Comprehensive identification of proteins interacting with long non-coding RNA TUG1 in R-loop regulation

Long non-coding RNAs (lncRNAs) regulate a wide array of cellular processes through interactions with RNA-binding proteins (RBPs). Taurine Upregulated Gene 1 (TUG1) is an lncRNA that is overexpressed in many types of cancer and has been implicated in resolving R-loops, thereby maintaining genomic integrity. However, the full spectrum of its protein interactions and stress-responsive dynamics remains unclear. Here, we employed CRISPR-assisted RNA-protein interaction detection (CARPID) combined with mass spectrometry to comprehensively identify the interacting proteins of TUG1 in HEK293T cells. Using three distinct single-guide RNAs (sgRNAs) targeting different regions of TUG1, we consistently identified 17 TUG1-interacting proteins under basal conditions. Upon camptothecin (CPT) treatment, which induces R-loop formation, the number of associated proteins increased to 25. Under these stress conditions, the protein sets identified by each sgRNA showed greater overlap, suggesting a more conserved pattern of TUG1-protein interactions in response to R-loop accumulation. Many of these proteins are known R-loop-associated factors, including DEAD/DEAH-box RNA helicases, poly(ADP-ribose) polymerase 1 (PARP1) and heterogeneous nuclear ribonucleoproteins (HNRNPs), indicating that TUG1 engages R-loop regulatory machinery to maintain genome integrity. Our study provides new insights into lncRNA-mediated R-loop regulation and its role in genome maintenance.