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

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Developing a new method called rPAL (RNA periodate oxidation and aldehyde ligation), the paper detects and characterizes glycoRNAs, which are RNAs modified with N-linked glycans. GlycoRNAs were recently discovered but the direct chemical linkage between the glycans and RNA was not previously identified.

Using rPAL coupled with mass spectrometry, the authors identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of N-glycan attachment on RNAs. They confirmed this by synthesizing acp3U standards and matching retention times and fragmentation patterns to cell-derived acp3U released by PNGaseF digestion.

Additional evidence comes from synthesis of an acp3U-GlcNAc standard to mimic the glycan-acp3U linkage. This also matched the retention time and MS/MS spectra of material released from glycoRNAs by endoglycosidase digestion. Finally, knockout of the enzyme DTWD2 involved in acp3U biosynthesis reduced detection of glycoRNAs, further supporting acp3U as a key linker. The authors propose a model where acp3U in tRNAs is amidated to allow N-glycan attachment in the ER, with mature glycoRNAs trafficking to the plasma membrane.

This is the first detailed characterization of the molecular connection between glycans and RNAs in the newly discovered glycoRNA family. The improved rPAL method enabled more sensitive detection and de novo identification of acp3U as a linker for N-glycan modifications of RNA.

https://www.biorxiv.org/content/10.1101/2023.11.06.565735v3.abstract