GalNAc-modified Exosomal RNA Drives Cellular Interactions
RNA modification, especially base modification, plays a key role in regulating RNA function. In recent years, in addition to classic base modifications, glyco-modified RNAs (glycoRNAs) found on the cell surface has gradually attracted the attention of the scientific research community. However, the biological functions and regulatory mechanisms of glycoRNA are still poorly understood, especially glycoRNA in extracellular vesicles. This study aims to explore whether glycoRNA exists in extracellular vesicles and its possible biological significance.
On June 4, 2025, Nature Cell Biology published an article entitled "Extracellular exosomal RNAs are glyco-modified", published by Megerditch Kiledjian's team at Rutgers University.
Different from the N-Azidoacetylmannosamine-tetraacylated (Ac4ManNAz) used by the glycoRNA discovery team, the authors used a similar N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz) metabolic labeling method to discover new N-Acetylgalactosamine (GalNAc)-modified glycoRNA in Exosomes. The study found that these exosomes containing glycoRNA are resistant to RNase. The study further confirmed that glycoRNA can be delivered to recipient cells through exosome-mediated intercellular communication. In addition, inhibiting protein glycosylation pathways affects RNA glycosylation levels, suggesting a potential regulatory link between protein and RNA glycosylation. Both ESCRT-dependent and -independent pathways are involved in regulating the release of exosomal glycoRNA.
In order to explore whether there is UDP-GlcNAc cap-modified RNA in cells and identify the glycosylation modification of small RNAs, the authors first confirmed that DXO family proteins can hydrolyze the GlcNAc cap. Subsequently, using Ac4GalNAz metabolic labeling combined with click chemistry technology, the authors discovered a new type of GalNAc Glycosylation Modification that mainly exists on small RNAs. Compared with the reported Ac4ManNAz-labeled glycoRNA, Ac4GalNAz-labeled glycoRNA migrates faster, suggesting the existence of different types of glycoRNAs. These glycoRNAs do not contain polyA tails and are insensitive to RNase A, but are degraded by RNase I and micrococcal nuclease (MNase), indicating that they are located inside the cell and may have specific secondary structures.
SpRai1 treatment experiments further confirmed that glycosylation modification occurred inside the RNA rather than at the 5' end. Therefore, this study identified a new GalNAc glycosylation modification of intracellular small RNAs, which is different from the 5'-end UDP-GlcNAc cap and reported Cell Surface GlycoRNAs, expanding the diversity of glycoRNAs.
In order to identify the types of RNA modified by GalNAc glycosylation, the authors performed RNA sequencing and analysis (nanopore-based) of small RNAs in HeLa and HEK293T cells labeled with Ac4GalNAz. Considering that Oxford Nanopore sequencing mainly targets poly(A) RNA, and RNA labeled with Ac4GalNAz does not contain a poly(A) tail, the RNA was poly(A) tailed before sequencing.
Fig. 1 Affinity purification of glycoRNA and Oxford Nanopore sequencing. (Sharma, et al., 2025)
The sequencing results showed that a variety of non-coding small RNAs (including snoRNA, snRNA, vault RNA, and Y RNA) were GalNAc-glycosylated. Northern blot analysis further verified the glycosylation of RNY3 and 5S rRNA. In addition, these glycoRNAs highly overlap with previously reported exosomal RNAs, suggesting that glycoRNAs may be closely related to membrane organelles such as exosomes. This study revealed the GalNAc glycosylation modification spectrum of small RNA at the transcriptome level for the first time.
The authors used two biochemical methods. First, by separating the nucleus and cytoplasm and combining IR-800 signal detection, it was found that Ac4GalNAz-labeled glycoRNA was mainly present in the cytoplasm, not the nucleus. Further separation of membrane components and soluble components in the cytoplasm revealed that glycoRNA was mainly enriched in the membrane component, suggesting that it may be associated with the cell membrane or organelle membrane. Unlike the previously reported Ac4ManNAz-labeled glycoRNA located on the cell surface, the glycoRNA discovered in this study was not located on the cell surface, because MNase treatment of intact cells did not significantly reduce the detection of glycoRNA. In addition, given the presence of small RNAs in the endoplasmic reticulum and the high overlap between the identified glycoRNAs and exosome RNAs, it is speculated that these glycoRNAs may be associated with exosomes. Therefore, glycoRNAs may participate in intercellular communication through exosomes, which can be considered as biomarkers for disease diagnosis.
To verify whether glycoRNA is localized in exosomes, the authors isolated exosomes from HeLa cells and verified the presence of exosome marker CD9 by western blot. After SPAAC analysis of the extracted exosomal RNA, it was found that Ac4GalNAz-labeled glycoRNA was almost entirely present inside the exosomes, rather than on the surface of the exosomes. MNase treatment of exosomes also did not affect the glycoRNA content, further confirming this.
Sequencing Analysis of purified exosomal glycoRNA showed that the types of exosomal glycoRNA and intracellular glycoRNA were highly similar. In addition, the study also found that the proportion of glycoRNA in exosomes was higher than that in intracellular small RNA, suggesting that glycosylation modification may play an important role in exosomal RNA sorting.
To explore the effect of the ESCRT pathway on the release of exosomal glycoRNA, the authors treated cells with siRNA targeting HGS, ESCRT-dependent pathway inhibitor Manumycin-A (MA), and ESCRT-independent pathway inhibitor GW4869, respectively. The results showed that knocking down HGS or using MA and GW4869 significantly reduced the level of glycoRNA in exosomes and led to the accumulation of intracellular glycoRNA levels. This indicates that both ESCRT-dependent and -independent pathways are involved in regulating the release of glycoRNA-rich exosomes.
Fig. 2 Schematic diagram of the ESCRT pathway. (Sharma, et al., 2025)
The authors compared the expression profiles of exosomal glycoRNAs in human induced pluripotent stem cells (iPSCs) and theirinduced neuron cells (iNs). After Ac4GalNAz labeling, the size distributions of exosomal glycoRNAs from iPSCs and iNs were significantly different, and similar differences were observed for intracellular RNA. Exosomal glycoRNAs from iPSCs migrated slower, while those from iNs migrated faster, similar to the expression profiles of more differentiated HeLa and HEK293T cells. This suggests that the expression pattern of exosomal glycoRNAs may be associated with the differentiation state of cells.
The authors co-incubated unlabeled HeLa cells with Ac4GalNAz-labeled exosomes from HeLa cells for 10 and 20 hours. After sufficient washing to remove residual exosomes, RNA of recipient cells was extracted and subjected to SPAAC-mediated biotin labeling. The results showed that labeled glycoRNA was detected in recipient cells, proving that glycoRNA can be transferred between cells through exosomes.
The authors also investigated whether acp3U modification is involved in GalNAc glycosylation. After Knocking Down acp3U-related enzymes DTWD2 and TSR3, the level of Ac4GalNAz-labeled glycoRNA did not change significantly, and the article showed that the modification may not be mediated by acp3U.
The authors turned their attention to GALE and GNE, the key enzymes in the GalNAc metabolic pathway. After knocking down GALE and GNE, the level of Ac4GalNAz-labeled glycoRNA in cells increased significantly, indicating that Ac4GalNAz is a sugar donor for glycoRNA modification, and GALE and GNE may affect the abundance of glycoRNA by regulating the steady-state level of Ac4GalNAz.
In addition, after treating cells with the oligosaccharyltransferase (OST) inhibitor NGI-1, the levels of glycoRNA in cells and exosomes decreased in a dose-dependent manner. This indicates that blocking the protein glycosylation pathway affects RNA glycosylation, suggesting that there may be a common regulatory mechanism between protein and RNA glycosylation.
In summary, this study revealed a new form of glycosylation modification of exosomal RNA - GalNAc modification, which is enriched in the exosome cavity, which is different from the previously reported cell surface glycoRNA and RNA cap structure in the form of UDP-GlcNAc. At the same time, multiple types of GalNAc-modified non-coding small RNAs (snoRNA, snRNA, vault RNA, and Y RNA, etc.) were identified at the transcriptome level, and it was confirmed that these glycoRNAs can be transferred between cells through exosomes and participate in intercellular communication. In addition, the study also found that there is a correlation between the protein glycosylation pathway and RNA glycosylation, and that both ESCRT-dependent and -independent pathways are involved in the release of glycoRNA exosomes. The expression profiles of exosomal glycoRNAs also differ under different cell differentiation states. These findings reveal important characteristics of glycoRNA, such as its enrichment in exosomes, intercellular transmission, and association with protein glycosylation pathways.
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