An integrative platform for detection of RNA 2′-O-methylation reveals its broad distribution on mRNA

• Published in: Cell reports methods 2024-03, Vol.4 (3), p.100721-100721, Article 100721
• Main Authors: Tang, Yao, Wu, Yifan, Wang, Sainan, Lu, Xiaolan, Gu, Xiangwen, Li, Yong, Yang, Fan, Xu, Ruilin, Wang, Tao, Jiao, Zichen, Wu, Yan, Liu, Liwei, Chen, Jian-Qun, Wang, Qiang, Chen, Qihan
• Format: Article
• Language: English
• Subjects: 2-O-methylation; alternative splicing; diagnose marker; epigenetics; fibrillarin methyltransferase; mRNA regulation; RNA modification; translation regulation; virus response
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2024.100721

Ribose 2′-O-methylation is involved in critical biological processes, but its biological functions and significance in mRNAs remain underexplored. We have developed NJU-seq, a sensitive method for unbiased 2′-O-methylation (Nm) profiling, and Nm-VAQ, a site-specific quantification tool. Using these tools in tandem, we identified thousands of Nm sites on mRNAs of human and mouse cell lines, of which 68 of 84 selected sites were further validated to be more than 1% 2′-O-methylated. Unlike rRNA, most mRNA Nm sites were from 1% to 30% methylated. In addition, mRNA Nm was dynamic, changing according to the circumstance. Furthermore, we show that fibrillarin is involved as a methyltransferase. By mimicking the detected Nm sites and the context sequence, the RNA fragments could be 2′-O-methylated and demonstrated higher stability but lower translation efficiency. Last, profiling of Nm sites in lung surgery samples revealed common signatures of lung cancer pathogenesis, providing potential new diagnostic markers. [Display omitted] •A platform for high-throughput and site-specific detection of RNA 2′-O-methylation (Nm)•Nominates thousands of mRNA Nm sites, with methylation ratios ranging from 1% to 30%•Demonstrates dynamic response of Nm to viral infection in cells•Profiling of Nm sites in patient samples reveals signatures of lung cancer pathogenesis 2′-O-methylation (Nm) modification is a widely distributed and abundant modification, playing crucial roles in RNA structural stability, protein translation rates, and alternative splicing types. However, research on the location information, catalytic mechanisms, and functions of Nm on mRNAs would benefit from the development of effective and reliable detection tools. In addition, it is also difficult for existing methods to provide an accurate measure of the proportion of modification at the Nm site. To address these gaps, we have developed an integrated methodological strategy to screen and validate Nm sites, especially on mRNA. Tang et al. present an integrative platform for detection of RNA 2′-O-methylation (Nm), including high-throughput and site-specific Nm detection technologies. Using these methods in tandem, they identify precise mRNA Nm sites and ratios and explore molecular mechanisms and disease-related occurrences.