AGO3 (Argonaute RISC catalytic component 3) is a key component of the RNA-induced silencing complex (RISC) that mediates RNA-mediated gene silencing through multiple mechanisms. AGO3 possesses RNA slicer activity, but unlike AGO2, this endonuclease activity is highly substrate-specific and requires both 5'- and 3'-flanking sequences around the guide-target complementarity region 1. The protein binds to small RNAs including microRNAs and tinyRNAs (≤17-nt guide RNAs), with binding affinity influenced by specific nucleotide sequence features, particularly YRHB tetranucleotides near the 3'-end 2. AGO3 plays crucial roles in stem cell regulation by processing DICER-dependent repeat-induced RNAs derived from retinoic acid-activated DR2 Alu repeats, leading to degradation of stem cell mRNAs like Nanog through recruitment of decapping complexes 3. The protein also functions with cityRNAs (14-nt cleavage-inducing tinyRNAs) to enhance endonuclease activity and mediate gene silencing primarily through target cleavage rather than translational repression 4. AGO3 has been implicated in neurodevelopmental disorders, with de novo variants identified in unsolved NDD cohorts 5. Additionally, AGO3 participates in muscle regeneration by mediating tRNA-derived small RNA functions in inflammatory responses 6.