Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis

Abstract

In animal gonads, PIWI-clade Argonaute proteins repress transposons sequence-specifically via bound Piwi-interacting RNAs (piRNAs). These are processed from single-stranded precursor RNAs by largely unknown mechanisms. Here we show that primary piRNA biogenesis is a 3'-directed and phased process that, in the Drosophila germ line, is initiated by secondary piRNA-guided transcript cleavage. Phasing results from consecutive endonucleolytic cleavages catalyzed by Zucchini, implying coupled formation of 3' and 5' ends of flanking piRNAs. Unexpectedly, Zucchini also participates in 3' end formation of secondary piRNAs. Its function can, however, be bypassed by downstream piRNA-guided precursor cleavages coupled to exonucleolytic trimming. Our data uncover an evolutionarily conserved piRNA biogenesis mechanism in which Zucchini plays a central role in defining piRNA 5' and 3' ends.

Figure 1. Aub/AGO3 mediated slicing triggers phased piRNA biogenesis.

(A) Normalized piRNA populations from Rhino depleted ovaries or from control ovaries mapping to the row mRNA. (B) As in (A) but piRNAs bound to Aub/AGO3/Piwi (22) are shown individually. (C) Alignment of the 1360 trigger-piRNA with the row mRNA (triangle: slicer cleavage position). (D) Metaplots showing profiles of Aub/AGO3/Piwi-bound piRNAs (from Rhino-depleted ovaries) at genic trigger sites (profiles represent the median of normalized values; responder peak: 100%). (E) Heatmap indicating piRNA levels (Σ Aub/AGO3 5′ ends) mapping antisense (≤6 mismatches) to 570 mRNAs with trigger events (position 0: responder-piRNA 5′ end). The binary histograms show the percentage of transcripts with a cloned 5′ end of indicated piRNAs mapping in sense/antisense orientation at nucleotide resolution. (F) Heatmaps indicating AGO3/Aub/Piwi-bound piRNA 5′ end levels from Rhino depleted ovaries in a window centered on 570 genic responder-piRNAs. The corresponding binary histograms indicate the percentage of transcripts that exhibit a cloned piRNA 5′ end at a given position.

Figure 2. Biogenesis of flanking primary piRNAs is coupled.

(A-C) Top: piRNA length histograms; bottom: heatmaps showing length groups for piRNAs in Piwi/Aub/AGO3 (5′ ends with similar length profiles were grouped using K-means clustering). (D) Sequence logos indicating nucleotide biases for piRNA length clusters defined in (A) (grey box: piRNA body; for Aub/AGO3 see Fig. S3). (E) Sequence logos indicating nucleotide biases for Piwi/Aub/AGO3-bound piRNAs at 5′ ends, position 10 and 3′ ends (only 3′ ends where ≥50% of piRNAs terminate were considered). (F) Heatmaps displaying somatic Piwi-bound piRNA 5′ or 3′ ends in a window around the most abundant TE piRNAs (sum of each line scaled to 100%; sorted according to piRNA level at position 0). (G) Heatmaps displaying 5′ or 3′ end counts of piRNAs mapping to positions 20-32 downstream of major piRNA 5′ ends (detail from (F)). The left plot is sorted as in (F), the right plot is resorted for dominant piRNA length species (see also Fig. S4B). (H) Sequence logos indicating uridine frequency along piRNA sequences of different length cohorts (defined in S4B; black line: dominant piRNA 3′ ends).

Figure 3. Primary piRNA biogenesis is continuous and guided by uridine residues.

(A) Cartoon of the piRNA biogenesis reporter with target site for an Aub/AGO3-bound piRNA. (B, C) Normalized small RNA 5′ end profiles from reporter lacking a piRNA target site (B) or from reporter with target site for an Aub/AGO3-bound piRNA (C; grey: antisense reads; red: sense reads; blue bars: U residues; numbers: normalized piRNA counts; histograms: length profile and 1U bias of trail-piRNAs). (D) Similar to (C), but reporter contains 4 Us every 26nt. (E) Similar to (C), but reporter contains single Us in indicated intervals.

Figure 4. Zucchini is involved in 3′ end formation of Drosophila and mouse piRNAs.

(A) Sequence logos for nucleotides at 5′ end, 10^th position and 3′ end of Aub-bound or AGO3-bound piRNAs in control or Zucchini depleted ovaries. (B) Normalized wildtype small RNA 5′ end profile for reporter (similar to Fig. 3D) with single target site for a Zucchini-independent piRNA. (C) As in (B) but piRNAs isolated from Zucchini depleted ovaries. (D, E) As in (C) but reporter contains target sites for two (D) or three (E) Zucchini-independent piRNAs. (F) Heatmaps showing length cohorts of Mili-bound piRNAs from Tdrkh heterozygous mouse testes. (G) Sequence logos showing nucleotide composition around dominant piRNA 3′ ends for length cohorts defined in (G); for individual length clusters see Fig. S6. (H) Frequency of Mili-bound piRNA 5′ ends around aligned dominant piRNA 3′ ends from Tdrkh heterozygous testes. (I-K) As in panels F-H but from Tdrkh mutant testes.