Proteins are a source of glycans found in preparations of glycoRNA

Recent discoveries suggesting that RNA can be modified with sialylated glycans (termed glycoRNA) could broaden our understanding of cellular glycosylation beyond traditional proteins and lipids. However, the pathway of RNA-glycosylation and its biological function remain elusive. Following the original glycoRNA isolation protocol, we also detect labeled glycans in small RNA preparations. However, glycosylated molecules showed resistance to treatment with RNase A/T1 but were sensitive to proteinase K digestion under denaturing conditions. Using liquid chromatography-mass spectrometry-based proteomics we here detect various proteins that copurify with small but not large RNA preparations isolated from human or murine cells, including the glycosylated membrane protein LAMP1. Importantly, we further demonstrate that recombinant soluble LAMP1 can be purified following the glycoRNA isolation method. These findings suggest that glycoproteins copurify with RNA using current glycoRNA purification protocols, thus representing a considerable source of glycans in samples of glycoRNA.