Ago HITS-CLIP decodes miRNA-mRNA interaction maps. MicroRNAs (miRNAs) regulate gene expression by binding to mRNA, but identifying their targets is challenging due to the short interaction length. The study uses Ago HITS-CLIP to covalently crosslink Ago protein-RNA complexes in mouse brain, generating datasets of Ago-miRNA and Ago-mRNA binding sites. These datasets, combined with bioinformatics, identify miRNA-mRNA interaction sites. The method was validated for miR-124 and other abundant miRNAs in mouse brain. Ago HITS-CLIP provides a general platform for exploring miRNA action in vivo and identifying precise sequences for targeting miRNA-mRNA interactions. The study highlights the complexity of RNA regulation and the role of miRNAs in controlling diverse cellular functions. Despite their biological importance, determining miRNA targets remains a challenge due to the limited number of nucleotides involved in binding. Bioinformatic analysis has improved prediction accuracy, but false positives remain high. The study also shows that miRNAs are often part of families, complicating loss-of-function studies. Ago HITS-CLIP overcomes these challenges by directly identifying protein-RNA interactions in living tissues. The method uses UV-irradiation to covalently crosslink RNA-protein complexes, allowing for stringent purification and high-throughput sequencing. This generates genome-wide maps and functional insights. The study shows that Ago HITS-CLIP can identify miRNA-mRNA interaction sites with high specificity and accuracy. The study focuses on miR-124, a brain-specific miRNA, and identifies its targets in mouse brain. Ago HITS-CLIP reveals that miR-124 binds to specific regions of mRNAs, particularly in the 3' UTR. The method also identifies new miRNA targets and confirms their functional relevance. The study demonstrates that Ago HITS-CLIP provides a reliable way to identify miRNA targets and understand their biological functions. The study also shows that Ago HITS-CLIP can predict miRNA functional networks. By analyzing the 20 most abundant miRNAs in mouse brain, the study identifies binding maps and functional annotations. The results show that miRNAs regulate diverse neuronal functions and that Ago HITS-CLIP provides a more accurate prediction than bioinformatic methods alone. The study concludes that Ago HITS-CLIP is a powerful tool for understanding miRNA action in vivo. It provides a detailed map of miRNA-mRNA interactions and identifies precise sequences for targeting miRNA-mRNA interactions. The method has important implications for understanding miRNA biology and for developing therapeutic strategies targeting miRNA activity.Ago HITS-CLIP decodes miRNA-mRNA interaction maps. MicroRNAs (miRNAs) regulate gene expression by binding to mRNA, but identifying their targets is challenging due to the short interaction length. The study uses Ago HITS-CLIP to covalently crosslink Ago protein-RNA complexes in mouse brain, generating datasets of Ago-miRNA and Ago-mRNA binding sites. These datasets, combined with bioinformatics, identify miRNA-mRNA interaction sites. The method was validated for miR-124 and other abundant miRNAs in mouse brain. Ago HITS-CLIP provides a general platform for exploring miRNA action in vivo and identifying precise sequences for targeting miRNA-mRNA interactions. The study highlights the complexity of RNA regulation and the role of miRNAs in controlling diverse cellular functions. Despite their biological importance, determining miRNA targets remains a challenge due to the limited number of nucleotides involved in binding. Bioinformatic analysis has improved prediction accuracy, but false positives remain high. The study also shows that miRNAs are often part of families, complicating loss-of-function studies. Ago HITS-CLIP overcomes these challenges by directly identifying protein-RNA interactions in living tissues. The method uses UV-irradiation to covalently crosslink RNA-protein complexes, allowing for stringent purification and high-throughput sequencing. This generates genome-wide maps and functional insights. The study shows that Ago HITS-CLIP can identify miRNA-mRNA interaction sites with high specificity and accuracy. The study focuses on miR-124, a brain-specific miRNA, and identifies its targets in mouse brain. Ago HITS-CLIP reveals that miR-124 binds to specific regions of mRNAs, particularly in the 3' UTR. The method also identifies new miRNA targets and confirms their functional relevance. The study demonstrates that Ago HITS-CLIP provides a reliable way to identify miRNA targets and understand their biological functions. The study also shows that Ago HITS-CLIP can predict miRNA functional networks. By analyzing the 20 most abundant miRNAs in mouse brain, the study identifies binding maps and functional annotations. The results show that miRNAs regulate diverse neuronal functions and that Ago HITS-CLIP provides a more accurate prediction than bioinformatic methods alone. The study concludes that Ago HITS-CLIP is a powerful tool for understanding miRNA action in vivo. It provides a detailed map of miRNA-mRNA interactions and identifies precise sequences for targeting miRNA-mRNA interactions. The method has important implications for understanding miRNA biology and for developing therapeutic strategies targeting miRNA activity.