A UV-Based ChIRP Method For The Verifiable Identification of Proteins That Directly Interact With LncRNA: From Cultured Cells To Mouse Testis

Summary

A UV-based ChIRP method to identify direct lncRNA-protein interactions.

Abstract

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.

Introduction

Recent high-throughput transcriptomic studies have uncovered thousands of long non-coding RNAs (lncRNAs) that participate in diverse biological processes, ranging from chromatin organization to cellular signaling^1,2. Despite their widespread expression and functional importance, the molecular mechanisms of most lncRNAs remain poorly defined. A major obstacle is the identification of their physical protein partners, particularly RNA-binding proteins (RBPs)^3,4,5.

Current RNA-centric methods p....

Protocol

All solutions were prepared using nuclease-free, protease-free ultrapure water and reagents. All reagents and equipment are listed in the Table of Materials.

All animal experiments were approved by the Animal Ethics Committee of Nanjing Medical University (Approval No.: IACUC-1812003-4) and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 8th edition). Maintain male C57BL/6 mice (4 weeks old) under a 12 h light/12 h dark cycle with ad libitum access to food and water. Minimize animal suffering by using the fewest animals necessary to achieve statisti....

Results

To identify Tug1-binding proteins, UV crosslinking was incorporated into the ChIRP workflow, establishing a method designated cPDiRT (Figure 1). Conventional ChIRP was first performed in GC2 cells to enrich Tug1-interacting proteins (Figure 2A, B). Mass spectrometry analysis identified more than 190 proteins enriched relative to the negative control (Figure 2C; Supplementary Table 2

Discussion

cPDiRT was developed as a modified ChIRP-based protocol that replaces paraformaldehyde (PFA) crosslinking with UV crosslinking to selectively capture direct lncRNA-protein interactions. By combining enzymatic dissociation of fresh testicular tissue into single cells with UV crosslinking, direct-interaction mapping was extended from cultured cell lines to native tissues. Parallel experiments in cultured cells enabled a side-by-side comparison of PFA-based and UV-based approaches, and UV-derived interactions were independe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA Solution	Gibco	25200056	for tissue dissociation and single-cell suspension preparation
100% Ethanol	Sigma-Aldrich	459844	for RNA cleanup
AffinityScript Reverse Transcriptase	Agilent	600107	for eCLIP cDNA synthesis
Anti-CCT3 Antibody	Abcam	Ab225878	antibody for eCLIP
Anti-PABPC1 Antibody	Abcam	Ab312314	antibody for eCLIP
Chloroform	Sigma-Aldrich	C2432	for RNA extraction
Collagenase Type IV	Sigma-Aldrich	C5138	for enzymatic dissociation of testicular tissue
Dephosphorylation reagents (T4 PNK)	New England Biolabs	M0201S	for eCLIP RNA dephosphorylation
DMEM High Glucose Medium	Gibco	11965092	for cell culture and tissue dissociation buffer
Dynabeads MyOne Streptavidin C1 Magnetic Beads	Thermo Fisher Scientific	65001	for capture of biotinylated probe-RNA-protein complexes
DynaMag-15 Magnet	Thermo Fisher Scientific	12301D	for magnetic bead separation
DynaMag-2 Magnet	Thermo Fisher Scientific	12321D	for magnetic bead separation
EDTA	Sigma-Aldrich	E9884	for chelating divalent cations in lysis and wash buffers
Exonuclease I	New England Biolabs	M0293S	for eCLIP cDNA purification
Fetal Bovine Serum (FBS)	Gibco	10099141C	for stopping trypsin digestion
Formamide	Thermo Fisher Scientific	AM9342	for hybridization buffer preparation
Hybridization Oven	TUOHE	LF-I	for probe hybridization and beads washing
Low-Melting-Temperature Agarose	Sigma-Aldrich	A9414	for eCLIP library size selection
MinElute Gel Extraction Kit	Qiagen	28604	for eCLIP library size selection
miRNeasy Mini Kit	Qiagen	217004	for RNA purification
NaCl	Sigma-Aldrich	S7653	for preparing high-salt wash buffer
Nitrocellulose Membrane	GE Healthcare / Amersham	10600002	for eCLIP western blot transfer
NP-40	Thermo Fisher Scientific	85125	for wash buffer detergent
Nuclease- and protease-free water	Thermo Fisher Scientific	10977035	for preparing nuclease-free solutions
NuPAGE 4-12% Bis-Tris Gel	Thermo Fisher Scientific	NP0322BOX	for eCLIP size selection
NuPAGE Bis-Tris Precast Gels	Thermo Fisher Scientific	NP0321BOX	for SDS-PAGE protein separation
PBS (pH 7.4)	Thermo Fisher Scientific	10010023	for cell washing and resuspension
Phenylmethylsulfonyl fluoride (PMSF)	Sigma-Aldrich	P7626	serine protease inhibitor for cell lysis buffer; added fresh before use
Protease Inhibitor Cocktail	Roche	4693132001	broad-spectrum protease inhibitor for cell lysis buffer; added fresh before use
Proteinase K	Sigma-Aldrich	P4850	for protein digestion in RNA extraction
qRT-PCR Master Mix	Thermo Fisher Scientific	11704044	for quantitative RT-PCR to confirm RNA enrichment
RNase/Protease-Free DNase I Solution	Thermo Fisher Scientific	EN0521	for tissue dissociation
RNeasy Mini Kit	Qiagen	74104	for RNA purification
SDS-PAGE Sample Loading Buffer	Beyotime	P0286	for protein sample preparation
Shrimp Alkaline Phosphatase	New England Biolabs	M0371S	for eCLIP cDNA purification
Silver Stain Kit	Beyotime	P00175	for visualizing proteins in SDS-PAGE gels
Sodium Deoxycholate	Sigma-Aldrich	D6750	for wash buffer detergent
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	L3771	for cell lysis and protein denaturation
Superase-in RNase Inhibitor	Thermo Fisher Scientific	AM2694	specialized RNase inhibitor; added fresh before use
T4 RNA Ligase	New England Biolabs	M0204	for eCLIP adapter ligation
Tris–HCl (pH 7.0)	Sigma-Aldrich	T5941	for preparing lysis, hybridization, and wash buffers
TRIzol Reagent	Thermo Fisher Scientific	15596026CN	for RNA extraction
Ultrasonic cell crusher	ATPIO	ATPIO-650D	for cell lysis and DNA fragmentation
Ultraviolet Crosslinker	Analytik Jena	UVP CL-1000	for UV crosslinking of RNA-protein interactions
urea	Sigma-Aldrich	U5128	for eCLIP protein degradation on membrane