An Expanded View of RNA Modification with Carbohydrate-Based Metabolic Probes.

The discovery of glycosylated RNA (glycoRNA) revealed an unexpected association between glycans and RNA. This discovery, facilitated by the metabolic carbohydrate reporter per-O-acetylated N-azidoacetyl-mannosamine, underscores the potential for biological roles of previously unknown RNA modifications. Our study builds upon the detection of glycoRNA with other metabolic probes such as per-O-acetylated N-azidoacetyl-glucosamine, N-azidoacetyl-galactosamine, and 6-azidofucose. This broader analysis uncovered potential new glycosylated transcripts that warrant further study. However, our results also revealed unexpected resilience of the unidentified carbohydrate-RNA linkage to enzymatic cleavages by glycosidases. Therefore, we investigated nonenzymatic formation of carbohydrate-RNA linkages in vitro using per-acetylated probes. From these studies, we draw two main conclusions. First, signals arising from metabolic incorporation using acetylated carbohydrate probes represent the main source of detectable glycoRNA species. Second, sequencing reveals that the most likely candidates for carbohydrate-modified RNAs are likely tRNAs. We demonstrated that an expanded repertoire of metabolic reporters can be incorporated into glycoRNA, opening new perspectives on the nature of post-transcriptionally modified RNA.