O-Glycosylated RNA Identification and Prediction by Solid-Phase Chemoenzymatic TnORNA Method and PONglyRNA Tool

作者：Jiajia Li, Linshu Wang, Jianbo Deng, Xinyu Miao, Xiaodong Yang, Yuan Zhou, Longjiang Xu, Shuang Yang · 发表于：Analytical Chemistry · 年份：2025 · DOI：10.1021/acs.analchem.5c02197 · 被引用次数：17 · 研究领域：Glycosylation and Glycoproteins Research、RNA modifications and cancer、RNA and protein synthesis mechanisms

Recent studies have shown that the cell surface undergoes post-transcriptional modification by N-linked glycosylation. However, the question of whether RNA can be glycosylated by O-glycans remains to be explored. The presence of O-glycosylation in cells and tissues is indirectly revealed by the presence of O-glycans on RNAs following treatment with O-glycoproteases. To identify RNA O-glycosylation, we have developed a chemoenzymatic method for capturing and enriching O-glycosylated RNA (O-glycoRNA) using covalent immobilization on a solid support. GalNAcEXO selectively releases Tn-containing O-glycosylated RNAs (TnORNA). Following the confirmation of small RNA integrity after galactosidase oxidation and GalNAcEXO digestion, we employed this method to compare the expression of O-glycoRNAs and N-glycoRNAs in pancreatic cancer cell lines and tissues. We found that glycosylated miR-103a-3p, miR-122–5p, and miR-4492 regulate pancreatic cancer cell growth and proliferation through the PI3K-Akt pathway. In vitro assays and PDAC tissue analysis confirmed the potential regulatory roles of Tn- O -glycosylated miRNAs in pancreatic tumor growth and metastasis. Furthermore, a significant number (131) of miRNAs carrying both N- and Tn- O -glycosylation were identified, indicating the potential co-occurrence of N-linked and O-linked glycosylation on small RNAs. We have also developed PONglyRNA, an online bioinformatic tool for the prediction of glycosylated RNAs, which showed competitive pe...