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Published on: May 10, 2019
A UV-Based ChIRP Method For The Verifiable Identification of Proteins That Directly Interact With LncRNA: From
Lingxiu Tong1, Yiting Ye1, Wanyin Jiang1
1State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University; Key Laboratory of Systems Biomedicine (Ministry of Education), Institute of Systems Biomedicine, Shanghai Jiao Tong University.
Journal of Visualized Experiments : Jove
|July 27, 2026
Summary
UV crosslinking within ChIRP (comprehensive identification of RNA-binding proteins) enriches direct long non-coding RNA-protein interactions. This method, cPDiRT, identifies these interactions in cells and native tissues.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- Long non-coding RNAs (lncRNAs) regulate biological processes, but their functions are poorly understood.
- Identifying interacting proteins, especially RNA-binding proteins (RBPs), is crucial for elucidating lncRNA function.
- Comprehensive identification of RNA-binding proteins (ChIRP) is a common method, but conventional protocols using paraformaldehyde (PFA) crosslinking capture indirect interactions.
Purpose of the Study:
- To compare UV crosslinking with PFA crosslinking within the ChIRP framework to identify direct lncRNA-protein interactions.
- To develop and validate a UV-based ChIRP workflow, termed cPDiRT (capturing proteins directly interacting with an RNA target), for studying lncRNA interactomes.
- To apply cPDiRT to identify direct Tug1-binding proteins in both cultured cells and mouse testes.
Main Methods:
- Compared UV crosslinking and PFA crosslinking within the ChIRP framework using the lncRNA Tug1 in cultured cells.
- Developed a UV-based ChIRP workflow (cPDiRT) that includes stringent washing conditions to preserve direct interactions.
- Extended the cPDiRT workflow to mouse testes by dissociating tissues into single-cell suspensions prior to UV crosslinking.
Main Results:
- UV-based ChIRP preferentially enriches direct lncRNA-protein interactions compared to PFA-based ChIRP.
- cPDiRT successfully identified Tug1-binding proteins in both cultured cells and mouse testes.
- Comparison of interactomes revealed shared and tissue-specific Tug1-binding proteins, with two shared interactions validated by eCLIP.
Conclusions:
- The UV-based cPDiRT approach effectively identifies direct lncRNA-protein interactions in both cell cultures and native tissues.
- Tissue dissociation combined with UV crosslinking offers a broadly applicable strategy for studying direct lncRNA-protein interactions across various organ systems.
- cPDiRT provides a valuable tool for advancing the understanding of lncRNA function through precise interactome mapping.