PT  - JOURNAL ARTICLE
AU  - Daniel Maticzka
AU  - Ibrahim Avsar Ilik
AU  - Tugce Aktas
AU  - Rolf Backofen
AU  - Asifa Akhtar
TI  - uvCLAP: a fast, non-radioactive method to identify <em>in vivo</em> targets of RNA-binding proteins
AID  - 10.1101/158410
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 158410
4099  - http://biorxiv.org/content/early/2017/07/01/158410.short
4100  - http://biorxiv.org/content/early/2017/07/01/158410.full
AB  - RNA-binding proteins (RBPs) play important and essential roles in eukaryotic gene expression regulating splicing, localization, translation and stability of mRNAs. Understanding the exact contribution of RBPs to gene regulation is crucial as many RBPs are frequently mis-regulated in several neurological diseases and certain cancers. While recently developed techniques provide binding sites of RBPs, they are labor-intensive and generally rely on radioactive labeling of RNA. With more than 1,000 RBPs in a human cell, it is imperative to develop easy, robust, reproducible and high-throughput methods to determine in vivo targets of RBPs. To address these issues we developed uvCLAP (UV crosslinking and affinity purification) as a robust, reproducible method to measure RNA-protein interactions in vivo. To test its performance and applicability we investigated binding of 15 RBPs from fly, mouse and human cells. We show that uvCLAP generates reliable and comparable data to other methods. Unexpectedly, our results show that despite their different subcellular localizations, STAR proteins (KHDRBS1-3, QKI) bind to a similar RNA motif in vivo. Consistently a point mutation (KHDRBS1Y440F) or a natural splice isoform (QKI-6) that changes the respective RBP subcellular localization, dramatically alters target selection without changing the targeted RNA motif. Combined with the knowledge that RBPs can compete and cooperate for binding sites, our data shows that compartmentalization of RBPs can be used as an elegant means to generate RNA target specificity.