a, Peak length distribution of small RNAs derived from the randomized sequence library. b, RNA substrates used in c. The top 6 nucleotides in the BmZuc motif are shown in colour and their mutations are shown in black. c, Siwi-loaded 111750-derived RNAs were incubated with Tri-KO 1,000g ppt. overexpressing BmZuc and BmArmi, or BmZuc(HN). Each gel image was adjusted to equalize the loading signal. ppt., pellet. d, Schematic representation of the randomized sequence library analysis for Siwi- or BmAgo3-loaded pre-piRNAs cleaved by BmZuc. e, Peak length distribution of Siwi- or BmAgo3-bound 2′-O-methylated small RNAs derived from the corresponding randomized sequence library. For Siwi immunoprecipitation, the same plasmid library as in a was used. f, Nucleotide composition around the 3′ ends of mature piRNAs in naive BmN4 cells (right) or type-E pre-piRNAs in Tri-KO cells (left), separately analysed for Siwi-dominant (top) and BmAgo3-dominant (bottom) piRNA loci. The 6 nucleotides in the BmZuc motif are highlighted. g, Schematic explanation for the weighted BmZuc motif (top) and the calculation of the BmZuc score in the 17-nt sliding window analysis (bottom). h, Similarity scores with the weighted BmZuc motif (BmZuc score) for Siwi were calculated for 111750 RNA and their mutant sequences in sliding windows and plotted as in c. i, Box plots show the maximum similarity scores with the weighted BmZuc motif for Siwi or BmAgo3 within the positions of 19−45 nt of Siwi-dominant (top) or BmAgo3-dominant (bottom) type-N or type-E piRNA loci or the shuffled control sequences (a pool of 3,236 species of 27-nt scrambled sequences that have the average nucleotide composition of the silkworm genome). Type-E piRNA loci have significantly higher BmZuc scores than the shuffled control sequences for both Siwi- and BmAgo3-dominant piRNAs (Mann–Whitney U test). Centre line, median; box limits, upper and lower quartiles; whiskers, 1.5 × interquartile range; points, outliers.