Evidence of target-mediated miRNA degradation in Drosophila ovarian cell culture

The target-mediated miRNA degradation (TDMD) is a recently discovered process of the post-transcriptional regulation of miRNA stability in animals. TDMD is induced by the formation of the non-canonical duplex of Ago-bound miRNAs with the specialized RNA target and, as suggested by studies on human cell culture, such complex is recognized by the ZSWIM8 receptor protein of the Cullin-RING-ligase complex CRL3. CRL3 ubiquitinates Ago resulting in the Ago proteolysis and the degradation of the released miRNAs. To date, the molecular mechanism of the TDMD process was not supported by studies in other animal species. In this paper, we investigated protein Dora, the Drosophila ortholog of ZSWIM8, in the culture of Drosophila ovarian somatic cells (OSCs). We show that Dora in OSCs localizes in protein granules that are not related to P- and GW-bodies. The knock-out of Dora up-regulated multiple miRNAs, including miR-7-5p. Also, we show that Dora associates with proteins of the CRL3 complex, and the depletion of its major component Cul3 up-regulates miR-7-5p. We concluded that the mechanism of TDMD is conserved in humans and Drosophila. The knock-out of Dora also down-regulated the putative protein-coding targets of miRNAs. One of them is Tom from the Brd-C gene family, which is known to repress the Notch signaling pathway. Indeed, in cells lacking Dora, we have observed the down-regulation of cut, the marker of the activated Notch pathway. This data indicates that TDMD in OSCs may be involved in the modulation of the Notch pathway.