RNA-binding preference of the Roquin-1 CCCH-type zinc finger

Zinc finger (ZnF) domains are widespread RNA-binding domains, recognizing single stranded and double stranded RNAs. Together with other RNA-binding domains, ZnFs enhance the affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing RBP Roquin controls mRNA decay by recognizing stem-loop elements in the 3'-UTR of its target mRNAs. Stem-loop elements are bound in a shape-specific manner by the unique ROQ domain with nanomolar affinity. The ZnF was suggested to play an essential role in RNA decay by Roquin. However, its RNA motif preference was not investigated so far. Here, we use RBNS to elucidate the RNA binding preference of the isolated Roquin-1 ZnF domain. We find that the ZnF preferentially recognizes single-stranded AU-rich elements (ARE). RNA Bind-n-Seq (RBNS) to systematically explore the binding specificity of Roquins ZnF domain. A 20-nt random RNA pool (1 µM) flanked by short constant adapter sequences was incubated with different concentrations of the Strep-tagged ZnF domain (1, 5, 20 µM).