Molecular Cell | Jinkai Wang's group developed a single-molecule m6A detection algorithm based on endogenous labeling to reveal the complexity of m6A on isomers
Eukaryotic pre-mRNA can be selectively processed to form different RNA isomers, which include variable splicing and variable caudation. In addition, the same gene also has variable promoters, thus producing RNA isomers with different 5 'ends. These types of complex RNA isomers are widespread in mammalian cells. However, it is not clear whether m6A can selectively label these RNA isomers, especially whether the same m6A site on different RNA isomers can also produce different m6A modifications. The lack of understanding of this scientific problem is mainly due to a lack of technology. Because existing m6A detection techniques require interruption of RNA, and second-generation high-throughput sequencing can only read short sequences, it is difficult to distinguish m6A on different isomers. Oxford nanopore sequencing (ONT), a third-generation sequencing technology, can detect m6A modifications using direct RNA sequencing (DRS) to directly read changes in the current of full-length RNA passing through the nanopore. However, the nanopore reads the electrical signals of multiple bases at once, and the detection of m6A by electrical signals relies on deep learning models. Due to the lack of large-scale m6A positive signal at the single molecule level, although the current deep learning model can accurately identify the m6A site, it is difficult to reach the resolution at the single molecule level, and it is still unable to accurately distinguish the m6A modification state on different isomers.
On February 7, 2025, Wang Jinkai's research group at the Sun Yat-sen School of Medicine at Sun Yat-sen University published an online study entitled "Single-molecule m6A detection empowered by endogenous labeling unveils" in the journal Molecular Cell complexities across RNA isoforms ". In this paper, APOBEC1-YTH was used to generate C-to-U mutations near m6A of endogenous RNA in cells, and the third generation of Oxford nanopore direct RNA sequencing (ONT DRS) was performed, so that m6A could be endogenous labeled at the single-molecule level by C-to-U mutations of 10-100 nt near the known m6A site. After data cleaning through semi-supervised learning, m6A electrical signals of about 1 million single molecule level 5-mer were obtained, and a deep learning model m6Aiso was trained to accurately detect m6A modifications at the single molecule level. This revealed that the same m6A site can also produce extensive differences in different isomers through at least three different mechanisms. Especially in the epithelial-stromal transformation model, after TGF-β induction, the activated transcription factor SMAD3 selectively promotes m6A modification of downstream RNA with SMAD3 as promoter by recruiting METTL3/METTL14/WTAP, resulting in selective m6A up-regulation of the same gene using isomers of different promoters. This study provides a new perspective for understanding the complexity of m6A in isomers and the selective regulation mechanism of m6A.
The corresponding author of the paper is Wang Jinkai, professor of Sun Yat-sen School of Medicine, Sun Yat-sen University. Guo Wenbing, Ren Zhijun and Huang Xiang, postdoctoral fellows of the research group, are co-first authors of the paper. This work was supported by the National Natural Science Foundation of China, the Science Foundation of Guangdong Province, the Postdoctoral Fund and the Interdisciplinary Team project of Sun Yat-sen University.
Original link:
https://www.cell.com/molecular-cell/abstract/S1097-2765(25)00049-8
