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. 2024 Sep 19;187(19):5228-5237.e12.
doi: 10.1016/j.cell.2024.07.044. Epub 2024 Aug 21.

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

Yixuan Xie  1 , Peiyuan Chai  2 , Nicholas A Till  3 , Helena Hemberger  2 , Charlotta G Lebedenko  2 , Jennifer Porat  2 , Christopher P Watkins  2 , Reese M Caldwell  2 , Benson M George  2 , Jonathan Perr  2 , Carolyn R Bertozzi  4 , Benjamin A Garcia  5 , Ryan A Flynn  6
Affiliations

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

Yixuan Xie et al. Cell. .

Abstract

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 (acp3U) 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.

Keywords: RNA modifications; acp3U; cell surface; glycoRNA.

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Conflict of interest statement

Declaration of interests R.A.F. is a stockholder of ORNA Therapeutics and is a board of directors’ member and stockholder of Chronus Health and Blue Planet Systems. C.R.B. is a cofounder of Firefly Biologics and a cofounder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), ReNAgade Therapeutics, and InterVenn Biosciences.

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