Regulome

The analysis of RNA-protein interactions allows a better understanding of the post-transcriptional status of RNAs (their stability, localisation, circulation, translation level/efficiency, etc.). ). CLIP Seq (Crosss-Linking ImmunoPrecipitation Sequencing) is a method used to identify target sites for protein binding (PBR) on RNA. This technology combines the immunoprecipitation of relevant PBRs and high throughput RNA sequencing.

The principle of the methodology is based on covalently binding RBPs proteins to RNA by UV irradiation or enzymatic reaction. After cell lysis, RNA-protein complexes are isolated by immunoprecipitation with specific antibodies. The next step consists in RNA trimming. After ligation of the adaptors, protein removal and purification, RBPS binding sites are sequenced by RNAseq technology.

This complex protocol is detailed in C.Y. Flora et al. 2018

Recent technological advances have profoundly transformed this landscape:

1. Nucleotide-Resolution Mapping: Enhanced methods such as eCLIP (enhanced CLIP) and iCLIP2 streamline library preparation efficiency and pinpoint exact binding sites via reverse transcription truncation or cross-link-induced mutations.
2. Antibody-Free and In Vivo Approaches: Proximity-labeling technologies (e.g., TRIBE, APEX-seq) employ fusion proteins to directly edit or mark adjacent RNA in living cells without immunoprecipitation.
3. Single-Cell and Spatial Multi-Omics: Integrating CLIP-seq principles with single-cell sequencing and spatial transcriptomics now enables mapping of the RBP-RNA interactome at single-cell resolution within native tissue contexts.

These innovations open unprecedented avenues in fundamental biology and targeted therapeutic strategies (mRNA therapeutics, gene therapy).

Update: Aug 2026