Hierarchical length and sequence preferences establish a single major piRNA 3′-end

PIWI-interacting RNAs (piRNAs) guard germline genomes against the deleterious action of mobile genetic elements. PiRNAs use extensive base-pairing to recognize their targets and variable 3′ends could change the specificity and efficacy of piRNA silencing. Here, we identify conserved rules that ensure the generation of a single major piRNA 3′end in flies and mice. Our data suggest that the PIWI proteins initially define a short interval on pre-piRNAs that grants access to the ZUC-processor complex. Within this Goldilocks zone, the preference to cut in front of Uridine determines the ultimate processing site. We observe a mouse-specific roadblock that relocates the Goldilocks zone and generates an opportunity for consecutive trimming. Our data reveal a conserved hierarchy between length and sequence preferences that controls the piRNA sequence space. The unanticipated precision of 3′end formation bolsters the emerging understanding that the functional piRNA sequence space is tightly controlled to ensure effective defense.