Transcriptome-wide Identification of GlycoRNAs by Clier-seq Pipeline

RNA molecules can undergo modification by N–glycans and be displayed on the cell surface. However, recent studies have focused primarily on N–glycan modifications on small RNAs less than 200 nt in length; the transcriptome–wide subtypes of glycosylated RNAs (glycoRNAs) remain poorly characterized. A validation system, which represents only a fraction of the total transcripts, has yet to be established to analyze glycoRNAs accurately. In this study, we aimed to comprehensively characterize transcriptome–wide global glycoRNAs and novel glycoRNA subtypes in both epithelial cells and B cells. Using metabolic labeling and density gradient centrifugation methods, we identified glycoRNAs predominantly below 2000 nt in both epithelial cells and B cells. We then developed the Clier–seq (Click chemistry–based enrichment of glycoRNAs sequencing) method to maximize the coverage of glycoRNAs (ranging from 50 to 2000 nt) and utilized the HISAT–StringTie–Ballgown pipeline to predict novel glycoRNA subtypes. We also established Clier–qPCR assays (click chemistry–based enrichment of glycoRNAs RT–qPCR) to validate the specificity of the candidate glycoRNAs. We demonstrated that transfer RNAs (tRNAs), particularly tRNAs (Ser), tRNAs (Thr), tRNAs (Val), and tRNAs (lys), are the primary targets of glycosylation. Additionally, we found that vault RNAs (vtRNAs), specifically vtRNA2–1, are glycosylated. Furthermore, we discovered several novel glycosylated long noncoding RNAs ranging from 200–400 nt in length. Herein, we propose a standardized bioinformatics pipeline for glycoRNA research, enabling accurate and comprehensive identification of glycoRNAs throughout the transcriptome.