The repression machinery controlling <i>ZAM</i> and <i>Idefix</i> acts post-transcriptionally, before translation.

<p>A: Transcripts from the pGFP-ZU transgene were examined in northern blot experiments. A typical result is shown in A. GFP transcripts revealed by a riboprobe complementary to GFP mRNAs are detected in the U line and not in the S line. Actin is used as a loading control. B: Northern blots and quantification based on three northern blot experiments performed on flies containing pGFP-IdU and pGFP-IdUAS transgenes. Their structures are presented above the graph. No GFP transcripts synthesized from the pGFP-IdU transgene are detected by the GFP riboprobe in an S background, whereas their amount is high in a U background. An even higher amount of GFP transcripts is observed in an S or U background when the <i>Idefix</i> fragment is inserted in the opposite orientation (pGFP-IdUAS transgenes). C and D- RNase protection assays reveal the presence of small RNAs (20 to 30 nt long) that are homologous to <i>ZAM</i> and <i>Idefix.</i> These RNAs are detected in S lines and, at a much lower level, in the U line. Small RNAs homologous to the antisense strand of the 5′UTR of <i>ZAM</i> are presented in C. 20 to 30 nt long antisense strand RNAs (−) homologous to the 5′UTR or the <i>gag</i> gene of <i>Idefix</i> are detected. Sense strands (+) are absent or present in very small amounts. A typical experiment is presented in D. Signs (+) and (−) indicate respectively sense-strand and anti-sense strand RNAs of ZAM or Idefix revealed by the riboprobes.</p

The silencing mechanism targeting ZAM and Idefix is active in somatic tissues throughout fly development.

<p>A) In an S/S genetic background, the pGFP-IdU sensor transgene driven by 24B-Gal4 is not expressed in embryos, larvae, or adults (top, middle and bottom panels, respectively). Only a very faint level of fluorescence, corresponding to the background expression of GFP, is detected. B) In a U/U genetic background, the GFP-IdU transgene silencing is released and GFP fluorescence is clearly observed in the three stages analyzed. The fluorescence pattern recapitulates the expression of the HOW gene in muscle and tendon cells, as expected for the 24B-Gal4 driver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001526#pone.0001526-Brand1" target="_blank">[22]</a>. C) In an S/S genetic background, the pGFP-IdUAS sensor transgene carrying the 5′UTR of <i>Idefix</i> in the opposite orientation is not subjected to the silencing exerted on the <i>Idefix</i> sequences. pGFP-IdUAS is correctly expressed and GFP is detected in embryos, larvae, and adult flies.</p

<i> ZAM</i> and <i>Idefix</i> are regulated by a PIWI-dependent pathway in the reproductive apparatus.

<p><i>In situ</i> hybridization experiments reveal <i>ZAM</i> and <i>Idefix</i> expression in female gonads from third instar larvae. <i>ZAM</i> and <i>Idefix</i> transcripts are not detected in S flies with a wild-type <i>piwi</i> gene (left). As shown by the black staining, <i>ZAM</i> and <i>Idefix</i> mRNAs are detected in U flies with a wild-type <i>piwi</i> gene (middle). In S lines homozygous for the <i>piwi</i>³ allele, <i>ZAM</i> or <i>Idefix</i> transcripts are no longer repressed, and their transcription is visualised in gonads (right). Probes used in these experiments are indicated on the left.</p

Transgenes with a GFP reporter gene fused to a <i>ZAM</i> sequence act as sensors of the repression.

<p>The genomic structures of the transgenes pGFP-Zenv and pGFP-Idgag used in this study are presented at the tops of both panels: The grey boxes correspond to the UASt promoter, the dotted boxes to the GFP gene, and the white box to the <i>env</i> fragment of <i>ZAM</i> or the <i>gag</i> fragment of <i>Idefix</i>. Triangles indicate the FRT sites. Focal plane of the follicles dissected from a line in which the pGFP-Zenv transgene is driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Zenv transgene in an S genetic background before (A) or after (B) <i>flp</i>-recombinase action, or in a U genetic background before the <i>flp</i> treatment (C). GFP expression in the ovarioles of a transgenic line bearing the pGFP-Idgag transgene driven by the ubiquitous Actin-Gal4 driver. Expression of the pGFP-Idgag transgene in an S genetic background before (D) or after (E) <i>flp</i>-recombinase action, or in a U genetic background before the <i>flp</i> treatment (F). No GFP is detected in ovaries of the S lines. Its expression is recovered after the flp treatment or when the COM locus is mutated, as in the U genetic background.</p

Transgenes bearing <i>ZAM</i> or <i>Idefix</i> sequences placed in an antisense orientation are not targeted by the repression.

<p>Expression of sensor transgenes carrying <i>ZAM</i> or <i>Idefix</i> fragments inserted in a sense and an antisense orientation. The genomic structure of the so-called pGFP-Zenv, pGFP-ZenvAS, pGFP-IdU, pGFP-IdUAS transgenes are depicted on the left. The orientation of the fragment is indicated by an arrow. The repression mechanism is able to discriminate between sense and antisense targeted sequences. In an S/S genetic background, only transgenes with ZAM and Idefix in an antisense orientation are correctly expressed. Clear fluorescence due to GFP expression is detected in the ovarian follicles of pGFP-ZenvAS and pGFP-IdUAS transgenes, as illustrated on the right.</p

The U5, but not U3, region of the <i>ZAM</i> LTR is required for repression.

<p>The genomic structure of the <i>ZAM</i> retrotransposon is depicted at the top. Structures of the <i>lacZ</i> reporter trangenes used in this study are shown below on the left, and their expression in follicle cells from the S or U background are indicated at right. Transcripts initiated from the endogenous transcription initiation site of <i>ZAM</i> (black arrow) in transgenes pZ499 and pZ475 are homologous to <i>ZAM</i> over 173 and 149 bp, respectively. These transgenes are sensitive to the S or U status of the line as illustrated by the histochemical detection of β-galactosidase activity in the ovarioles. pZ310 contains the U3 sequence of the element and is expressed from a minimal heat shock promoter (white arrow) so that no sequence homologous to <i>ZAM</i> is present within the p310 transcript. Its expression is not under the control of the S or U status of the lines and is thus observed in the ovarioles from both the S and U backgrounds, as illustrated on the right.</p

<i> ZAM</i> and <i>Idefix</i> are regulated by a PIWI-independent pathway outside of the reproductive apparatus.

<p>The pGFP-ZU sensor transgene driven by 24B-Gal4 is not expressed in larvae, pupae, or adult stages in a [S/S; piwi+/+] genetic background (right panel). Only a very faint level of fluorescence, corresponding to the background, is detected. A clear GFP expression is observed in these stages of development in a [U/U; piwi+/+] line (middle panel). In <i>piwi</i> mutant backgrounds, in homozygous [S/S; piwi3/3] lines, the silencing of the sensor transgene is not released. A very faint fluorescence level similar to that observed in homozygous [S/S; piwi+/+] lines is observed (left panel).</p