a, Unlike miRNAs (for example, Bantam), hpRNA biogenesis in S2 cells is highly dependent on Dcr-2 and AGO2; Loqs and AGO1 suppression affect both miRNA and hpRNA biogenesis. b, miRNA and hpRNA biogenesis in pharate adult Drosophila. miR-8 was affected only in the loqs mutant, whereas hpRNA products were strongly decreased in the Dcr-2 and loqs mutants (<10%), and significantly affected in the AGO2 and hen1 mutants. c, Modification of hpRNA-derived small RNAs is mediated by Hen1. Note that hpRNA products from hen1 mutants run as a range of faster-migrating species after β-elimination. CS, Canton S (a strain of fruitfly); KO, knockout deletion strain.

a, hp-CG18854 contains a >400-bp duplex separated by a large loop; the enlarged region highlights the phased nature of small RNA duplexes. Northern probes were designed against the RNAs labelled in blue. b, The hp-CG4068 locus consists of 20 tandem repeats that partially overlap the 3′ UTR of CG4068 (Supplementary Fig. 3) and generate phased small RNA duplexes. Each repeat adopts a hairpin structure, but higher-order hairpins are possible because repeats are complementary to each other; ‘1-repeat’ and ‘2-repeat’ isoforms are shown. Distinct small RNAs were cloned from related repeats with minor sequence differences (for example, RNAs highlighted in red and green).

a, In S2 cells, both single-repeat (1×) and double-repeat (2×) hp-CG4068 constructs specifically repressed the hp-CG4068B, hp-CG4068C and mus308 sensors; the hp-CG4068D sensor was unaffected. b, Both genomic (g) and cDNA (c) hp-CG18854 expression constructs specifically repressed the hp-CG18854B sensor. c, The 2′-O-methyl ASO against hp-CG18854B specifically derepressed the hp-CG18854B sensor, whereas ASO-CG4068B specifically relieved endogenous repression of the hp-CG4068B sensor (d). e, Compared to a perfect hp-CG4068B sensor, mutant sensors with a central bulge or central bulge plus seed mutations exhibited the same level of derepression. Error bars depict the standard deviation of eight transfections; statistical comparisons were performed with the unequal variance Students t-test; **P < 6 × 10⁻⁵, ***P < 1 × 10⁻⁸. f, Compensatory covariation (pink shaded box) between hp-CG4068B and mus308 target sites of D. melanogaster (Dm) and D. sechellia (Dsec). Red font, nucleotides that have evolved; sens, sensor. g, Endogenous RNA-induced silencing complex from S2R cleaved both perfect hp-CG4068B and mus308 sensors, and this activity was specifically competed away by cognate ASO.