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.

表观转录组修饰在调节 RNA 稳定性、定位和功能方面起着关键作用。最近，糖基化也作为一种 RNA 修饰出现，尽管其功能意义仍不清楚。在这里，我们报道了在哺乳动物细胞中用 N-叠氮基乙酰半乳糖胺四酰化生物正交探针进行的代谢标记揭示了小的、非编码的、糖修饰的 RNA （glycoRNA），这些 RNA 表现出由于对 RNase 的抗性而赋予的不寻常的稳定性。这些 glycoRNA 主要作为腔内货物存在于外泌体囊泡中，与最近报道的细胞表面 glycoRNA 不同。重要的是，外泌体 glycoRNA 可以转移到幼稚细胞中，突出了在细胞间 RNA 通讯中的作用。外泌体生物发生的抑制导致细胞内 glycoRNA 积累，而阻断聚糖向蛋白质的转移会减少 glycoRNA 向外泌体的分选。这些发现表明外泌体货物选择中蛋白质和 RNA 糖基化之间存在调节联系。我们的研究支持糖基化在将 RNA 靶向外泌体中的功能作用，并揭示了基于外泌体的诊断和 RNA 治疗应用的潜在途径。