The expansion of the cell genetic code relies on post-transcriptional regulatory mechanisms that shape the expression and translation of different mRNA transcripts into functional proteins. RNA-binding proteins (RBPs) control the splicing, editing, stability, and translation of mRNAs in a timely manner, and constitute an important layer of regulation that is often underestimated in genomic studies. Here we described individual nucleotide resolution UV-crosslinking immunoprecipitation and sequencing (iCLIP), a method that allows precise in vivo annotation of RBP–RNA interactions for identifying not only mRNA targets but also the mechanisms of action by which a given RBP controls the expression of single genes or gene-operons regulating cellular functions.