Extracellular Exosomal RNAs are Glyco-Modified

Epitranscriptomic modifications play pivotal roles in regulating RNA function, encompassing base alterations and the addition of both canonical m 7 G and noncanonical nucleotide metabolite caps. Recently, the spectrum of modifications has extended to include glyco modification at the 5’ cap or within the RNA. Despite this expansion, the functional implications of glyco modification on RNA remain elusive. Our study reveals that mammalian cells labeled with N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz) produce small noncoding and nonpolyadenylated glyco-modified RNA (glycoRNA), primarily localized within exosome vesicles. The resistance of glycoRNA-containing exosomes to RNase treatment suggests that Ac4GalNAz-derived glycoRNA constitutes intraluminal cargo, distinct from recently reported cell surface glycoRNAs. Furthermore, we demonstrate that exosome cargo can be transferred to naïve cells, underscoring exosome-mediated intercellular communication of glycoRNAs. Inhibition of exosome biogenesis leads to the accumulation of intracellular glycoRNA while blocking glycan transfer to proteins concomitantly reduced the targeting of glycoRNA within exosomes. These findings highlight a correlation between protein and RNA glycosylation, suggesting that the accumulation of glycoRNAs within exosomes is a regulated process. Our results support a functional role for glyco-modification in mediating RNA targeting into exosomes, offering new insights for enhancing recent advances in exosome-directed diagnostics and therapeutic applications.