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

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 performance in validation tests and potentials in improving glycosylation site prediction (http://ponglyrna.797000.xyz:8880/). In conclusion, this study establishes robust experimental and computational tools for identifying O-linked glycoRNAs. Additionally, it uncovers the novel role of glycosylation in PDAC development and progression through altered glycosylation of oncogenic miRNAs.