Abstract
Silencing of Stellate genes in Drosophila melanogaster testes is caused by antisense piRNAs produced as a result of transcription of homologous Suppressor of Stellate (Su(Ste)) repeats. Mechanism of piRNA-dependent Stellate repression remains poorly understood. Here, we show that deletion of Su(Ste) suppressors causes accumulation of spliced, but not nonspliced Stellate transcripts both in the nucleus and cytoplasm, revealing post-transcriptional degradation of Stellate RNA as the predominant mechanism of silencing. We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction. Immunostaining of isolated nuclei revealed co-localization of a portion of cellular Aub with the nuclear lamina. We suggest that the piRNA-Aub complex is potentially able to perform Stellate silencing in the cell nucleus. Also, we revealed that the level of the Stellate protein in Su(Ste)-deficient testes is increased much more dramatically than the Stellate mRNA level. Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of beta-gal activity. In cell culture, exogenous Su(Ste) dsRNA dramatically decreases beta-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level. We suggest that piRNAs, similarly to siRNAs, degrade only unmasked transcripts, which are accessible for translation.
