uvCLAP is a fast and non-radioactive method to identify in vivo targets of RNA-binding proteins
Maticzka D, Ilik IA, Aktas T, Backofen R, Akhtar A.
RNA-binding proteins (RBPs) play important and essential roles in eukaryotic gene expres-sion regulating splicing, localization, translation, and stability of mRNAs. We describeultraviolet crosslinking and affinity purification (uvCLAP), an easy-to-use, robust, repro-ducible, and high-throughput method to determine in vivo targets of RBPs. uvCLAP is fast anddoes not rely on radioactive labeling of RNA. We investigate binding of 15 RBPs fromfly,mouse, and human cells to test the method’s performance and applicability. Multiplexing ofsignal and control libraries enables straightforward comparison of samples. Experiments formost proteins achieve high enrichment of signal over background. A point mutation and anatural splice isoform that change the RBP subcellular localization dramatically alter targetselection without changing the targeted RNA motif, showing that compartmentalization ofRBPs can be used as an elegant means to generate RNA target specificity.
