RNA binding proteins play a central role in how cells control gene expression. They interact with RNA molecules to influence processes such as splicing, stability, and translation. However, identifying exactly which RNA molecules these proteins bind to inside cells has been difficult due to technical limitations in sensitivity and reproducibility.

Researchers at the University of Chinese Academy of Sciences have developed a new method called CuCLIP-seq to improve how these interactions are studied. This approach enables direct capture of RNA binding protein and RNA interactions within cells using a chemical crosslinking strategy.

Development of CuCLIP-seq

a, Schematic diagram of the principle of CuCLIP reaction. Proteins and RNAs are labeled with Nhydroxysuccinimide- azidebutyrate (B-NSA) and 5-alkynyl uridine (5-EU), respectively. The labeled RBPs and RNAs are rapidly chemically crosslinked by CuAAC to generate a stable RBPRNA complex. b, Schematic diagram of CuAAC-Crosslinking and Immunoprecipitation Sequencing (CuCLIP-seq) method. The RBP with SBP tag is overexpressed in cells. The RNAs and RBPs are labeled with 5-EU and B-NSA, respectively. Then, the CuAAC catalytic reagent is added for biorthogonal reaction to generate the crosslinked RBP-RNA complex. The RBP-RNA complex is enriched with streptavidin magnetic beads, followed by fragmentation and 3’terminal dephosphorylation of the RNA. Finally, the RBP is digested, and purified RNA is extracted for library construction and sequencing.

The technique works by forming a stable connection between RNA binding proteins and their target RNA molecules through a specialized chemical reaction. Once these complexes are captured, they can be isolated and analyzed using RNA sequencing, allowing researchers to identify the exact RNA targets associated with each protein.

One of the key strengths of CuCLIP-seq is its ability to detect low-abundance RNA targets that are often missed by other methods. This makes it especially useful for studying rare or transient interactions that still play important roles in cellular function.

The researchers demonstrated the method by mapping RNA targets of several well-known RNA binding proteins, including those involved in alternative splicing and RNA editing. The results showed improved specificity and consistency compared to existing approaches.

By providing a more reliable way to study RNA protein interactions, this method helps researchers better understand how gene expression is regulated at the RNA level. These insights could contribute to advances in areas such as cancer biology, neurobiology, and RNA-based therapeutics.

Availability – All original code has been deposited on Zenodo (https://zenodo.org/records/17820375).