NMR-derived secondary structure of the full-length Ox40 mRNA 3′UTR and its multivalent binding to the immunoregulatory RBP Roquin

Control of posttranscriptional mRNA decay is a crucial determinant of cell homeostasis and differentiation. mRNA lifetime is governed by cis-regulatory elements in their 3′ untranslated regions (UTR). Despite ongoing progress in the identification of cis elements we have little knowledge about the functional and structural integration of multiple elements in 3′UTR regulatory hubs and their recognition by mRNA-binding proteins (RBPs). Structural analyses are complicated by inconsistent mapping and prediction of RNA fold, by dynamics, and size. We here, for the first time, provide the secondary structure of a complete mRNA 3′UTR. We use NMR spectroscopy in a divide-and-conquer strategy complemented with SAXS, In-line probing and SHAPE-seq applied to the 3′UTR of Ox40 mRNA, which encodes a T-cell co-receptor repressed by the protein Roquin. We provide contributions of RNA elements to Roquin-binding. The protein uses its extended bi-modal ROQ domain to sequentially engage in a 2:1 stoichiometry with a 3′UTR core motif. We observe differential binding of Roquin to decay elements depending on their structural embedment. Our data underpins the importance of studying RNA regulation in a full sequence and structural context. This study serves as a paradigm for an approach in analysing structured RNA-regulatory hubs and their binding by RBPs.