Figure 1.

dentification of the mRNA interactome. RNA-binding proteins are covalently cross-linked to RNAs using 254 nM UV-C light [2] or 365 nM UV light in conjunction with photoactivatable ribonucleosides (PARs) such as 4-thiouridine (4SU) [2,3]. The RNA is used as bait in a pull-down with oligo-dT-coated beads and purified under stringent conditions to eliminate contamination from non-cross-linked proteins. (a) Proteins are released by RNAse digestion and analyzed by mass spectrometry. (b) RNAs are blotted onto nitrocellulose, released by proteinase K, and analyzed with high-throughput sequencing. The diagnostic T to C changes in 4SU-labelled RNA identifies cross-link sites. PAR-CLIP, photoactivatable ribonucleoside UV cross-linked immunoprecipitation; X-link, cross-link.