Detaillierte Analyse der Bindepräferenz von Roquin an RNA‐Stammschleifen

Abstract

The cellular levels of mRNAs are controlled post‐transcriptionally by cis‐regulatory elements located in the 3'‐untranslated region. These linear or structured elements are recognized by RNA‐binding proteins (RBPs) to modulate mRNA stability. The Roquin‐1 and ‐2 proteins specifically recognize RNA stem‐loop motifs, the trinucleotide loop‐containing constitutive decay elements (CDEs) and the hexanucleotide loop‐containing alternative decay elements (ADEs), with their unique ROQ domain to initiate mRNA degradation. However, the RNA‐binding capacity of Roquin towards different classes of stem‐loops has not been rigorously characterized, leaving its exact binding preferences unclear. Here, we map the RNA‐binding preference of the ROQ domain at nucleotide resolution introducing sRBNS (structured RNA Bind‐n‐Seq), a customized RBNS workflow with pre‐structured RNA libraries. We found a clear preference of Roquin towards specific loop sizes and extended the consensus motifs for CDEs and ADEs. The newly identified motifs are recognized with nanomolar affinity through the canonical RNA‐ROQ interface. Using these new stem‐loop variants as blueprints, we predicted novel Roquin target mRNAs and verified the expanded target space in cells. The study demonstrates the power of high‐throughput assays including RNA structure formation for the systematic investigation of (structural) RNA‐binding preferences to comprehensively identify mRNA targets and elucidate the biological function of RBPs.