MicroRNAs (miRNA) load onto AGO proteins to target mRNAs for translational repression or degradation. However, miRNA degradation can be triggered when extensively base-paired with target RNAs, which induces confirmational change of AGO and recruitment of ZSWIM8 ubiquitin ligase to mark AGO for proteasomal degradation. This target RNA-directed miRNA degradation (TDMD) mechanism appears to be evolutionarily conserved, but recent studies have focused on mammalian systems. Here, we performed AGO1-CLASH in Drosophila S2 cells, with Dora (ortholog of vertebrate ZSWIM8) knockout mediated by CRISPR-Cas9 to identify five TDMD triggers (sequences that can induce miRNA degradation). Interestingly, one trigger in the 3′ UTR of AGO1 mRNA induces miR-999 degradation. CRISPR-Cas9 knockout of the AGO1 trigger in S2 cells and in Drosophila specifically elevates miR-999, with concurrent repression of the miR-999 targets. AGO1 trigger knockout flies respond poorly to hydrogen peroxide-induced stress, demonstrating the physiological importance of this TDMD event.

MicroRNAs (miRNAs) interact with the target mRNAs to induce translational repression and mRNA degradation. Interestingly, miRNAs themselves can turnover rapidly when binding to a target RNA with extensive complementarity, a phenomenon called target-directed miRNA degradation (TDMD). To date, all validated TDMD “triggers” can induce miRNA degradation reside in non-coding regions of the RNA. We found that TDMD triggers placed in the 3′ untranslated region (UTR) of a reporter degraded miRNAs more effectively than those in the coding sequence (CDS). Inhibiting translation of the reporter enhanced miRNA degradation by the CDS trigger, indicating that ribosome-free CDS triggers are more accessible to miRNAs. By small RNA sequencing, we explored mammalian miRNAs sensitive to global translation status. Yet, no endogenous CDS trigger could be confidently assigned to these miRNAs. Our work revealed the intricate relationship between translation and TDMD, and explains the paucity of effective TDMD triggers in the CDS.

Up to 60% of patients with small cell lung cancer (SCLC) continue to smoke, which is associated with worse clinical outcomes. Platinum-based chemotherapies, in combination with topoisomerase inhibitors, are first-line therapies for SCLC, with rapid chemoresistance as a major barrier. We provided evidence in this study that nicotine and its major metabolite, cotinine, at physiologically relevant concentrations, reduced the efficacy of platinum-based chemotherapies and facilitated chemoresistance in SCLC cells. Mechanistically, nicotine or cotinine reduced chemotherapy-induced DNA damage by modulating cellular redox processes, with nAChRs as the upstream targets. Surprisingly, cisplatin treatment alone also increased the levels of nAChRs in SCLC cells, which served as a self-defense mechanism against platinum-based therapies. These discoveries were confirmed in long-term in vitro and in vivo studies. Collectively, our results depicted a novel and clinically important mechanism of chemoresistance in SCLC treatment: nicotine exposure significantly compromises the efficacy of platinum-based chemotherapies in SCLC treatment by reducing therapy-induced DNA damage and accelerating chemoresistance acquisition. The results also emphasized the urgent need for tobacco cessation and the control of NRT use for SCLC management.