MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, China.
College of Animal Sciences, Key Laboratory of Animal Nutrition & Feed Sciences, Ministry of Agriculture, Zhejiang University, Hangzhou, China.
Nat Chem Biol. 2020 Aug;16(8):887-895. doi: 10.1038/s41589-020-0526-9. Epub 2020 Apr 27.
Transcriptome-wide mapping of N-methyladenosine (mA) at base resolution remains an issue, impeding our understanding of mA roles at the nucleotide level. Here, we report a metabolic labeling method to detect mRNA mA transcriptome-wide at base resolution, called 'mA-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 N-allyladenosine (aA) at supposed mA-generating adenosine sites. We pinpointed the mRNA aA locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. We identified a few thousand mRNA mA sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of mA-label-seq with available mA sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered mA sites and holds promise to locate nuclear nascent RNA mA modifications.
基于碱基分辨率的 N6-甲基腺苷（m6A）转录组图谱绘制仍是一个难题，这阻碍了我们在核苷酸水平上对 m6A 功能的理解。在此，我们报道了一种代谢标记方法，可在碱基分辨率上全面检测 mRNA m6A 转录组，称为“m6A-label-seq”。人类和小鼠细胞可以用蛋氨酸类似物 Se-allyl-L-selenohomocysteine 喂养，该类似物将酶辅因子 SAM 上的甲基替换为烯丙基。因此，细胞 RNA 可以在假定的 m6A 产生腺苷位点上被 N-烯丙基腺苷（aA）代谢修饰。我们根据在互补 DNA 反转录过程中碘代反应在相反位置的错误掺入，确定了 mRNA aA 的位置。我们在人类 HeLa、HEK293T 和小鼠 H2.35 细胞中鉴定了几千个 mRNA m6A 位点，与现有的 m6A 测序方法进行了平行比较，并通过正交方法验证了选定的位点。该方法在检测聚集的 m6A 位点方面具有优势，有望定位核新生 RNA m6A 修饰。
