Identification of RNA helicase target sites by UV cross-linking and analysis of cDNA.
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.
Cluster of Excellence Macromolecular Complexes, Institute for Molecular Biosciences, Goethe University Frankfurt, Frankfurt, Germany.
Markus T
MT
Bohnsack
David
D
Tollervey
Sander
S
Granneman
D001665:Binding Sites; D018076:DNA, Complementary / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020365:RNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D014466:Ultraviolet Rays
D018076:DNA, Complementary; D016601:RNA-Binding Proteins; D020365:RNA Helicases