In vivo detection of protein binding in the transcriptome

Jungkamp, A.-C. et al. Mol. Cell 44, 828–840 (2011).

Immunoprecipitation of RNA-binding proteins cross-linked to RNA and sequencing of bound fragments is a commonly used method to determine binding sites in the transcriptome. To achieve nucleotide resolution, photoactivatable ribonucleosides can be used to enhance cross-linking and reduce background. Jungkamp and colleagues now optimize the technique for use in the transparent nematode Caenorhabditis elegans (in vivo photoactivatable ribonucleoside cross-linking and immunoprecipitation or iPAR-CLIP). The group tested parameters for efficient and uniform labeling of RNA and demonstrated tissue-specific results. They performed iPAR-CLIP on epitope-tagged GLD-1, a protein required for normal oogenesis, and recovered 439 binding sites including all known sites with high reproducibility. They extensively validated the sites, which included using a proteomic approach to confirm the effect of gld-1 knockdown on the amounts of protein targets.