{"doi":"10.1101/2023.11.04.565616","title":"A minimal sequence motif drives selective tRNA dihydrouridylation by hDUS2","abstract":"The post-transcriptional reduction of uridine to dihydrouridine (D) by dihydrouridine synthase (DUS) enzymes is among the most ubiquitous transformations in RNA biology. D is found at multiple sites in tRNAs and studies in yeast have proposed that each of the four eukaryotic DUS enzymes modifies a different site, however the molecular basis for this exquisite selectivity is unknown and human DUS enzymes have remained largely uncharacterized. Here we investigate the substrate specificity of human dihydrouridine synthase 2 (hDUS2) using mechanism-based crosslinking with 5-bromouridine (5-BrUrd)-modified oligonucleotide probes and in vitro dihydrouridylation assays. We find that hDUS2 modifies U20 in the D loop of diverse tRNA substrates and identify a minimal GU motif within the tRNA tertiary fold required for directing its activity. Further, we use our mechanism-based platform to screen small molecule inhibitors of hDUS2, a potential anti-cancer target. Our work elucidates the principles of substrate modification by a conserved DUS and provides a general platform to studying RNA modifying enzymes with sequence-defined activity-based probes.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":413677,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9547,"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":1088132,"name":"Nathan J. Yu","orcid":null,"position":1,"is_corresponding":false},{"id":638904,"name":"Ralph E. Kleiner","orcid":"0000-0003-0508-9975","position":2,"is_corresponding":false},{"id":1194932,"name":"Jingwei Ji","orcid":"0000-0002-9527-8296","position":0,"is_corresponding":true}],"reference_count":27,"raw_metadata":null,"created_at":"2026-07-19T01:21:57.641539Z","pmid":"37961591","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":[]}