A metabolic labeling method detects m6A transcriptome-wide at single base resolution

Autor: Xiao Shu, Zhike Lu, Yizhen Wang, Xinhua Feng, Mohan Cheng, Ting Li, Lijia Ma, Jianzhao Liu, Qing Dai, Xiaolong Cui, Jie Cao, Siying Xiang, Chuan He, Xiner Ying, Minsong Gao, Fengqin Wang, Yanan Yue
Rok vydání: 2020
Předmět:
Zdroj: Nature Chemical Biology. 16:887-895
ISSN: 1552-4469
1552-4450
DOI: 10.1038/s41589-020-0526-9
Popis: Transcriptome-wide mapping of N6-methyladenosine (m6A) at base resolution remains an issue, impeding our understanding of m6A roles at the nucleotide level. Here, we report a metabolic labeling method to detect mRNA m6A transcriptome-wide at base resolution, called ‘m6A-label-seq’. Human and mouse cells could be fed with a methionine analog, Se-allyl-l-selenohomocysteine, which substitutes the methyl group on the enzyme cofactor SAM with the allyl. Cellular RNAs could therefore be metabolically modified with N6-allyladenosine (a6A) at supposed m6A-generating adenosine sites. We pinpointed the mRNA a6A locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. We identified a few thousand mRNA m6A sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of m6A-label-seq with available m6A sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered m6A sites and holds promise to locate nuclear nascent RNA m6A modifications. The authors developed a metabolic labeling method via incorporation of allyl-SAM analogs to profile transcriptome-wide m6A at base resolution, which enables identification of m6A motifs and clustered m6A sites.
Databáze: OpenAIRE
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