear, phase-separated compartments, also named condensates. Amongst others, they contain the mRNA surveillance machinery responsible for transposon silencing and fertility. In the nematode Caenorhabditis elegans, three condensates, P granules, Z granules and Mutator foci, are home to RNA interference-related pathways, driven by a highly diversified Argonaute sub-clade (WAGO) that mediates gene silencing. Intriguingly, it has been shown that WAGO-mediated gene silencing can be inherited via both oocyte and sperm. Especially the inheritance via sperm is remarkable, since significant amounts of cellular material, including Argonaute proteins, are expelled from maturing spermatids into so-called residual bodies. How then does sperm-mediated inheritance of cytoplasmic RNAi work? We genetically identify WAGO-3 as a major Argonaute protein required for the paternal inheritance of endogenous small RNAs. Just like other Argonaute proteins, like WAGO-1 and ALG-3, WAGO-3 localizes to P granules in naive germ cells. During spermatogenesis, however, P granules disappear and WAGO-3, but not WAGO-1 and ALG-3, accumulates in a newly identified condensate, the PEI granule. In contrast to P granules, PEI granules remain stable during later stages of spermatogenesis. They are retained within maturing spermatids and selectively keep WAGO-3 from accumulating in the residual body. Using immunoprecipitation experiments followed by label-free quantitative mass spectrometry, we identified two uncharacterized proteins: PEI-1 and PEI-2. Both proteins are specifically expressed during spermatogenesis and we dissect their roles in PEI granule transport and function. Based on correlative light and electron microscopy (CLEM) and genetic studies, proper segregation of PEI granules in mature sperm is coupled, likely via S-palmitoylation, to the myosin-driven transport of membranous organelles. Our results identify a new sperm-specific condensate, which we call PEI granules. While not essential for spermatogenesis, PEI granules are required for paternal inheritance of small RNAs and we reveal a novel mechanism for the subcellular sorting of condensates through coupling to transport of membranous structures. pei-like genes are also found in human and often expressed in testis, suggesting that the here identified mechanism of subcellular transport of membraneless organelles may be more broadly conserved.