Nuclear RNAi is necessary to initiate synthesis of tertiary siRNAs.

<p>A) The principle of the <i>operon</i> transgene expression. Straight line represents the DNA locus (bottom), curved lines pre-mRNA and mRNA (before and after nuclear export). Diamonds and snowflakes are mCherry protein (red) and GFP protein (green), respectively (top). B) Schematic representation of the transgenes in the <i>operon; piRNA sensor</i> strain. The <i>piRNA sensor</i> (bottom) generates 22G-RNAs (black lines) against GFP that can confer trans-silencing of the <i>operon</i>-derived GFP. In case a spreading of 22G-RNAs occurs in the nucleus, silencing of mCherry would be expected. C) Representative fluorescence images of somatic and germline GFP (top row) and mCherry (middle row) expression and DIC images (bottom row) of the parental <i>operon</i> strain (left), silenced wild type <i>operon; piRNA sensor</i> animals (middle) and de-silenced <i>operon; piRNA sensor; hrde-1</i> mutant (right) animals. D) Small RNA high-throughput sequencing reads with unique matches antisense to the <i>operon</i> from animals as indicated in C). The values of the <i>y</i>-axes correspond to reads matching the <i>operon</i> normalised to reads matching Histone 2B (<i>his-58</i>). The x-axes represent the relative position of reads in the <i>operon</i> transgene with numbers representing nucleotides from the start codon (set as 0). The transgene structure is schematically represented at the bottom. Colour code: red = mCherry, dark grey = <i>gpd-2</i> trans-splicing linker (L), green = GFP, light grey = <i>par-5</i> 3′UTR.