GlycoRNAs are glycosylated small non-coding RNAs, including small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), small nucleolar RNAs (snoRNAs), transfer RNAs (tRNAs), Y-RNAs[1], and microRNAs [2]. Glycosylated mRNAs are not known. Particularly, the N-glycans on glycoRNAs are highly sialylated and fucosylated. GlycoRNAs are displayed on the cell surface and can bind Siglec receptors important in immune response modulation (Fig.1).
While lipids and proteins are believed to be the only biopolymers modified with sugars, RNAs were not thought to be glycosylated. In 2021 in Nobel laureate Bertozzi lab, by metabolic tagging of glycan sugar precursors, mammalian small noncoding RNAs were discovered to link with sialylated glycans, challenging the old beliefs and making significant advancement in the RNA and glycobiology fields [1].
Fig.1 GlycoRNA is generated by RNA glycosylation with sialylated glycan, displayed on cell membrane, and recognized by Sialic acid-binding immunoglobulin-type lectins (Siglec) receptors [1,5].
The mechanism underlying RNA glycosylation is not yet clear. GlycoRNA biogenesis depends on existing canonical N-glycan biosynthetic machinery [1]. ldlD mutant CHO cells and CRISPR-GALE knockout K562 cells defective in the N-glycan biosynthetic pathway have stunted glycoRNA production. Introduction of external glycans can reverse this inhibition. Furthermore, inhibition of oligosaccharyltransferase (OST), an enzyme that mediates protein N-glycosylation, causes diminished glycoRNA production. For N- and O-linked glycans in glycoproteins, the sugars are appended primarily within the endoplasmic reticulum followed by additional modifications in the Golgi before final packaging into vesicles for transport or secretion. However, paradoxically, RNA is typically absent in these cellular compartments. Taken together, RNA glycosylation uses at least in part the same machinery and mechanism as protein glycosylation, while not excluding an independent pathway.
Glycosylation Linkage
The precise nature of the glycan-RNA chemical linkage is being actively investigated. Non-covalent linkage is unlikely, as glycoRNAs are robust enough to withstand stringent denaturing conditions such as organic phase separation, proteinase K treatment, silica-based RNA purification, and heating in high concentrations of formamide. GlycoRNAs are sensitive to PNGase F that cleaves the linkage between asparagine and the proximal GlcNAc of N-glycans in glycoproteins. However, native RNA nucleobases do not have such an amide bond-containing linker. Additional guanosine modification in RNA must be necessary to establish an RNA-glycan link similar to glycoproteins. Another possibility is that the RNA and preassembled N-glycan are linked together by a small, either peptidic or non-peptidic, linker. Defining the chemical and structural nature of RNA-glycan linkage will be critical in future glycoRNA studies.
By RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) has been determined as the primary site of attachment of N-glycans in glycoRNA [7].
Fig.2 Chemical linkage between N-glycan and RNA via modified RNA base acp3U [7].
GlycoRNA Functions and Diseases
GlycoRNAs are predominantly located on the cell surface as shown by cell fractionation and immunohistochemical imaging and can be removed from the cell surface by sialic acid-cleaving enzyme treatment [1]. The cell surface localization suggests their role in mediating extracellular interactions. Particularly, glycoRNAs have binding affinities with sialic acid-binding immunoglobulin-like lectin (Siglec) receptor family (Fig.1), highlighting their involvement as direct ligands in cell adhesion, cell signaling, and immune response modulation with the target cells [1]. Moreover, Siglec receptor family ligand partners are largely unknown, in part due to the past research limited to glycolipids and glycoproteins only. The new found glycoRNAs may be the long sought-after ligands for these Siglec receptors and other orphan glycan-binding receptors [5-6].
Aberrant protein and lipid glycosylation has long been established hallmarks of various human diseases. Similarly, glycoRNAs have the potential for disrupted glycan networks in disease contexts. For example, among the small noncoding glycoRNAs, Y-RNAs stand out being particularly interesting, because their binding proteins and ribonucleoproteins (RNPs) are known antigens associated with autoimmune diseases such as systemic lupus erythematosus (SLE)[1]. Other glycoRNAs have been found involved in cancer, cardiovascular, neurological, immune, and respiratory diseases.
As a new avenue for therapeutic research, modifications to glycoRNA glycans could influence responsiveness to immunotherapeutic agents. Antisense targeting the RNA sequence moieties is amenable to rational drug design and can be highly selective. Notably, large molecule treatment modalities can access extracellularly to react with the glycoRNA targets exposed on the cell surface, without the difficulties to enter the cells. For biomarker applications, the glycan and RNA moieties have the biochemical properties for both immunochemical and sequence based detection methods, allowing highly sensitive and specific diagnosis and prognosis of diseases [3-4].
GlycoRNAs as a new biomolecule class hold transformative potential in transcriptomics, epitranscriptomics, RNA biology, glycobiology, cell biology, biochemistry, signal transduction, immunology, fundamental biology, and biomedical/clinical sciences. Arraystar GlycoRNA Array can profile and identify glycoRNAs in broad scientific research areas and many clinical diseases.
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References
1. Flynn RA. et al (2021) "Small RNAs are modified with N-glycans and displayed on the surface of living cells." Cell 184(12):3109-3124.e22 [PMID:34004145]
2. Li, J. et al (2024) "O-Glycosylated RNA Identification and Site-specific Prediction by Solid-phase Chemoenzymatic TnORNA method and PONglyRNA tool" bioRxiv [doi: https://doi.org/10.1101/2024.06.18.599663]
3. Angata K. et al (2020) "Glycogene Expression Profiling of Hepatic Cells by RNA-Seq Analysis for Glyco-Biomarker Identification." Front Oncol 10:1224 [PMID:32850363]
4. Nishimura S (2011) "Toward automated glycan analysis." Adv Carbohydr Chem Biochem 65:219-71 [PMID:21763513]
5. Disney MD (2021) "A glimpse at the glycoRNA world." Cell 184(12):3080-3081 [PMID:34115968]
6. Clyde D (2021) "Sugar-coated RNAs." Nat Rev Genet 22(8):480 [PMID:34168329]
7. Xie Y. et al (2024) "The modified RNA base acp(3)U is an attachment site for N-glycans in glycoRNA." Cell 187(19):5228-5237.e12 [PMID:39173631]