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

Abstract

PIWI-interacting RNAs (piRNAs) guard germline genomes against the deleterious action of retroviruses and other mobile genetic elements. How piRNAs faithfully discriminate between self and non-self to restrict all mobile elements while sparing essential genes remains a key outstanding question in genome biology. 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 of the ZUC-processor to cut in front of a Uridine determines the ultimate processing site. We observe a mouse-specific roadblock that relocates the Goldilocks zone and generates an opportunity for consecutive trimming by PNLDC1. 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.

Target-specific requirements for RNA interference can arise through restricted RNA amplification despite the lack of specialized pathways

“…In support of such exonucleolytic trimming in conjunction with Argonaute binding, the 3'-5' exonuclease SND1 has been shown to trim the 3' ends of miRNAs bound to AGO1 in Arabidopsis (46). Furthermore, piRNA maturation in Drosophila and mice suggests a model where piwi-type Argonautes bind the 5' end of the pre-piRNA followed by endonucleolytic cutting and exonucleolytic trimming to generate consistently sized mature piRNAs (47).…”

CRISPR‐Cas9 Genome Editing and Rapid Selection of Cell Pools

“…These 5′ monophosphorylated RNA fragments are then loaded into Piwi and become pre‐piRNAs. Zucchini cuts again at the extended piRNA 3′ end to generate a final piRNA length that resembles a footprint of the PIWI protein and follows sequence preferences of the ZUC‐processor complex (Stoyko, Genzor, & Haase, 2022). RNA helicases contribute to piRNA processing and the formation of piRNA silencing complexes, but their precise functions and interplay with Zucchini and Piwi remain largely elusive (Ishizu, Kinoshita, Hirakata, Komatsuzaki, & Siomi, 2019; Yamashiro et al., 2020).…”