The repression machinery controlling ZAM and Idefix acts post-transcriptionally, before translation.
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 Idefix 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 ZAM and Idefix. 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 ZAM are presented in C. 20 to 30 nt long antisense strand RNAs (−) homologous to the 5′UTR or the gag gene of Idefix 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.