HITS-CLIP is a high-throughput sequencing method that maps protein-RNA interactions in vivo. This study used HITS-CLIP to analyze Nova2, a neuron-specific splicing factor, in mouse brains, revealing genome-wide RNA binding sites. These maps confirmed that Nova binding determines alternative splicing outcomes and can predict Nova's function. HITS-CLIP identified Nova-RNA interactions in 3' UTRs, showing Nova regulates alternative polyadenylation in the brain. The method is robust and unbiased, allowing identification of functional protein-RNA interactions. Nova2 binds to YCAY motifs in RNA, which are enriched in Nova binding sites. HITS-CLIP showed that Nova binding sites are highly reproducible across multiple mouse brains, with clusters showing significant YCAY enrichment. Analysis of Nova-regulated exons revealed that Nova binding sites are closely associated with splicing outcomes, with Nova-dependent splicing regulation confirmed by experimental validation. HITS-CLIP also identified Nova binding near poly(A) sites, revealing a new function in regulating alternative polyadenylation in the brain. Nova was shown to influence poly(A) site usage, affecting mRNA stability and processing. This was validated using reporter constructs and RNA analysis, demonstrating that Nova binding to YCAY elements directly regulates poly(A) site utilization. The study highlights the importance of Nova in brain-specific RNA processing, including alternative splicing and polyadenylation. HITS-CLIP provides a powerful tool for studying RNA regulation in vivo, offering insights into the mechanisms of protein-RNA interactions and their roles in gene expression. The method's reproducibility and unbiased nature make it a valuable resource for discovering new regulatory elements and understanding the function of RNA-binding proteins.HITS-CLIP is a high-throughput sequencing method that maps protein-RNA interactions in vivo. This study used HITS-CLIP to analyze Nova2, a neuron-specific splicing factor, in mouse brains, revealing genome-wide RNA binding sites. These maps confirmed that Nova binding determines alternative splicing outcomes and can predict Nova's function. HITS-CLIP identified Nova-RNA interactions in 3' UTRs, showing Nova regulates alternative polyadenylation in the brain. The method is robust and unbiased, allowing identification of functional protein-RNA interactions. Nova2 binds to YCAY motifs in RNA, which are enriched in Nova binding sites. HITS-CLIP showed that Nova binding sites are highly reproducible across multiple mouse brains, with clusters showing significant YCAY enrichment. Analysis of Nova-regulated exons revealed that Nova binding sites are closely associated with splicing outcomes, with Nova-dependent splicing regulation confirmed by experimental validation. HITS-CLIP also identified Nova binding near poly(A) sites, revealing a new function in regulating alternative polyadenylation in the brain. Nova was shown to influence poly(A) site usage, affecting mRNA stability and processing. This was validated using reporter constructs and RNA analysis, demonstrating that Nova binding to YCAY elements directly regulates poly(A) site utilization. The study highlights the importance of Nova in brain-specific RNA processing, including alternative splicing and polyadenylation. HITS-CLIP provides a powerful tool for studying RNA regulation in vivo, offering insights into the mechanisms of protein-RNA interactions and their roles in gene expression. The method's reproducibility and unbiased nature make it a valuable resource for discovering new regulatory elements and understanding the function of RNA-binding proteins.