piRNAs and Piwi clade Argonautes arose in the primordial metazoan ancestor 
and are generally restricted to the germline, where they act in an RNA-induced silencing complex (RISC) to silence foreign genetic elements. From protozoa to mammals, loss of Piwi proteins, and consequently piRNAs, results in abnormal fertility phenotypes or sterility, revealing their highly conserved and essential role in animal reproduction 2
. piRNAs are incredibly diverse, with tens of thousands of unique sequences expressed in any single organism. While piRNAs in many organisms map to large, broadly syntenic genomic clusters, the sequences are not conserved among even closely related species, and no unifying sequence features have been identified beyond a bias among primary piRNAs for a 5′ uridine 
The mechanisms of de novo piRNA biogenesis remain elusive. In fly and mouse, primary piRNAs appear to be processed from long, single-stranded RNA precursors 
. This long transcript is cleaved by the endoribonuclease Zucchini with little or no sequence specificity to generate candidate piRNA 5′ ends 
, which are likely subsequently purified according to the binding preferences of the Piwi proteins that bind primary piRNAs 
. Silkworm data suggest that the 3′ ends of these piRNA precursors are then trimmed by a 3′ to 5′ exonuclease until the 3′ end is sufficiently short for anchoring by Piwi to protect against further trimming 
. The 3′ end is then methylated to prevent degradation 
. While recent studies have shed light on the biogenesis of primary piRNAs in many animal models, little is known in any organism about how primary piRNA expression is regulated or how specific sequences are designated as piRNAs.
21U RNAs, a class of germline-enriched small RNAs, represent the piRNAs of Caenorhabditis elegans
. They are terminally methylated 
, show a 5′ uridine bias 
, and are dependent upon and bound by the Piwi Argonaute PRG-1 
, which is required for normal fertility 
. Yet C. elegans
piRNAs exhibit some unusual features. While the vast majority of 21U RNAs map to two large genomic clusters on chromosome IV, the loci do not exhibit prominent strand biases 
. The 21U RNAs also do not appear to play a prominent role in silencing transposable elements, a main function of mouse and fly piRNAs, nor do they engage a ping-pong amplification mechanism 
. Rather, PRG-1 and the 21U RNAs target aberrant and coding transcripts broadly via imperfect complementarity, triggering production of secondary endogenous siRNAs 
. These 21U RNA-dependent 22G RNAs can induce chromatin changes to establish dominant, heritable target silencing 
. 21U RNAs evolve rapidly, presumably constrained only by selection against sequences that silence mRNAs; thus, mismatch-tolerant 21U RNAs constitute an epigenetic memory of self versus non-self. Finally, a conserved motif lies upstream of 21U RNA genomic loci 
. This stretch of sequence, which includes an eight-nucleotide (nt) core motif approximately 40 nt upstream of the 21U RNA locus, is conserved across divergent nematodes 
. Recently, Cecere et al. found that this motif is bound by forkhead family transcription factors and that deletion of the core motif abrogates 21U RNA expression 
, but it is still unknown how 21U RNA sequences are defined and how their expression is regulated.
Here, we demonstrate that piRNAs are expressed autonomously in C. elegans. Combining computational and transgenic approaches, we find that the conserved core motif defines the piRNA transcriptional cassette, specifying expression of 21U RNAs from genomic thymidines situated at an optimal distance downstream to determine which genomic sequences are expressed as C. elegans piRNAs. Core motifs also encode information dictating germline-specific expression of 21U RNAs. We show that more than 70% of C. elegans piRNAs are preferentially enriched in male or female germline. Unexpectedly, this germline enrichment appears to be enforced by a single nucleotide position within the core motif. We demonstrate autonomous expression of synthetic 21U RNAs from multiple minimal transgenic cassettes consisting only of the 8 nt core motif, the ~40 nt intervening genomic spacer, the 21U RNA sequence, and ~50–100 nt of flanking genomic context. Finally, we use single-copy transgenes integrated in genomic isolation to show that the clustered organization of endogenous piRNA loci is entirely dispensable for robust piRNA expression. Together, our results suggest that each 21U RNA locus encodes all of the information necessary for driving independent, autonomous transcription from more than 15,000 unique piRNA loci in C. elegans.