propiR1 targeting of lnc027353 initiates responder and trailer piRNA production. (A) Genomic coordinates, annotation and small RNA coverage of the lnc027353 locus (top), with the position of the propiR1 target site indicated with a gray box. A magnification on the x-axis and y-axis is shown in the inset. The putative slice site (t10/11) is highlighted in red. Black vertical lines indicate distances of 30 nt from the putative slice site. The genomic sequence of the locus is represented with colored boxes (red: G; orange: C; light blue: A; dark blue: T). The sequence of propiR1 and the AAEL027353-derived responder piRNA are shown at the bottom. (B) Northern blot analysis of the lnc027353-derived responder piRNA in cells treated with propiR1 or control antisense oligonucleotides (AO). EtBr stained rRNA serves as a loading control. (C) Sanger sequencing results of 5′ RACE products of lnc027353, including the number of clones obtained (bottom panel) and schematic representation of generated piRNAs bound to different PIWI proteins (top panel). The expected slicer product as well as a putative Zucchini cleavage product serving as a precursor for trailer piRNA production are indicated with dashed vertical lines. Asterisks indicate 5′ RACE products corresponding to the 5′ ends of piRNAs detected by small RNA sequencing. One clone harboring the RACE adapter ligated 77 nt downstream from the slice site is not depicted. Green: 5′ RACE adapter; blue: propiR1 target site; red: slice site (t10/11), black: lnc027353 transcript. (D) Northern blot analysis of the lnc027353-derived responder piRNA in RNA extracted from immunoprecipitations (IP) of the indicated PIWI proteins. No ab indicates a control IP in which the antibody was omitted. The blot was reused from Figure 1C and reprobed for the lnc027353 responder piRNA.
