Comprehensive profiling of Roquin binding preferences for RNA stem-loops

Roquin proteins target specific RNA stem-loop motifs with their unique ROQ domain for mRNA degradation. The cellular levels of mRNAs are controlled by these post-transcriptional cis-regulatory stem-loop elements in the 3'-untranslated region (3'-UTR). Here, we first provide an RBNS (RNA Bind-n-Seq) experiment using pre-structured RNA libraries as input. This sRBNS (structured RBNS) was used to map Roquin's RNA binding preferences at nucleotide resolution, revealing preferences for loop sizes and consensus motifs for CDEs and ADEs. Structured RNA Bind-n-Seq (sRBNS) experiment to test the ability of Roquin's unique ROQ domain to interact with specific RNA structures. Different pre-structured RNA pools (1 µM) with random loop nucleotides flanked by constant stem sequences were incubated with different concentrations of the coreROQ domain (10, 50, 500 nM).