Pseudo-Rna Binding Domains Mediate Rna Structure Specificity in Upstream of N-Ras

RNA binding proteins (RBPs) commonly feature multiple RNA binding domains (RBDs), which provides these proteins with a modular architecture. Accumulating evidence supports that RBP architectural modularity and adaptability defines 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 now 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 canonical/non-canonical CSD interactions impacted RNA binding, Unr-mediated translation regulation, and the Unr-dependent RNA interactome. Taken together, our studies revealed 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.