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<p>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 <b>C</b>hemical <b>R</b>eduction <b>A</b>ssisted <b>C</b>ytosine <b>I</b>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.</p>

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Quantitative CRACI reveals transcriptome-wide distribution of RNA dihydrouridine at base resolution

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月6日

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  • 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

摘要

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.