In Caenorhabditis elegans, the piRNA (21U RNA) pathway is required to establish proper gene regulation and an immortal germline. To achieve this, PRG‐1‐bound 21U RNAs trigger silencing mechanisms mediated by RNA‐dependent RNA polymerase (RdRP)‐synthetized 22G RNAs. This silencing can become PRG‐1‐independent and heritable over many generations, a state termed RNA‐induced epigenetic gene silencing (RNAe). How and when RNAe is established, and how it is maintained, is not known. We show that maternally provided 21U RNAs can be sufficient for triggering RNAe in embryos. Additionally, we identify PID‐2, a protein containing intrinsically disordered regions (IDRs), as a factor required for establishing and maintaining RNAe. PID‐2 interacts with two newly identified and partially redundant eTudor domain‐containing proteins, PID‐4 and PID‐5. PID‐5 has an additional domain related to the X‐prolyl aminopeptidase APP‐1, and binds APP‐1, implicating potential N‐terminal proteolysis in RNAe. All three proteins are required for germline immortality, localize to perinuclear foci, affect size and appearance of RNA inheritance‐linked Z granules, and are required for balancing of 22G RNA populations. Overall, our study identifies three new proteins with crucial functions in C. elegans small RNA silencing.

Transgenerational epigenetic inheritance (TEI) describes the transmission of gene-regulatory information across generations without altering DNA sequences, and allows priming of offspring towards transposable elements (TEs) and changing environmental conditions. One important mechanism that acts in TEI is based on small non-coding RNAs. Whereas factors for maternal inheritance of small RNAs have been identified, paternal inheritance is poorly understood, as much of the cellular content is extruded during spermatogenesis. We identify a phase separation-based mechanism, driven by the protein PEI-1, which is characterized by a BTB-BACK domain and an intrinsically disordered region (IDR). PEI-1 specifically secures the Argonaute protein WAGO-3 within maturing sperm in C. elegans. Localization of PEI granules in mature sperm is coupled, via Spalmitoylation, to myosin-driven transport of membranous organelles. pei-1-like genes are also found in human and often expressed in testis, suggesting that the here identified mechanism may be broadly conserved.

Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

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