RNA-associated glycoconjugates highlight potential ambiguities in glycoRNA analysis

Sungchul Kim; Zeshi Li; Yong Geun Choi; Kirsten Janssen; Jan Willem H. Langenbach; Daan J. van den Brink; Christian Büll; Bhagyashree S. Joshi; Adam Pomorski; Vered Raz; Marvin E. Tanenbaum; Pascal Miesen; Chirlmin Joo

doi:10.1038/s12276-025-01585-z

RNA-associated glycoconjugates highlight potential ambiguities in glycoRNA analysis

Sungchul Kim^*
, Zeshi Li^*
, Yong Geun Choi
, Kirsten Janssen
, Jan Willem H. Langenbach
, Daan J. van den Brink
, Christian Büll
, Bhagyashree S. Joshi
, Adam Pomorski
, Vered Raz
, Marvin E. Tanenbaum
, Pascal Miesen^*
, Chirlmin Joo^*

^*Corresponding author for this work

Chemical Biology and Drug Discovery

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

A recent ground-breaking study suggested that small RNA from mammalian cells can undergo N-glycan modifications (termed glycoRNA)¹. The discovery relied upon a metabolic glycan labeling strategy in combination with commonly used phase-separation-based RNA isolation. Following the reported procedure, here we likewise identify an N-glycosylated species in the RNA fraction. However, our results suggest that the reported RNase sensitivity of the glycosylated species depends on the specific RNA purification method. This suggests the possibility of copurifying unexpected RNase-insensitive N-glycoconjugates during glycoRNA isolation. The co-existence of two independent, yet highly similar molecular entities, complicates biochemical assays on glycoRNA and calls for more specific approaches for glycoRNA analysis. To address this, we propose a control experiment that can help distinguish genuine glycoRNA species from copurified glycoconjugates.

Original language	English
Journal	Experimental and Molecular Medicine
DOIs	https://doi.org/10.1038/s12276-025-01585-z
Publication status	E-pub ahead of print - 15 Nov 2025

Bibliographical note

Publisher Copyright:
© The Author(s) 2025.

Funding

This work was supported by Young Scientist Fellowship program of the Institute for Basic Science from the Ministry of Science and ICT of Korea (grant no. IBS-R008-D1 to S.K.); by TU Delft-Leiden Health Initiative (to V.R. and C.J.); by the Basic Research Laboratory Program (grant no. NRF-2023R1A2C2004745 to C.J.); by the European Research Council (ERC Consolidator grant no. 819299 to C.J.); by the Ministry of Science and ICT (Bio&Medical Technology Development Program of the National Research Foundation, RS-2025-02217909 to C.J.); by a Dutch Research council (NWO) ENW-XS grant (grant no. OCENW.XS21.4.046 to P.M.); and by NWO VENI talent program (grant no. VI.Veni.222.272 to Z.L.). We thank G.-J. Boons (Utrecht University, the Netherlands) for critical discussions and providing important resources.

Funders	Funder number
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research)	OCENW.XS21.4.046, VI.Veni.222.272
TU Delft-Leiden Health Initiative
EC \| EC Seventh Framework Programm \| FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activiti	819299
Young Scientist Fellowship program of the Institute for Basic Science from the Ministry of Science and ICT of Korea (IBS-R008-D1)

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Kim, S., Li, Z., Choi, Y. G., Janssen, K., Langenbach, J. W. H., van den Brink, D. J., Büll, C., Joshi, B. S., Pomorski, A., Raz, V., Tanenbaum, M. E., Miesen, P., & Joo, C. (2025). RNA-associated glycoconjugates highlight potential ambiguities in glycoRNA analysis. Experimental and Molecular Medicine. Advance online publication. https://doi.org/10.1038/s12276-025-01585-z

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abstract = "A recent ground-breaking study suggested that small RNA from mammalian cells can undergo N-glycan modifications (termed glycoRNA)1. The discovery relied upon a metabolic glycan labeling strategy in combination with commonly used phase-separation-based RNA isolation. Following the reported procedure, here we likewise identify an N-glycosylated species in the RNA fraction. However, our results suggest that the reported RNase sensitivity of the glycosylated species depends on the specific RNA purification method. This suggests the possibility of copurifying unexpected RNase-insensitive N-glycoconjugates during glycoRNA isolation. The co-existence of two independent, yet highly similar molecular entities, complicates biochemical assays on glycoRNA and calls for more specific approaches for glycoRNA analysis. To address this, we propose a control experiment that can help distinguish genuine glycoRNA species from copurified glycoconjugates.",

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doi = "10.1038/s12276-025-01585-z",

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T1 - RNA-associated glycoconjugates highlight potential ambiguities in glycoRNA analysis

AU - Kim, Sungchul

AU - Li, Zeshi

AU - Choi, Yong Geun

AU - Janssen, Kirsten

AU - Langenbach, Jan Willem H.

AU - van den Brink, Daan J.

AU - Büll, Christian

AU - Joshi, Bhagyashree S.

AU - Pomorski, Adam

AU - Raz, Vered

AU - Tanenbaum, Marvin E.

AU - Miesen, Pascal

AU - Joo, Chirlmin

PY - 2025/11/15

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N2 - A recent ground-breaking study suggested that small RNA from mammalian cells can undergo N-glycan modifications (termed glycoRNA)1. The discovery relied upon a metabolic glycan labeling strategy in combination with commonly used phase-separation-based RNA isolation. Following the reported procedure, here we likewise identify an N-glycosylated species in the RNA fraction. However, our results suggest that the reported RNase sensitivity of the glycosylated species depends on the specific RNA purification method. This suggests the possibility of copurifying unexpected RNase-insensitive N-glycoconjugates during glycoRNA isolation. The co-existence of two independent, yet highly similar molecular entities, complicates biochemical assays on glycoRNA and calls for more specific approaches for glycoRNA analysis. To address this, we propose a control experiment that can help distinguish genuine glycoRNA species from copurified glycoconjugates.

AB - A recent ground-breaking study suggested that small RNA from mammalian cells can undergo N-glycan modifications (termed glycoRNA)1. The discovery relied upon a metabolic glycan labeling strategy in combination with commonly used phase-separation-based RNA isolation. Following the reported procedure, here we likewise identify an N-glycosylated species in the RNA fraction. However, our results suggest that the reported RNase sensitivity of the glycosylated species depends on the specific RNA purification method. This suggests the possibility of copurifying unexpected RNase-insensitive N-glycoconjugates during glycoRNA isolation. The co-existence of two independent, yet highly similar molecular entities, complicates biochemical assays on glycoRNA and calls for more specific approaches for glycoRNA analysis. To address this, we propose a control experiment that can help distinguish genuine glycoRNA species from copurified glycoconjugates.

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SN - 1226-3613

JO - Experimental and Molecular Medicine

JF - Experimental and Molecular Medicine

ER -

RNA-associated glycoconjugates highlight potential ambiguities in glycoRNA analysis

Abstract

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