The study introduces HITS-CLIP, a high-throughput sequencing method for mapping protein-RNA binding sites in vivo, to investigate the role of the neuron-specific splicing factor Nova2 in brain alternative RNA processing. HITS-CLIP revealed highly reproducible RNA binding maps in multiple mouse brains, confirming that Nova binding positions determine the outcome of alternative splicing. The method also identified a large number of Nova-RNA interactions in 3' UTRs, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP provides a robust and unbiased approach to identify functional protein-RNA interactions in vivo, offering insights into the mechanisms of RNA regulation and the role of Nova in tissue-specific RNA metabolism.The study introduces HITS-CLIP, a high-throughput sequencing method for mapping protein-RNA binding sites in vivo, to investigate the role of the neuron-specific splicing factor Nova2 in brain alternative RNA processing. HITS-CLIP revealed highly reproducible RNA binding maps in multiple mouse brains, confirming that Nova binding positions determine the outcome of alternative splicing. The method also identified a large number of Nova-RNA interactions in 3' UTRs, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP provides a robust and unbiased approach to identify functional protein-RNA interactions in vivo, offering insights into the mechanisms of RNA regulation and the role of Nova in tissue-specific RNA metabolism.