Fig. 6

Identification of piRNAs in Pirmy and Pirmy-like transcripts. A Short stretches of homology identified in the UTRs are represented on the set of 28 Pirmy-like RNAs. The boxes highlighted in black match within exons and boxes highlighted in red match the exon-exon junctions. Line 1 indicates the nucleotide positions as in Fig. 3. B Pie chart shows ~ 81% of the matches within exons and ~ 19% at exon-exon junctions. C Use of sequences homologous to 3′ UTRs of a hypothetical protein (SpotA4), Sod, Bche, PLA2G12B and Spn as probes (Additional file 13: Fig. S6) on small RNA northern blots (3 experiments) shows testis-specific signals of ~ 30 nt size (indicated by arrows), which correspond to the size of piRNAs. Control blot using a scrambled oligonucleotide probe shows no signal at ~ 30 nt size. D Hybridization using probes from sense (S) and antisense (AS) strands of Sod and Bche (Additional file 13: Fig. S6) shows differential transcription from the two strands under identical conditions. Lower panels in C, D show loading control using U6 probe (B—brain, K—kidney, T—testis, Ladder—decade marker). E, F EMSA using RNA oligonucleotide sequences from FJ541077 (E) and FJ541118 (F) that have homology to UTRs of the genes of hypothetical protein spot A (A4) and Sod respectively. These oligonucleotides and piR1, a known piRNA, showed the shift in mobility with recombinant MIWI protein. Gel shifts obtained with RNA oligonucleotides from spotA4 and Sod were competed out by cold piR1 and vice versa. Pre-incubation with MIWI antibody abolished the gel shift whereas pre-incubation with argonaute 3 antibody (ARG) did not, indicating specificity of binding. The small RNA northern blots, EMSA experiments were repeated three times each. G Schematic representation of 3′UTR reporter constructs. H The concentration-dependent reduction in luciferase expression when target UTRs of Sod and PLA2G12B are treated with antagopirs (0–20 nM). A non-target UTR (Cdc2l1) did not show a difference (n = 3; *** P < 0.001; **P < 0.01; *P < 0.05)