Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach,Molecular Cell

当前位置： X-MOL 学术 › Mol. Cell › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach
Molecular Cell ( IF 14.5 ) Pub Date : 2024-07-12 , DOI: 10.1016/j.molcel.2024.06.021
Liang Lu ₁ , Xiaoting Zhang ₂ , Yuenan Zhou ₃ , Zuokun Shi ₃ , Xiwen Xie ₄ , Xinyue Zhang ₅ , Liaoliao Gao ₄ , Anbo Fu ₄ , Cong Liu ₂ , Bo He ₆ , Xushen Xiong ₇ , Yafei Yin ₅ , Qingqing Wang ₈ , Chengqi Yi ₉ , Xiaoyu Li ₄

Affiliation

Department of Biochemistry and Department of Gastroenterology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, China.
State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.
Department of Biochemistry and Department of Gastroenterology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; Department of Cell Biology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China.
Department of Biochemistry and Department of Gastroenterology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China.
Department of Cell Biology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China.
Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
The Second Affiliated Hospital and Liangzhu Laboratory, Zhejiang University School of Medicine, Hangzhou 311121, China.
Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, China.
State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.

5-methylcytosine (mC) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive mC sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report mC-TAC-seq, a bisulfite-free approach combining TET-assisted mC-to-fC oxidation with selective chemical labeling, therefore enabling direct base-resolution mC detection through pre-enrichment and C-to-T transitions at mC sites. With mC-TAC-seq, we comprehensively profiled the mC methylomes in human and mouse cells, identifying a substantially larger number of confident mC sites. Through perturbing potential mC methyltransferases, we deciphered the responsible enzymes for most mC sites, including the characterization of NSUN5’s involvement in mRNA mC deposition. Additionally, we characterized mC dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in mC-TAC-seq allow mC detection in chromatin-associated RNAs. The accurate and robust mC-TAC-seq will advance research into mC methylation functional investigation.

中文翻译：

通过无亚硫酸氢盐酶辅助化学标记方法对哺乳动物转录组进行碱基分辨率 m5C 分析

5-甲基胞嘧啶 (mC) 是一种常见的 RNA 修饰，对于基因表达调控至关重要。然而，由于重亚硫酸盐测序 (BS-seq) 过程中 RNA 严重降解和序列复杂性降低，准确且灵敏的 mC 位点识别仍然具有挑战性。在这里，我们报告了 mC-TAC-seq，这是一种无亚硫酸氢盐的方法，将 TET 辅助的 mC 到 fC 氧化与选择性化学标记相结合，因此能够通过预富集和 C 到 T 转换来直接进行碱基分辨率 mC 检测。 mC 站点。通过 mC-TAC-seq，我们全面分析了人类和小鼠细胞中的 mC 甲基化组，识别出大量的可信 mC 位点。通过干扰潜在的 mC 甲基转移酶，我们破译了大多数 mC 位点的负责酶，包括 NSUN5 参与 mRNA mC 沉积的特征。此外，我们还对 mESC 分化过程中的 mC 动力学进行了表征。值得注意的是，mC-TAC-seq 中温和的反应条件和核苷酸组成的保存允许在染色质相关 RNA 中进行 mC 检测。准确而稳健的 mC-TAC-seq 将推进 mC 甲基化功能研究的研究。

更新日期：2024-07-12

点击分享查看原文

点击收藏

阅读更多本刊新发论文本刊介绍/投稿指南