Mapping the GlycoRNA-Protein Interactome: Insights into Glycan-Modified RNA Binding Partners and Functional Networks

Abstract Glycosylated RNAs (glycoRNAs) are a recently discovered class of biomolecules, but their functions remain poorly understood. Here, we comprehensively map the glycoRNA landscape and protein interactome in acute myeloid leukemia (AML) cells. Using bioorthogonal labeling and small RNA sequencing, we identified glycan-modified small non-coding RNAs enriched in pathways linked to glycan biogenesis, signaling, and apoptosis regulation. Two complementary labeling strategies, rPAL (targeting terminal sialic acids) and rGAL (targeting galactose/N-acetylgalactosamine), combined with protein pull-downs and mass spectrometry revealed a broad, glycan-specific set of glycoRNA-binding proteins (glyR-BPs). Functional enrichment analyses showed conserved associations across human and mouse cells, implicating glyR-BPs in protein folding, intracellular trafficking, mitochondrial regulation, and redox homeostasis. Notably, these interactions mapped largely to physiological networks rather than pathogenic drivers. Together, this work provides the first glycan-specific map of the glycoRNA–protein interactome, revealing conserved functional signatures and offering a framework for dissecting glycoRNA roles in cellular homeostasis and disease.