Quantitative 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 cis-regulation of adjacent D20 sites. Application of CRACI to human mRNA reveals that D modifications are present but rare and occur at very low stoichiometry. CRACI thus provides a powerful platform for investigating D biology across species.