Placentino, Maria et al. (2021) International Worm Meeting "Intrinsically disordered protein PID-2 modulates Z granules and is required for heritable piRNA-induced silencing in the Caenorhabditis elegans embryo"

Butter, Falk, Isolehto, Ida, Placentino, Maria, Dietz, Sabrina, Ketting, Rene, de Jesus Domingues, Antonio Miguel, Hellmann, Svenja, de Albuquerque, Bruno FM, Schreier, Jan

[International Worm Meeting, 2021]

Argonaute proteins (AGOs) are involved in many gene regulatory processes and use small RNAs as guides to find their target. C. elegans employs several small RNA mediated pathways to initiate and maintain proper gene regulation throughout development. In germ cells, 21U RNAs and associated Argonautes are particularly important to maintain an immortal germline. 21U RNAs trigger a silencing mechanism that is dependent of amplification of 22G RNAs by RNA dependent RNA polymerase. Strikingly, silencing responses can become independent of triggering 21U RNAs and maintained across generations, a mechanism called RNA epigenetic memory (RNAe). Components of small RNA pathways often localize to perinuclear phase separated condensates termed germ granules. The exact functions of germ granules are not clear, but it is known that they are necessary for proper function of small RNA pathways and inheritance of small RNAs. Intrinsically disordered regions (IDRs) can mediate multivalent protein-protein interactions and are often important for phase separation. We show that maternally provided 21U RNAs are sufficient to initiate RNAe in embryos and that the IDR containing protein PID-2/ZSP-1 is required for this process. PID-2/ZSP-1 localizes to and affects the appearance and number of Z-granules[1][2]. Furthermore, we found that PID-2/ZSP-1 mutually exclusively interacts with PID-4 and PID-5. Mutants lacking PID-4 and PID-5 partially phenocopy pid-2 mutants. Interestingly, even though PID-4 and PID-5 interact with PID-2, they localize to the adjacent P-granules rather than Z-granules. We identify distinct interacting proteins for PID-4 and PID-5. For instance, PID-5 interacts with the X-prolyl aminopeptidase APP-1 and PID-5 itself has an APP-1 related domain that is likely catalytically inactive. Several AGOs contain intrinsically disordered N-terminal tails and are predicted substrates for APP-1, suggesting a role for N-terminal processing in small RNA pathways and germ granule dynamics. Taken together, we have found a protein complex that affects small RNA mediated regulation and germ granules, potentially providing a mode of communication between different granules, and are on the route to understand the functional relationship between small RNA mediated gene regulation, epigenetic inheritance, and phase separation. [1] Placentino et al. EMBO J, 2021 [2] Wan et al. EMBO J, 2021

Schreier, Jan et al. (2021) International Worm Meeting "A novel sperm-specific compartment secures a cytoplasmic Argonaute protein for paternal epigenetic inheritance of small RNA-mediated gene silencing"

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.