This study presents a novel paradigm of how multi-RBD proteins can achieve RNA target specificity by selecting certain RNA tertiary structures and provides a basis for reconstituting an entire RNP for future structure determination efforts.. Non-RNA binding domains of Upstream of N-Ras mediate RNA tertiary structure specificity

RNA binding proteins (RBPs) commonly feature multiple RNA binding domains (RBDs) which are often predicted to be connected by flexible linker regions. How these multiple RBDs recognize their cognate single-stranded RNA sequences in concert is poorly understood. In this study, we show that regions connecting RBDs can contribute to RNA tertiary structure selectivity, exemplified by Upstream of N-Ras (Unr), an RBP that reportedly contains five single-stranded RNA binding cold shock-domains (CSDs). We solved several NMR and crystal structures of various Unr fragments encompassing the major part of Unr, allowing us to draw a structural model of the almost full-length protein. Surprisingly, we discovered four additional non-canonical CSDs in direct interaction with their neighboring canonical CSDs. Using NMR titration, NMR relaxation and SAXS experiments we show that these novel domains do not bind RNA independently, but are involved in mediating RNA tertiary structure specificity by reducing the conformational heterogeneity of Unr. Functional assays and RNA immunoprecipitation followed by sequencing, showed that disruption of canonical-non-canonical CSD interactions influences RNA binding and Unr-mediated translation regulation. Furthermore, by using quantitative proteomics of wild type and inter-domain mutant Unr, we could further identify components of the Unr-dependent ribonucleoprotein (RNP) interactome. Thus, our work presents a novel paradigm of how multi-RBD proteins can achieve RNA target specificity by selecting certain RNA tertiary structures and provides a basis for reconstituting an entire RNP for future structure determination efforts.