{"doi":"10.1101/2025.05.09.653160","title":"Toward a comprehensive modification landscape of yeast mitochondrial tRNAs using Nanopore direct RNA sequencing and Dihydrouridine sequencing","abstract":"ABSTRACT Saccharomyces cerevisiae is an invaluable model in the study of mitochondrial tRNA biology. Yet the positions of modified bases in all yeast mitochondrially-encoded tRNAs (mt-tRNAs) are still not fully mapped. We performed Nanopore direct RNA sequencing (DRS) on tRNAs from the crude mitochondrial fraction of yeast to map base modifications across all 24 mt-tRNA isoacceptors. Additionally, we developed a method to detect dihydrouridine sites in tRNAs, tD-seq, where chemical reduction of dihydrouridine causes disruptions to reverse transcription. We mapped dihydrouridine, pseudouridine, and N2-dimethylguanosine sites in mt-tRNAs using DRS, tD-seq, and knockouts of five conserved tRNA-modifying enzymes. Our results establish Dus1 and Dus2 as the enzymes responsible for D 14 , D 16 , D 17 , D 17a , and D 20 formation in S. cerevisiae mt-tRNAs, and revealed interactions between Dus1, Dus2, and Trm1-catalyzed modifications. We provide a comprehensive analysis of S. cerevisiae mt-tRNA base modifications, and identify novel modification “circuits” in yeast mt-tRNAs, in which the loss of a single enzyme’s activity can change modification levels at sites catalyzed by other enzymes. These findings expand our understanding of mt-tRNA base modifications and their interdependence, and advance opportunities for the yeast model for investigating defects in human mt-tRNA function. GRAPHICAL ABSTRACT","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":556274,"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":0.5214,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":575537,"name":"David M. Garcia","orcid":"0000-0003-0600-9527","position":1,"is_corresponding":false},{"id":1454696,"name":"James W. Reinsch","orcid":"0009-0004-6102-6594","position":0,"is_corresponding":true}],"reference_count":67,"raw_metadata":null,"created_at":"2026-07-19T02:55:08.896385Z","pmid":"40654949","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":[]}