The study introduces TREX (targeted RNase H-mediated extraction of crosslinked RBPs), a highly sensitive method for identifying proteins that directly bind to specific RNA regions in living cells. TREX outperforms existing methods in identifying known interactors of U1 snRNA and reveals endogenous region-specific interactors of NORAD long noncoding RNA. Using TREX, the authors generate a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and previously unknown interactions that regulate ribosome biogenesis. TREX is an RNA-centric tool that can map endogenous RNA-protein interactions in living cells, providing a versatile approach for studying RNA regulation and function.The study introduces TREX (targeted RNase H-mediated extraction of crosslinked RBPs), a highly sensitive method for identifying proteins that directly bind to specific RNA regions in living cells. TREX outperforms existing methods in identifying known interactors of U1 snRNA and reveals endogenous region-specific interactors of NORAD long noncoding RNA. Using TREX, the authors generate a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and previously unknown interactions that regulate ribosome biogenesis. TREX is an RNA-centric tool that can map endogenous RNA-protein interactions in living cells, providing a versatile approach for studying RNA regulation and function.