biostudies-literatureJu CWNHGRI NIH HHS8863https://www.ebi.ac.uk/biostudies/studies/S-EPMC12501074biostudies-literatureUnknown16(1)Dihydrouridine (D) is an abundant RNA modification, yet its roles in mammals remain poorly understood due to limited detection methods. We even do not have a comprehensive profile of D site location and modification stoichiometry in tRNA. Here, we introduce Chemical Reduction Assisted Cytosine Incorporation sequencing (CRACI), a highly sensitive, quantitative approach for mapping D at single-base resolution. Using CRACI, we generate the transcriptome-wide maps of D in both cytoplasmic and mitochondrial tRNAs from mammals and plants. We uncover D sites in mitochondrial tRNAs and identify DUS2L as the 'writer' protein responsible for human mitochondrial tRNAs. Furthermore, we demonstrate that most D modifications have a limited impact on tRNA stability, except for D20a, which also exhibits cNature communicationsQuantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution.PMC12501074RM1 HG008935Ju CWLi HZou JHe CCui LYe CXia PHan ZZhong YLiu SShah HYang FZhang LSLiu YGe RJiang BZhu XShen SLiu BJi YWei JXu YfalseQuantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution.Dihydrouridine (D) is an abundant RNA modification, yet its roles in mammals remain poorly understood due to limited detection methods. We even do not have a comprehensive profile of D site location and modification stoichiometry in tRNA. Here, we introduce Chemical Reduction Assisted Cytosine Incorporation sequencing (CRACI), a highly sensitive, quantitative approach for mapping D at single-base resolution. Using CRACI, we generate the transcriptome-wide maps of D in both cytoplasmic and mitochondrial tRNAs from mammals and plants. We uncover D sites in mitochondrial tRNAs and identify DUS2L as the 'writer' protein responsible for human mitochondrial tRNAs. Furthermore, we demonstrate that most D modifications have a limited impact on tRNA stability, except for D20a, which also exhibits c2025-01-01T00:00:00Z2025 Oct2026-06-04T05:35:58.278Z2026-05-05T03:13:01.54ZS-EPMC125010744105306510.1038/s41467-025-63918-w