Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production

Abstract

PIWI-interacting RNAs (piRNAs) protect the animal germ line by silencing transposons. Primary piRNAs, generated from transcripts of genomic transposon "junkyards" (piRNA clusters), are amplified by the "ping-pong" pathway, yielding secondary piRNAs. We report that secondary piRNAs, bound to the PIWI protein Ago3, can initiate primary piRNA production from cleaved transposon RNAs. The first ~26 nucleotides (nt) of each cleaved RNA becomes a secondary piRNA, but the subsequent ~26 nt become the first in a series of phased primary piRNAs that bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. The ping-pong pathway increases only the abundance of piRNAs, whereas production of phased primary piRNAs from cleaved transposon RNAs adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence.

Fig. 1

Zuc-dependent phasing of primary piRNAs. (A) Ping-Pong analysis of all piRNAs from w¹, aub^HN2/QC42; ago3^t2/t3 and zuc^HM27/Df. (B) Distance from the 3′ ends of upstream piRNAs to the 5′ ends of downstream piRNAs on the same genomic strand. (C) Nucleotide composition immediately after the 3′ ends of uniquely mapping 42AB piRNAs. Z₁₀: Ping-Pong; Z₁: phasing.

Fig. 2

Contribution of maternal and secondary piRNAs to phasing. (A) Ping-Pong, phasing, and +1U percentage were analyzed for P{GSV6}42A18-derived piRNAs inherited paternally or maternally, with or without Vasa. (B) Length distribution of Piwi-bound, uniquely mapping P{GSV6}42A18 piRNAs. Reads were normalized to flamenco-derived, uniquely mapping piRNAs.

Fig. 3

Phasing of Piwi-bound piRNAs after sites of Aub- or Ago3-cleavage. Distance was measured from the 5′ ends of Piwi-, Ago3-, and Aub-bound piRNAs to sites of Aub- or Ago3-cleavage on the same genomic strand.

Fig. 4

Mouse piRNAs are phased. (A) Length distribution of Piwi-bound, uniquely mapping fly piRNAs derived from 42AB in w¹, aub^HN2/QC42, ago3^t2/t3 and vasa^D5/PH165. Reads were normalized to flamenco-derived, uniquely mapping piRNAs. (B) Distance from the 3′ ends of upstream piRNAs to the 5′ ends of downstream piRNAs on the same genomic strand for uniquely mapping piRNAs in Tdrkh^+/− and Tdrkh^−/− mouse testes at 11 dpp. (C) Genomic nucleotide composition 29 nt before and 1 nt after the 3′ ends of uniquely mapping mouse piRNAs.