The RNA-binding domain of hnRNP U extends beyond the RGG/RG motifs

Abstract

ABSTRACTHeterogeneous nuclear ribonucleoprotein U (hnRNP U) is a ubiquitously expressed protein that regulates chromatin architecture through its interactions with numerous DNA, protein, and RNA partners. The RNA-binding domain (RBD) of hnRNP U was previously mapped to an RGG/RG element within its disordered C-terminal region, but little is understood about its binding mode and potential for selective RNA recognition. Analysis of publicly available hnRNP U enhanced UV crosslinking and immunoprecipitation (eCLIP) data identified high-confidence binding sites within human RNAs. We synthesized a set of diverse RNAs encompassing eleven of these identified crosslink sites for biochemical characterization using a combination of fluorescence anisotropy and electrophoretic mobility shift assays. Thesein vitrobinding experiments with a rationally designed set of RNAs and hnRNP U domains revealed that the RGG/RG element is a small part of a more expansive RBD that encompasses most of the disordered C-terminal region. This RBD contains a second, previously experimentally uncharacterized RGG/RG element with RNA-binding properties comparable to the canonical RGG/RG element. These RGG/RG elements serve redundant functions, with neither serving as the primary RBD. While in isolation each RGG/RG element has modest affinity for RNA, together they significantly enhance the association of hnRNP U with RNA, enabling binding of most of the designed RNA set with low to mid-nanomolar binding affinities. Identification and characterization of the complete hnRNP U RBD highlights the perils of a reductionist approach to defining biochemical activities in this system and paves the way for a detailed investigation of its RNA-binding specificity.