Transcriptome-wide identification of glycoRNAs by Clier-seq pipeline

Area of Science:

• Biochemistry and Molecular Biology
• RNA Biology
• Glycobiology

Background:

• N-glycan modifications on RNA create cell surface glycosylated RNAs (glycoRNAs).
• Previous research focused on small glycoRNAs (<200 nt), leaving transcriptome-wide subtypes poorly understood.
• A robust validation system for accurate glycoRNA analysis is currently lacking.

Purpose of the Study:

• To comprehensively characterize transcriptome-wide glycoRNAs and identify novel glycoRNA subtypes in epithelial and B cells.
• To establish a standardized bioinformatics pipeline for glycoRNA research.

Main Methods:

• Metabolic labeling and density gradient centrifugation to identify glycoRNAs (<2,000 nt).
• Development of Clier-seq (click chemistry-based enrichment of glycoRNAs sequencing) for enhanced glycoRNA coverage (50–2,000 nt).
• HISAT-StringTie-Ballgown pipeline for novel glycoRNA subtype prediction and Clier-qPCR for validation.

Main Results:

• GlycoRNAs were predominantly identified below 2,000 nt in both cell types.
• Transfer RNAs (tRNAs), especially tRNAs (Ser), tRNAs (Thr), tRNAs (Val), and tRNAs (Lys), were primary glycosylation targets.
• Vault RNAs (vtRNAs), specifically vtRNA2-1, and novel glycosylated long noncoding RNAs (200–400 nt) were identified.

Conclusions:

• This study provides a comprehensive characterization of glycoRNAs, expanding beyond small RNAs.
• Transfer RNAs and vault RNAs are significant targets of RNA glycosylation.
• The proposed standardized bioinformatics pipeline facilitates accurate and extensive glycoRNA identification across the transcriptome.