Glycosylated RNA (glycoRNA) has recently emerged as a novel constituent of the glycocalyx on cell surfaces, yet its biological functions remain largely unexplored. In this report, we present the first analysis of glycoRNA expression and functionality in alveolar epithelial cells. To this end, we optimized new techniques for the detection of glycoRNA on living cell surfaces and in cell membrane-associated RNA samples through in-gel imaging after labeling with fluorescent dye conjugates. Specifically, we used conjugation of Cy5-hydrazide after mild oxidation with sodium periodate for detection of total cell surface sialoglycoRNA. Conjugation of dibenzocyclooctyne-sulfo-Cy5 in cells fed with tetraacetylated N-azidoacetyl-mannosamine or 6-azido-L-fucose detected de novo-formed sialoglycoRNA or fucoglycoRNA, respectively. Finally, biotinylated lectins in combination with infrared dye-conjugated streptavidin were used to differentiate between specific glycosidic linkages. Comparisons across primary alveolar epithelial cells and different alveolar epithelial-like cell lines revealed a cell-type-specific variation in glycoRNA abundance. Treatment of primary alveolar epithelial cells with an RNase cocktail reduced epithelial surface glycoRNA and was associated with a reduction in transepithelial electrical resistance and influenza A viral particle abundance. As such, the present work identifies glycoRNA as a novel component of the alveolar epithelial glycocalyx with potential relevance in epithelial barrier regulation and viral infection.
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