Epitranscriptomic modifications play pivotal roles in regulating RNA stability, localization and function. Recently, glycosylation has also emerged as an RNA modification, though its functional implications remain unclear. Here we report that metabolic labelling with a N-azidoacetylgalactosamine-tetraacylated bioorthogonal probe in mammalian cells reveals small, non-coding, glyco-modified RNAs (glycoRNAs) that exhibit unusual stability imparted by their resistance to RNases. These glycoRNAs are primarily found within exosome vesicles as intraluminal cargo, distinct from recently reported cell surface glycoRNAs. Importantly, exosomal glycoRNAs can be transferred to naive cells, highlighting a role in intercellular RNA communication. The inhibition of exosome biogenesis leads to intracellular glycoRNA accumulation, while blocking glycan transfer to proteins reduces glycoRNA sorting into exosomes. These findings suggest a regulatory link between protein and RNA glycosylation in exosome cargo selection. Our studies support a functional role for glycosylation in targeting RNA into exosomes and uncover potential avenues for exosome-based diagnostics and RNA therapeutic applications.
