Figure 1

Protein-centric methods for detecting RNA-protein interactions. (a) Schematic of native and denaturing methods. RNA-protein crosslinks are represented by red Xs. Non-specific interactions in solution are labeled (NS) and represented by blue RNA fragments. (b) Computational considerations for identification of interaction sites. The top panel depicts two transcripts - one low-abundance and one high-abundance - that both contain a region that is twofold enriched in the immunoprecipitated (IP) sample over a control. Enrichment measurements in the low-abundance case suffer from high variance. The bottom panel shows simulated enrichment values in a low-abundance region and a high-abundance region, which both have a twofold enrichment in the IP sample. For the low abundance region, the observed log-fold changes are often far from the true underlying value while the abundant transcript shows a more consistent enrichment estimation. (c) A schematic of methods for mapping the precise protein binding sites on RNA. PAR-CLIP takes advantage of U → C transitions induced by UV crosslinking after 4SU incorporation. iCLIP uses the occasional arrest of reverse transcription at crosslink sites and tags and sequences these positions. CRAC relies on reverse transcription errors (deletions and substitutions) at crosslink sites to map sites. CRAC, cross-linking and analysis of cDNA; iCLIP, individual-nucleotide resolution cross-linking and immunoprecipitation; PAR-CLIP, photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation.