A UV-Based ChIRP Method For The Verifiable Identification of Proteins That Directly Interact With LncRNA: From...

Long non-coding RNAs (lncRNAs) have emerged as important regulators of diverse biological processes. However, the molecular mechanisms underlying the functions of most lncRNAs remain poorly understood. Identifying interacting proteins, particularly RNA-binding proteins (RBPs), is a key strategy for elucidating lncRNA function. Among available approaches, comprehensive identification of RNA-binding proteins (ChIRP) coupled with mass spectrometry is widely used because of its straightforward probe design and simple workflow. Conventional ChIRP relies on paraformaldehyde (PFA) crosslinking, which captures both direct RNA-protein contacts and indirect interactions mediated by bridging factors. In contrast, UV crosslinking combined with stringent washing conditions preferentially preserves direct RNA-protein interactions. Using the lncRNA Tug1 as a model, UV crosslinking and PFA crosslinking were compared within the ChIRP framework in cultured cells, demonstrating that UV-based ChIRP enriches for direct lncRNA-protein interactions. This UV-based workflow was subsequently extended to mouse testes, in which tissues were dissociated into single-cell suspensions prior to UV crosslinking, enabling the identification of Tug1-binding proteins in a native tissue context. Comparison with cell line-derived interactomes revealed both shared and tissue-specific interactors. Two shared direct interactions were independently validated by enhanced crosslinking immunoprecipitation (eCLIP). This UV-based ChIRP approach is referred to as cPDiRT (capturing proteins directly interacting with an RNA target). Together, cPDiRT enables the identification of lncRNA-binding proteins in both cultured cells and native tissues. The combination of tissue dissociation and UV crosslinking provides a broadly applicable strategy for studying direct lncRNA-protein interactions across diverse organ systems.