Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) are distinct classes of small RNAs required for proper germline development. To identify the roles of piRNAs and siRNAs in regulating gene expression in Caenorhabditis elegans, we subjected small RNAs and mRNAs from the gonads of piRNA and siRNA defective mutants to high-throughput sequencing. We show that piRNAs and an abundant class of siRNAs known as WAGO-class 22G-RNAs are required for proper expression of spermatogenic and oogenic genes. WAGO-class 22G-RNAs are also broadly required for transposon silencing, whereas piRNAs are largely dispensable. piRNAs, however, have a critical role in controlling histone gene expression. In the absence of piRNAs, histone mRNAs are misrouted into the nuclear RNAi pathway involving the Argonaute HRDE-1, concurrent with a reduction in the expression of many histone mRNAs. We also show that high-level gene expression in the germline is correlated with high level 22G-RNA production. However, most highly expressed genes produce 22G-RNAs through a distinct pathway that presumably involves the Argonaute CSR-1. In contrast, genes targeted by the WAGO branch of the 22G-RNA pathway are typically poorly expressed and respond unpredictably to loss of 22G-RNAs. Our results point to broad roles for piRNAs and siRNAs in controlling gene expression in the C. elegans germline.

SUMMARY The germline contains an immortal cell lineage that ensures the faithful transmission of genetic and, in some instances, epigenetic information from one generation to the next. Here, we show that in Caenorhabditis elegans, the small RNA 3′-2′-O-methyltransferase henn-1/HEN1 is required for sustained fertility across generations. In the absence of henn-1, animals become progressively less fertile, becoming sterile after ~30 generations at 25°C. Sterility in henn-1 mutants is accompanied by severe defects in germline proliferation and maintenance. The requirement for henn-1 in transgenerational fertility is likely due to its role in methylating and, thereby, stabilizing Piwi-interacting RNAs (piRNAs). However, despite being essential for piRNA stability in embryos, henn-1 is not required for piRNA stability in adults. Thus, we propose that methylation is important for the role of piRNAs in establishing proper gene silencing during early stages of development but is dispensable for their role in the proliferated germline.

Highlights d Piwi/PRG-1 prevents aberrant gene silencing in C. elegans d Aberrant gene silencing in piwi/prg-1 mutants can occur for at least 50 generations d rRNAs are misrouted into the endogenous RNAi pathway in piwi/prg-1 mutants d Both gain and loss of siRNAs are stochastic in piwi/prg-1 mutants Authors

Piwi-interacting RNAs (piRNAs) are a largely germline-specific class of small RNAs found in animals.Although piRNAs are best known for silencing transposons, they regulate many different biological processes. Here we identify a role for piRNAs in preventing runaway amplification of small interfering RNAs (siRNAs) from certain genes, including ribosomal RNAs (rRNAs) and histone mRNAs. In Caenorhabditis elegans, rRNAs and some histone mRNAs are heavily targeted by piRNAs, which facilitates their entry into an endogenous RNA interference (RNAi) pathway involving a class of siRNAs called 22G-RNAs. Under normal conditions, rRNAs and histone mRNAs produce relatively low levels of 22G-RNAs. But if piRNAs are lost, 22G-RNA production is highly elevated. We show that 22G-RNAs produced downstream of piRNAs likely function in a feed-forward amplification circuit. Thus, our results suggest that piRNAs facilitate lowlevel 22G-RNA production while simultaneously obstructing the 22G-RNA machinery to prevent runaway amplification from certain RNAs. Histone mRNAs and rRNAs are unique from other cellular RNAs in lacking polyA tails, which may promote feed-forward amplification of 22G-RNAs. In support of this, we show that the subset of histone mRNAs that contain polyA tails are largely resistant to silencing in piRNA mutants.