PIWI-interacting RNAs (piRNAs), 24–30 nucleotide long RNAs found in the germ cells of animals, are unique among small silencing RNAs in that they require neither an RdRP nor Dicer for their production (Figure 1Figure 1C). Instead, they are thought to derive from single-stranded precursor RNAs tens or hundreds of thousands of nucleotides long. piRNAs bind a distinct subclade of Argonaute proteins, the PIWI proteins, which include Piwi, Aubergine, and Ago3 in flies, Smedwi in planaria, Siwi in sea urchins, and Hiwi in humans. In vitro, Piwi can cleave a target RNA, suggesting that piRNAs regulate their targets post-transcriptionally, but other evidence suggests they promote heterochromatin assembly in the nucleus. piRNAs were first identified in 2001 in flies, where they repress selfish genetic elements such as retrotransposons.piRNAs contain 5′ monophosphate and 2′-O-methyl, 3′ hydroxy termini. The 2′-O-methyl group is added, at least in flies, by the methyltransferase Hen1. In flies, piRNAs are thought to arise from ‘master loci’ rich in transposons, then act in trans to silence dispersed copies of the selfish genetic elements present in the original trigger locus. piRNAs have also been implicated in silencing transposons early in mammalian spermatogenesis. In mammals, however, many piRNAs map to genomic clusters that do not contain repetitive sequences. The functions of these piRNAs are unknown.piRNAs may be generated by reciprocal cycles of PIWI-protein-catalyzed slicing followed by 3′ trimming by an exonuclease. In Drosophila, for example, the first 10 nucleotides of many piRNAs bound to Aubergine, most of which are antisense to transposable element transcripts, can be paired to piRNAs associated with Ago3. (Recall that all Argonaute proteins known to cleave their RNA targets cut after small RNA nucleotide 10.) Nearly all Aubergine-bound piRNAs begin with uracil, whereas Ago3-associated piRNAs, which are almost all in the sense orientation, typically contain an adenosine at nucleotide 10 — reflecting their base pairing with the first nucleotide of an antisense piRNA. Hence the suggestion that piRNAs are amplified by reciprocal rounds of cleavage, in which Ago3 sense piRNAs direct cleavage of antisense transcripts producing the 5′ monophosphate end of Aub and Piwi antisense piRNAs. A 3′-to-5′ exonuclease could then trim the 3′ end of piRNA transcripts, perhaps acting together with the Hen1 methyltransferase, which might terminate the trimming process by adding a 2′-O-methyl group to the 3′ terminus of the mature piRNA.