The study presents iCLIP, a high-resolution method for mapping protein-RNA interactions, to investigate the role of hnRNP C in splicing regulation. iCLIP enables precise identification of hnRNP C binding sites on pre-mRNAs at the nucleotide level. The results show that hnRNP C preferentially binds to uridine-rich regions, with a defined spacing pattern consistent with hnRNP particle organization. These findings suggest that hnRNP particles assemble on both introns and exons but are generally excluded from splice sites. Integration of iCLIP data with alternative splicing profiles reveals how hnRNP particle positioning influences exon inclusion. The study demonstrates that hnRNP C binding to uridine tracts can either silence or enhance exon inclusion, depending on the location of the binding. The RNA map generated from iCLIP data shows that hnRNP C binding is associated with specific splicing outcomes, with silenced exons showing increased hnRNP C binding near splice sites. The study also highlights the structural implications of hnRNP C binding, including the formation of long-range spacing between cross-link sites, which reflects hnRNP particle organization. The findings suggest that hnRNP particles play a dual role in splicing regulation by either incorporating exons into the particle (silencing) or binding to introns preceding exons (enhancing inclusion). The high-resolution iCLIP data provides insights into the mechanism of splicing regulation and has potential applications for studying other ribonucleoprotein complexes. The study underscores the importance of hnRNP C in splicing regulation and highlights the utility of iCLIP in elucidating the functional roles of RNA-binding proteins in post-transcriptional regulation.The study presents iCLIP, a high-resolution method for mapping protein-RNA interactions, to investigate the role of hnRNP C in splicing regulation. iCLIP enables precise identification of hnRNP C binding sites on pre-mRNAs at the nucleotide level. The results show that hnRNP C preferentially binds to uridine-rich regions, with a defined spacing pattern consistent with hnRNP particle organization. These findings suggest that hnRNP particles assemble on both introns and exons but are generally excluded from splice sites. Integration of iCLIP data with alternative splicing profiles reveals how hnRNP particle positioning influences exon inclusion. The study demonstrates that hnRNP C binding to uridine tracts can either silence or enhance exon inclusion, depending on the location of the binding. The RNA map generated from iCLIP data shows that hnRNP C binding is associated with specific splicing outcomes, with silenced exons showing increased hnRNP C binding near splice sites. The study also highlights the structural implications of hnRNP C binding, including the formation of long-range spacing between cross-link sites, which reflects hnRNP particle organization. The findings suggest that hnRNP particles play a dual role in splicing regulation by either incorporating exons into the particle (silencing) or binding to introns preceding exons (enhancing inclusion). The high-resolution iCLIP data provides insights into the mechanism of splicing regulation and has potential applications for studying other ribonucleoprotein complexes. The study underscores the importance of hnRNP C in splicing regulation and highlights the utility of iCLIP in elucidating the functional roles of RNA-binding proteins in post-transcriptional regulation.