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 () and Chuan He ()
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Cheng-Wei Ju: The University of Chicago
Han Li: The University of Chicago
Bochen Jiang: The University of Chicago
Xuanhao Zhu: The University of Chicago
Liang Cui: Singapore-MIT Alliance for Research and Technology
Zhanghui Han: National University of Singapore
Junxi Zou: The University of Chicago
Yunzheng Liu: California Institute of Technology
Shenghai Shen: The Hong Kong University of Science and Technology
Hardik Shah: The University of Chicago
Chang Ye: The University of Chicago
Yuhao Zhong: The University of Chicago
Ruiqi Ge: The University of Chicago
Peng Xia: The University of Chicago
Yiyi Ji: The University of Chicago
Shun Liu: The University of Chicago
Fan Yang: The University of Chicago
Bei Liu: The University of Chicago
Yuzhi Xu: New York University
Jiangbo Wei: National University of Singapore
Li-Sheng Zhang: The Hong Kong University of Science and Technology
Chuan He: The University of Chicago

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-63918-w

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