Pseudo-RNA-Binding Domains Mediate RNA Structure Specificity in Upstream of N-Ras
Nele Merret Hollmann, Pravin Kumar Ankush Jagtap, Paweł Masiewicz, Tanit Guitart, Bernd Simon, Jan Provazník, Frank Stein, Per Haberkant, Lara Sweetapple, Laura Villacorta, Dylan Mooijman, Vladimı́r Beneš, Mikhail M. Savitski, Fátima Gebauer, Janosch Hennig
Abstract
RNA-binding proteins (RBPs) commonly feature multiple RNA-binding domains (RBDs), which provide these proteins with a modular architecture. Accumulating evidence supports that RBP architectural modularity and adaptability define the specificity of their interactions with RNA. However, how multiple RBDs recognize their cognate single-stranded RNA (ssRNA) sequences in concert remains poorly understood. Here, we use Upstream of N-Ras (Unr) as a model system to address this question. Although reported to contain five ssRNA-binding cold-shock domains (CSDs), we demonstrate that Unr includes an additional four CSDs that do not bind RNA (pseudo-RBDs) but are involved in mediating RNA tertiary structure specificity by reducing the conformational heterogeneity of Unr. Disrupting the interactions between canonical and non-canonical CSDs impacts RNA binding, Unr-mediated translation regulation, and the Unr-dependent RNA interactome. Taken together, our studies reveal a new paradigm in protein-RNA recognition, where interactions between RBDs and pseudo-RBDs select RNA tertiary structures, influence RNP assembly, and define target specificity.