Evidence of target-mediated miRNA degradation inDrosophilaovarian cell culture

AbstractTarget-mediated miRNA degradation (TDMD) is a recently discovered process of 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 for human cell culture, this complex is recognized by the ZSWIM8 receptor protein of the Cullin-RING-ligase complex CRL3. CRL3 ubiquitinates Ago, resulting in proteolysis of Ago and degradation of the released miRNAs. To date, the molecular mechanism of the TDMD process was not studied in other animal species. Here we investigated protein Dora, theDrosophilaortholog of ZSWIM8, in the culture ofDrosophilaovarian somatic cells (OSC). We show that Dora in OSCs localizes in protein granules that are not related to P-and GW-bodies. The knock-out ofDoraup-regulates multiple miRNAs, including miR-7-5p. Also, we show that Dora associates with proteins of the CRL3 complex, and the depletion of its main component Cul3 up-regulates miR-7-5p. We concluded that the mechanism of TDMD is conserved in humans andDrosophila. The knock-out ofDoraalso down-regulates the putative protein-coding targets of miRNAs. One of them isTomfrom 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 ofcut, the marker of the activated Notch pathway. This data indicates that TDMD in OSCs may contribute to modulation of the Notch pathway.