Quantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution
- Cheng-Wei Ju
- Han Li
- Bochen Jiang
- Xuanhao Zhu
- Liang Cui
- Zhanghui Han
- Junxi Zou
- Yunzheng Liu
- Shenghai Shen
- Hardik Shah
- Chang Ye
- Yuhao Zhong
- Ruiqi Ge
- Peng Xia
- Yiyi Ji
- Shun Liu
- Fan Yang
- Bei Liu
- Yuzhi Xu
- Jiangbo Wei
- Li-Sheng Zhang
- Chuan He
2025-10-06
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 C hemical R eduction A ssisted C ytosine I ncorporation 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.