Identification of RNA helicase target sites by UV cross-linking and analysis of cDNA

Abstract

Many RNA helicases have been implicated in one or more pathways of RNA metabolism, but only in a very few cases have their target sites on the RNA been identified. Here, we give a detailed description of the UV cross-linking and analysis of cDNA (CRAC) method, and its application to the identification of binding sites of RNA-interacting helicases. CRAC makes use of a bipartite tag on the protein of interest and includes a purification step under highly denaturing conditions. This is particularly important for the accurate mapping of binding sites within large RNA-protein complexes--such as spliceosomes or preribosomes. Partial RNase digestion leaves a footprint of the protein covering the interaction site, and the UV cross-linking sites are frequently highlighted by microdeletions in cDNA sequence reads. Deep sequencing of cDNA libraries generated from cross-linked RNA fragments allows a genome-wide analysis of the interactome of RNA-binding proteins. In the case of RNA helicases, this has proven to be an important step toward their functional analysis.