TREX is a highly sensitive method for identifying proteins that directly bind to specific RNA regions in living cells. The study introduces TREX, which uses targeted RNase H-mediated extraction of crosslinked RBPs to reveal endogenous RNA–protein interactions. 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 researchers generated 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 for unbiased positional mapping of endogenous RNA–protein interactions in living cells. The method involves organic phase separation and RNase H-mediated RNA degradation to isolate and identify proteins bound to specific RNA sequences. TREX was benchmarked against the full length of U1 snRNA, demonstrating its superiority over existing antisense oligonucleotide-based pulldown methods. TREX was also applied to the most conserved segment of NORAD lncRNA, highlighting its versatility in probing region-specific interactions in endogenous settings. The study also revealed the interactome of 18S, 5.8S, and 28S rRNA, as well as the 5'-ETS, ITS1, ITS2, and 3'-ETS spacer regions. TREX identified numerous proteins that interact with these regions, including ribosomal proteins, RBFs, RAFs, and previously unknown rRNA-binding proteins. The results demonstrate the effectiveness of TREX in identifying functional interactors of a given RNA region under endogenous settings. The study also highlights the importance of region-specific interactions in ribosome biogenesis and provides a comprehensive catalog of the direct interactions of each transcribed spacer region in human 45S rRNA. TREX is a powerful tool for unbiased region-specific exploration of direct RNA–protein interactions in their endogenous context.TREX is a highly sensitive method for identifying proteins that directly bind to specific RNA regions in living cells. The study introduces TREX, which uses targeted RNase H-mediated extraction of crosslinked RBPs to reveal endogenous RNA–protein interactions. 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 researchers generated 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 for unbiased positional mapping of endogenous RNA–protein interactions in living cells. The method involves organic phase separation and RNase H-mediated RNA degradation to isolate and identify proteins bound to specific RNA sequences. TREX was benchmarked against the full length of U1 snRNA, demonstrating its superiority over existing antisense oligonucleotide-based pulldown methods. TREX was also applied to the most conserved segment of NORAD lncRNA, highlighting its versatility in probing region-specific interactions in endogenous settings. The study also revealed the interactome of 18S, 5.8S, and 28S rRNA, as well as the 5'-ETS, ITS1, ITS2, and 3'-ETS spacer regions. TREX identified numerous proteins that interact with these regions, including ribosomal proteins, RBFs, RAFs, and previously unknown rRNA-binding proteins. The results demonstrate the effectiveness of TREX in identifying functional interactors of a given RNA region under endogenous settings. The study also highlights the importance of region-specific interactions in ribosome biogenesis and provides a comprehensive catalog of the direct interactions of each transcribed spacer region in human 45S rRNA. TREX is a powerful tool for unbiased region-specific exploration of direct RNA–protein interactions in their endogenous context.