{"doi":"10.1101/2023.09.20.558674","title":"The RNA-binding domain of hnRNP U extends beyond the RGG/RG motifs","abstract":"ABSTRACT Heterogeneous 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. These in vitro binding 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":397313,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9634,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":255313,"name":"Deborah S. Wuttke","orcid":"0000-0002-8158-8795","position":1,"is_corresponding":false},{"id":252273,"name":"Robert Batey","orcid":"0000-0002-1384-6625","position":2,"is_corresponding":false},{"id":1012582,"name":"Otto A. Kletzien","orcid":"0000-0003-4806-6757","position":0,"is_corresponding":true}],"reference_count":70,"raw_metadata":null,"created_at":"2026-07-19T01:19:35.497854Z","pmid":"37786719","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}