a, Schematic of different modes of silencing of the three small RNA silencing pathways in Drosophila. b, Top, sequences of tapiR1, target gene AAEL026349, an siRNA targeting the gene at the same position, and Sanger sequencing results of 5′ RACE of Aag2 cells treated with tapiR1 antisense oligonucleotide and siRNAs. The tapiR1 target site is indicated in blue, RACE-sequencing adaptor in yellow and gene sequence in dark grey. The predicted slice site between nucleotides 10 and 11 is marked with a red vertical line. Bottom, summary of the results from 5′ RACE in the indicated conditions. Numbers refer to the number of sequenced clones with the 5′ RACE adaptor ligated to the predicted slice site and the total number of sequenced clones is shown between brackets. c, Small RNA coverage in Aag2 cells (not normalized) and individual reads (direction of the arrow indicates the strand) on tapiR1 target genes. Red boxes indicate positions of tapiR1 target sites on the mRNA. d, Schematic (top) and luciferase expression (bottom) of IRES-containing reporter constructs. Depicted are mean, s.d. and individual measurements of a representative experiment performed in triplicate wells with two different reporter clones. Bottom right, firefly luciferase (FLuc) activity normalized to Renilla luciferase (RLuc); raw luciferase counts from the same experiment are shown in the left (FLuc) and middle (RLuc). e, Luciferase activity of a reporter harbouring the tapiR1 target site of AAEL001555 in the 3′UTR of FLuc upon dsRNA-mediated knockdown of the indicated genes. Symbols are colour-coded according to the indicated RNA decay pathways. FLuc expression was normalized to RLuc expression to control for differences in transfection efficiencies and expressed relative to non-targeting control dsRNA (Sindbis virus dsRNA). Depicted are mean and standard deviation of one experiment performed in triplicates. Horizontal lines indicate a fold change of 1 and 1.5. f, AAEL008511 target gene expression as measured by RT–qPCR upon knockdown of the indicated genes. Primer sets located 5′ (upstream) and 3′ (downstream) to the tapiR1 target site were used for PCR. Mean, s.d. and individual measurements of one out of three experiments performed in triplicate are shown. The other two experiments are presented in Supplementary Fig. 2f, g. The horizontal line indicates a twofold change. g, Schematic illustration of the two PAT assays and expected results of genes with increasing poly(A) tail lengths. LM-PAT, ligation mediated-PAT; RACE-PAT, rapid amplification of cDNA ends-PAT. h–j, Electrophoretic analysis with ethidium-bromide-stained agarose gels of a LM-PAT assay (h) and RACE-PAT assay (i) of different tapiR1 target genes upon treatment with two concentrations of tapiR1 or control antisense oligonucleotides. As positive control, poly(A)-tail length was measured from SINV RNA in vitro transcribed from a plasmid (IVT), or from infected Aag2 cells, in which the poly(A) tail is elongated during viral replication56 (j). White asteriks indicate primer dimers.