fig3

Figure 3. Overview of RNAi and AONs. (A) RNAi pathways (siRNAs on the left side; miRNAs on the right side). siRNAs are usually of exogenous origin, while miRNAs are typically derived from transcripts possessing internal hairpins. Following Dicer-mediated processing, the RNA is recruited into AGO2 to constitute the RISC. siRNAs target mRNAs with quite precise sequence complementarity, resulting in mRNA cleavage by AGO2. In contrast, miRNAs recognize target sites with imperfect complementarity, predominantly in the 3’UTR of mRNAs, resulting in translational repression, deadenylation, and destabilization of the target. Additionally, miRNAs with precise complementarity can induce endonucleolytic cleavage by AGO2. Drosha, an RNase III enzyme, cleaves miRNA precursors in the initial stages of miRNA production by processing pri-miRNAs into pre-miRNAs, which are further processed by Dicer to produce mature miRNAs. AGO proteins play a central role in the RISC complex, which combines siRNAs with AGO proteins and Dicer enzymes to achieve gene silencing through the cleavage of target mRNA by the antisense strand of siRNAs; (B) AONs pathways (ssAONs on the left side; Gapmer AONs on the right side). ssAONs are directed to the nucleus to bind to their target pre-mRNA, which disrupts the splicing process by blocking the spliceosome. The specific example highlighted involves an AON targeting exon 51 of the dystrophin gene, leading to exon skipping. On the other hand, Gapmer AONs, shown on the right side, can trigger RNase H-mediated cleavage of the target mRNA in both the nucleus and the cytoplasm. RNAi: RNA interference; AONs: antisense oligonucleotides; siRNAs: small interfering RNAs; miRNAs: microRNAs; AGO2: Argonaute protein 2; RISC: RNAi‐induced silencing complex; 3’UTR: 3’ untranslated region; ssAONs: splice-switching AONs.