The Modified RNA Base AcpU is an Attachment Site for N-glycans in GlycoRNA

Overview

Journal Cell

Publisher Cell Press

Specialty Cell Biology

Date 2024 Aug 22

PMID 39173631

Authors

Yixuan Xie

Peiyuan Chai

Nicholas A Till

Helena Hemberger

Charlotta G Lebedenko

Jennifer Porat

Christopher P Watkins

Reese M Caldwell

Benson M George

Jonathan Perr

Carolyn R Bertozzi

Benjamin A Garcia

Ryan A Flynn

Affiliations

Soon will be listed here.

Abstract

GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acpU) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.

Citing Articles

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References

Boccaletto P, Stefaniak F, Ray A, Cappannini A, Mukherjee S, Purta E . MODOMICS: a database of RNA modification pathways. 2021 update. Nucleic Acids Res. 2021; 50(D1):D231-D235. PMC: 8728126. DOI: 10.1093/nar/gkab1083. View

Zhang H, Li X, Martin D, Aebersold R . Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol. 2003; 21(6):660-6. DOI: 10.1038/nbt827. View

Reuter G, Schauer R, Szeiki C, Kamerling J, Vliegenthart J . A detailed study of the periodate oxidation of sialic acids in glycoproteins. Glycoconj J. 1989; 6(1):35-44. DOI: 10.1007/BF01047888. View

Chang Y, Herath J, Wang T, Chow C . Synthesis and solution conformation studies of 3-substituted uridine and pseudouridine derivatives. Bioorg Med Chem. 2007; 16(5):2676-86. PMC: 2295217. DOI: 10.1016/j.bmc.2007.11.039. View

Zhang N, Tang W, Torres L, Wang X, Ajaj Y, Zhu L . Cell surface RNAs control neutrophil recruitment. Cell. 2024; 187(4):846-860.e17. PMC: 10922858. DOI: 10.1016/j.cell.2023.12.033. View

Xie Y, de Luna Vitorino F, Chen Y, Lempiainen J, Zhao C, Steinbock R . SWAMNA: a comprehensive platform for analysis of nucleic acid modifications. Chem Commun (Camb). 2023; 59(83):12499-12502. PMC: 11006432. DOI: 10.1039/d3cc04402e. View

Gahmberg C, Andersson L . Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride. J Biol Chem. 1977; 252(16):5888-94. View

Juhling F, Morl M, Hartmann R, Sprinzl M, Stadler P, Putz J . tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic Acids Res. 2008; 37(Database issue):D159-62. PMC: 2686557. DOI: 10.1093/nar/gkn772. View

Liu T, Qian W, Gritsenko M, Camp 2nd D, Monroe M, Moore R . Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry. J Proteome Res. 2005; 4(6):2070-80. PMC: 1850943. DOI: 10.1021/pr0502065. View

10.

Lopez-Sambrooks C, Shrimal S, Khodier C, Flaherty D, Rinis N, Charest J . Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells. Nat Chem Biol. 2016; 12(12):1023-1030. PMC: 5393272. DOI: 10.1038/nchembio.2194. View

11.

Nainyte M, Amatov T, Carell T . Synthesis of an acpU phosphoramidite and incorporation of the hypermodified base into RNA. Chem Commun (Camb). 2019; 55(81):12216-12218. DOI: 10.1039/c9cc06314e. View

12.

Maley F, Trimble R, Tarentino A, PLUMMER Jr T . Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem. 1989; 180(2):195-204. DOI: 10.1016/0003-2697(89)90115-2. View

13.

Adams K, Pratt B, Bose N, Dubois L, John-Williams L, Perrott K . Skyline for Small Molecules: A Unifying Software Package for Quantitative Metabolomics. J Proteome Res. 2020; 19(4):1447-1458. PMC: 7127945. DOI: 10.1021/acs.jproteome.9b00640. View

14.

Li J, Yue S, Gao Z, Hu W, Liu Z, Xu G . Novel Approach to Enriching Glycosylated RNAs: Specific Capture of GlycoRNAs via Solid-Phase Chemistry. Anal Chem. 2023; 95(32):11969-11977. DOI: 10.1021/acs.analchem.3c01630. View

15.

Takakura M, Ishiguro K, Akichika S, Miyauchi K, Suzuki T . Biogenesis and functions of aminocarboxypropyluridine in tRNA. Nat Commun. 2019; 10(1):5542. PMC: 6895100. DOI: 10.1038/s41467-019-13525-3. View

16.

Ma Y, Guo W, Mou Q, Shao X, Lyu M, Garcia V . Spatial imaging of glycoRNA in single cells with ARPLA. Nat Biotechnol. 2023; 42(4):608-616. PMC: 10663388. DOI: 10.1038/s41587-023-01801-z. View

17.

Pedowitz N, Pratt M . Design and Synthesis of Metabolic Chemical Reporters for the Visualization and Identification of Glycoproteins. RSC Chem Biol. 2021; 2:306-321. PMC: 8323544. DOI: 10.1039/d1cb00010a. View

18.

Dong L, Shen S, Chen W, Lu H, Xu D, Jin S . Glycosyl triazoles as novel insect β-N-acetylhexosaminidase OfHex1 inhibitors: Design, synthesis, molecular docking and MD simulations. Bioorg Med Chem. 2018; 27(12):2315-2322. DOI: 10.1016/j.bmc.2018.11.032. View

19.

Liu F, Chen H, Armstrong Z, Withers S . Azido Groups Hamper Glycan Acceptance by Carbohydrate Processing Enzymes. ACS Cent Sci. 2022; 8(5):656-662. PMC: 9136970. DOI: 10.1021/acscentsci.1c01172. View

20.

Rillahan C, Antonopoulos A, Lefort C, Sonon R, Azadi P, Ley K . Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome. Nat Chem Biol. 2012; 8(7):661-8. PMC: 3427410. DOI: 10.1038/nchembio.999. View