RNA silencing pathways convert long RNA sequences, typically double-stranded RNA (dsRNA), into ~21-nt RNA signaling molecules such as small interfering RNAs (siRNAs) and microRNAs (miRNAs). These small RNAs guide protein complexes that repress mRNA transcription or translation, or catalyze mRNA destruction. The study of RNA silencing has focused on three key questions: the molecular nature of the silencing trigger, the silencing targets, and the proteins involved in the RNAi machinery. Silencing triggers, such as dsRNA, initiate the assembly of protein-RNA complexes that repress gene expression by reducing mRNA transcription, stability, or translation. These triggers identify their targets through sequence-specific base pairing. In animals, RNA silencing was first observed when dsRNA triggered gene silencing, leading to the discovery of RNA interference (RNAi). RNAi is part of a broader network of sequence-specific cellular responses to RNA, collectively known as RNA silencing. All RNA silencing pathways are triggered by 21–27-nt-long small RNAs, including siRNAs, rasiRNAs, and miRNAs. These pathways share a common set of proteins that produce or amplify small RNAs and couple them to regulatory outcomes. Small RNAs guide RNA silencing effector complexes, such as the RNA-induced silencing complex (RISC) or the RNA-induced initiation of transcriptional gene silencing (RITS) complex. Argonaute proteins are central to all RNA silencing effector complexes. The production of siRNAs and miRNAs involves distinct pathways. siRNAs are generated from dsRNA by Dicer, which cleaves dsRNA into siRNAs. miRNAs are produced from long primary miRNAs (pri-miRNAs) by Drosha and Dicer. Both siRNAs and miRNAs are initially double-stranded but function as single-stranded in complexes. The sequence and structure of small RNAs determine their function and target specificity. siRNAs and miRNAs can also trigger DNA methylation and the formation of repressive heterochromatin. The assembly of RISC involves the loading of one of the two siRNA strands into RISC. In Drosophila, Dcr-2 and R2D2 are involved in this process. The RISC assembly pathway includes several steps, with the RLC playing a central role in loading siRNA into RISC. The two strands of siRNA are distinguished by their thermodynamic stability, with the more stable strand being incorporated into RISC. The RLC contains both double-stranded and single-stranded siRNA, suggesting that siRNA unwinding initiates in this complex. The function of Argonaute proteins in RISC is critical for RNA silencing. Ago2 is involved in both mRNA cleavage and translational repression. The two-state model for Argonaute function suggests thatRNA silencing pathways convert long RNA sequences, typically double-stranded RNA (dsRNA), into ~21-nt RNA signaling molecules such as small interfering RNAs (siRNAs) and microRNAs (miRNAs). These small RNAs guide protein complexes that repress mRNA transcription or translation, or catalyze mRNA destruction. The study of RNA silencing has focused on three key questions: the molecular nature of the silencing trigger, the silencing targets, and the proteins involved in the RNAi machinery. Silencing triggers, such as dsRNA, initiate the assembly of protein-RNA complexes that repress gene expression by reducing mRNA transcription, stability, or translation. These triggers identify their targets through sequence-specific base pairing. In animals, RNA silencing was first observed when dsRNA triggered gene silencing, leading to the discovery of RNA interference (RNAi). RNAi is part of a broader network of sequence-specific cellular responses to RNA, collectively known as RNA silencing. All RNA silencing pathways are triggered by 21–27-nt-long small RNAs, including siRNAs, rasiRNAs, and miRNAs. These pathways share a common set of proteins that produce or amplify small RNAs and couple them to regulatory outcomes. Small RNAs guide RNA silencing effector complexes, such as the RNA-induced silencing complex (RISC) or the RNA-induced initiation of transcriptional gene silencing (RITS) complex. Argonaute proteins are central to all RNA silencing effector complexes. The production of siRNAs and miRNAs involves distinct pathways. siRNAs are generated from dsRNA by Dicer, which cleaves dsRNA into siRNAs. miRNAs are produced from long primary miRNAs (pri-miRNAs) by Drosha and Dicer. Both siRNAs and miRNAs are initially double-stranded but function as single-stranded in complexes. The sequence and structure of small RNAs determine their function and target specificity. siRNAs and miRNAs can also trigger DNA methylation and the formation of repressive heterochromatin. The assembly of RISC involves the loading of one of the two siRNA strands into RISC. In Drosophila, Dcr-2 and R2D2 are involved in this process. The RISC assembly pathway includes several steps, with the RLC playing a central role in loading siRNA into RISC. The two strands of siRNA are distinguished by their thermodynamic stability, with the more stable strand being incorporated into RISC. The RLC contains both double-stranded and single-stranded siRNA, suggesting that siRNA unwinding initiates in this complex. The function of Argonaute proteins in RISC is critical for RNA silencing. Ago2 is involved in both mRNA cleavage and translational repression. The two-state model for Argonaute function suggests that