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5 minutes

The authors thank Qiangfeng Zhang (Tsinghua University) and Ci Chu (Howard Chang laboratory) for helpful discussions regarding the original ChIRP protocol. K.Z. was supported by the National Natural Science Foundation of China (32170858) and the Excellent Academic Leader of Shanghai Oriental Talents Program (BJKJ2025057). L.Y. was supported by the National Natural Science Foundation of China (82571836, 32070843, and 82371617).

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	<li>Gr&#228;we C, Stelloo S, Van Hout FAH, Vermeulen M. RNA-centric methods: Toward the interactome of specific RNA transcripts. Trends Biotechnol. 2021;39(9):890-900.</li>
	<li>Chu C, Chang HY. ChIRP-MS: RNA-directed proteomic discovery. Methods Mol Biol. 2018;1861:37-45.</li>
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</ol>

All authors declare no competing financial interests.

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...

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 signaling1,2. 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 primarily rely on chemical crosslinking, which captures both direct and indirect RNA-protein interactions. As a result, these approaches are more suitable for global interactome mapping than for identifying bona fide direct contacts6. Comprehensive identification of RNA-binding proteins followed by mass spectrometry (ChIRP-MS) is a widely used technique for mapping lncRNA-RBP interactions because of its straightforward probe design and efficient workflow7,8. Conventional ChIRP-MS uses paraformaldehyde (PFA) crosslinking, which captures both direct RNA-protein contacts and indirect associations mediated by bridging factors9,10.
In contrast, UV crosslinking at 254 nm offers a critical advantage. It covalently links RNA and proteins only when they are in near-zero-distance contact (typically &#60;1 &#197;)11. This process converts hydrogen bonds into stable covalent bonds and enables stringent high-salt washing conditions that remove non-covalently bound indirect interactors. Consequently, UV-based approaches provide greater specificity for direct RNA-protein interactions than chemical crosslinking methods12,13. Nevertheless, UV-based approaches remain underutilized in tissue-derived lncRNA interactome studies, largely due to concerns about crosslinking efficiency and limited UV penetration.
To investigate direct lncRNA-protein interactions in a physiologically relevant context, the lncRNA Tug1 was selected as a model system. Tug1 represents an ideal benchmark for validating a tissue-compatible direct-interaction workflow because of its severe knockout phenotype and high enrichment in the testis. Genetic ablation of Tug1 results in complete male sterility, impaired spermatogenesis, and abnormal sperm morphology14. In addition, Tug1 supports Sertoli cell function and blood-testis barrier integrity in high-fat diet mice15. Despite these well-established phenotypes, the direct protein partners that mediate Tug1 function in the testis&#8212;an organ expressing large numbers of RBPs that have recently been experimentally profiled16&#8212;remain unknown. This knowledge gap largely reflects the fact that most lncRNA-protein interaction mapping methods have been developed for cultured cell systems.
To address this limitation, the ChIRP framework was modified by replacing PFA crosslinking with UV crosslinking to capture proteins that directly interact with Tug1. Candidate interactions were independently validated by enhanced crosslinking immunoprecipitation followed by deep sequencing (eCLIP-seq)17. The workflow was further extended to tissue applications by dissociating fresh mouse testicular tissue into single cells prior to UV crosslinking. This optimized approach was designated cPDiRT (capturing proteins directly interacting with an RNA target).
Application of cPDiRT to Tug1 in the mouse testis enabled direct-interaction mapping in a native tissue context. Parallel experiments in cultured cells provided a side-by-side comparison of PFA-based and UV-based interaction profiles. Combined with independent eCLIP validation, this approach generated a high-confidence set of molecular partners that may contribute to Tug1 function during spermatogenesis. The protocol presented here enables the capture of proteins that directly interact with a target lncRNA in both cultured cells and native tissues. By defining the direct interactome of Tug1 in the testis, this method advances mechanistic studies of a fertility-essential lncRNA and provides a generalizable platform for investigating lncRNA-protein interactions in reproductive and other complex tissues.

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

a uv-based chirp method for the verifiable identification of proteins that directly interact with lncrna: from cultured cells to mouse testis

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

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.

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</strong...

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

Long non-coding RNAs (lncRNAs) have emerged as important regulators of diverse biological processes. However, the molecular mechanisms underlying the ...

Our research developed cPDiRT, a UV cross-linking method to map directly in RNA and proteins interactions in both the cultured cells and the native tissues such as testes and carving TUG1's molecular mechanisms in spermatogenesis. This protocol enables direct RNA protein into action mapping a wider range of systems from cultured cells to intact native tissues. To begin, wash the GC2 cells three times with PBS to remove residual culture medium.Then, add three milliliters of PBS to the dish and distribute it evenly before cross-linking. Irradiate the cells in a 254-nanometer ultraviolet cross-linker at a dose of 4, 000 millijoules per square centimeter. Collect the cells in PBS and centrifuge the cell suspension at 3000G for five minutes at four degrees Celsius.After discarding the supernatant, weigh the pellet and confirm a minimum pellet mass of 500 milligrams per sample. Collect testes from four-week-old male C57BL6 mice. Wash the decapsulated testicular tissue twice with 5 to 10 milliliters of DMEM.Then, incubate 100 milligrams of tissue with 10 milliliters of DMEM containing one milligram per milliliter collagenase IV at 37 degrees Celsius. Continue the digestion for 5 to 15 minutes until the seminiferous tubules are released. Then, centrifuge the suspension at 1000G for one minute at four degrees Celsius and remove the supernatant.Wash the pellet with 5 to 10 milliliters of DMEM. Centrifuge the suspension again at 1000G for one minute at four degrees Celsius. And re-suspend the pellet in 0.25%trypsin containing one milligram per milliliter deoxyribonuclease I.Incubate the suspension at 37 degrees Celsius for 5 to 15 minutes.Then, add an equal volume of DMEM supplemented with 10%FBS to stop the enzymatic digestion. Filter the suspension through a 70-micrometer cell strainer. Centrifuge the cells at 2000G for five minutes at four degrees Celsius.Discard the supernatant and wash the pellet with 5 to 10 milliliters of PBS. Centrifuge the suspension again at 2000G for five minutes at four degrees Celsius. Re-suspend the single cell suspension in PBS and distribute it evenly onto a 15-centimeter culture dish.Cross-link the testicular cells as demonstrated earlier for GC2 cells. Omit irradiation for the control cells. Collect the cross-linked cells.Centrifuge the suspension at 3000G for five minutes at four degrees Celsius and discard the supernatant. Ensure a minimum pellet mass of 500 milligrams by pooling testicular cells isolated from at least 10 to 12 mice. After weighing the pellet, re-suspend it in cell lysis buffer at a ratio of 100 milligrams of pellet per one milliliter of buffer.After vortexing, sonicate the sample using a focused ultrasonicator or a water bath sonicator until the lysate becomes visually clear. Then, aliquot approximately 10 microliters of the lysate into microcentrifuge tubes for RNA and protein analysis. Add 30 microliters of pre-washed magnetic beads to the lysate.Incubate the samples at 37 degrees Celsius for 30 minutes with gentle mixing in a hybridization oven. Place the samples on a magnetic stand for approximately one minute or until the solution becomes clear. Transfer the cleared lysate to a fresh tube.Place the new tube on the magnetic stand. Then, transfer the lysate to another tube to remove any residual beads. Add two volumes of hybridization buffer tempered at 37 degrees Celsius to each sample.Add biotinulated DNA probes at a ratio of one microliter of 100 micromolar probe stock per one milliliter of lysate. Incubate the samples at 37 degrees Celsius with end-over-end rotation for 12 to 16 hours in a hybridization oven. Immediately before use, wash 100 microliters of streptavidin magnetic beads three times with cell lysis buffer.And remove the residual buffer after the final wash. Briefly centrifuge the hybridization reaction tubes. Then, add the hybridized sample to the washed beads and mix gently.Return the bead suspension to the original tube and incubate at 37 degrees Celsius with mixing for 30 to 40 minutes. After incubation, briefly centrifuge the samples. Place the tubes on a magnetic stand for one to two minutes, or until the solution becomes clear.Then, discard the supernatant. To wash the beads, add one milliliter of the wash buffer and incubate the tubes in the hybridization oven at 37 degrees Celsius for five minutes. After five washes, transfer 1-10%of the beads to a fresh tube for RNA analysis.Finally, add 40 microliters of 1X SDS sample buffer to the remaining beads. Boil the samples at 95 degrees Celsius for 30 minutes to elude the proteins and reverse the cross-links. Recover the proteins for downstream mass spectrometry analysis.ChIRP mass spectrometry analysis identified more than 190 proteins enriched relative to the negative control in GC2 cells. At a UV dose of 4, 000 millijoules per square centimeter, cPDiRT-MS identified a more selective protein set. More than two-thirds of these proteins overlapped with those identified by conventional ChIRP-MS.Most proteins identified with at least two unique peptides were reproducibly detected across biological replicates in both GC2 cells and mouse testes. Gene ontology analysis of the reproducibly identified proteins revealed biological processes broadly consistent with the known functions of TUG1. Comparison of the GC2 cell and mouse testes data sets identified 15 proteins that were commonly enriched.Enhanced cross-linking immunoprecipitation sequencing revealed clear and specific binding peaks on the TUG1 transcript for PABPC1 and CCT3. cPDiRT allows researchers to identify and measure proteins that directly interact with specific linked RNA in both the culture cells and the native tissues. The most important challenge is achieving efficient UV cross-linking while ensuring high specificity for direct RNA and the protein interactions.Our protocol will support future studies to apply this approach to other RNAs and the tissues in restating direct RNA protein into action in development and the reproduction.

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Research

JoVE Journal

Biochemistry

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Chromatin Isolation by RNA Purification (ChIRP)

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

This video demonstrates the enhanced crosslinking immunoprecipitation (eCLIP) method for identifying RNA-binding proteins in mouse testis. eCLIP enables the precise identification of binding sites where RNA-binding proteins (RBPs) interact with their target RNAs.

An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Luminescent identification of functional elements in 3’ untranslated regions (3’UTRs) (3’LIFE) is a technique to identify functional regulation in 3’UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

Here, we present an eCLIP protocol to determine major RNA targets of RBP candidates in testis.

Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis

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

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

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