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.