Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA.
Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
Cell. 2024 Sep 19;187(19):5228-5237.e12. doi: 10.1016/j.cell.2024.07.044. Epub 2024 Aug 21.
GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acpU) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.
糖基 RNA 由带有分泌型 N-聚糖的 RNA 组成，这些聚糖展示在细胞表面。尽管之前的研究支持 RNA 和聚糖之间的共价连接，但 RNA-聚糖连接的直接化学性质尚未被描述。在这里，我们开发了一种灵敏且可扩展的方案来检测和表征天然糖基 RNA。利用 RNA 优化的过碘酸钠氧化和醛基连接（rPAL）以及所有理论质谱的顺序窗口采集（SWATH-MS），我们鉴定了修饰的 RNA 碱基 3-（3-氨基-3-羧基丙基）尿嘧啶（acpU）作为糖基 RNA 中 N-聚糖附着的位点。rPAL 作为一种在细胞中鉴定直接糖基-RNA 键的方法具有灵敏性和稳健性，其灵活性将使糖基 RNA 生物学的进一步探索成为可能。
