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.

糖基化RNA（glycosylated RNA, glycoRNA）的发现，揭示了聚糖（glycans）与RNA之间此前未被认知的关联。该发现借助代谢糖类探针全-O-乙酰化N-叠氮乙酰甘露糖胺（per-O-acetylated N-azidoacetyl-mannosamine）得以实现，凸显了此前未知的RNA修饰可能具备的生物学功能。本研究基于此前利用其他代谢探针检测糖基化RNA的工作展开，所使用的探针包括全-O-乙酰化N-叠氮乙酰葡萄糖胺（per-O-acetylated N-azidoacetyl-glucosamine）、N-叠氮乙酰半乳糖胺（N-azidoacetyl-galactosamine）以及6-叠氮岩藻糖（6-azidofucose）。此次更全面的分析发现了潜在的新型糖基化转录本，有待进一步研究。但本研究结果同时显示，尚未被鉴定的糖类-RNA连接键对糖苷酶（glycosidases）的酶切具有意外的耐受性。为此，我们利用全乙酰化探针在体外开展了糖类-RNA连接键的非酶促形成实验。基于上述研究，我们得出两项核心结论：其一，经乙酰化糖类探针代谢掺入所产生的信号，是可检测到的糖基化RNA物种的主要来源；其二，测序结果显示，糖类修饰RNA的最具潜力候选者为转运RNA（transfer RNA, tRNA）。我们证实，可将更多种类的代谢示踪剂掺入糖基化RNA中，这为转录后修饰RNA的本质研究提供了全新视角。