Sensitive and Simultaneous Determination of Uridine Thiolation and Hydroxylation Modifications in Eukaryotic RNA by Derivatization Coupled with Mass Spectrometry Analysis

The discovery of dynamic and reversible modifications in RNA expands their functional repertoires. Now, RNA modifications have been viewed as new regulators involved in a variety of biological processes. Among these modifications, thiolation is one kind of special modification in RNA. Several thiouridines have been identified to be present in RNA, and they are essential in the natural growth and metabolism of cells. However, detection of these thiouridines generally is challenging, and few studies could offer the quantitative levels of uridine modifications in RNA, which limits the in-depth elucidation of their functions. Herein, we developed a chemical derivatization in combination with mass spectrometry analysis for the sensitive and simultaneous determination of uridine thiolation and hydroxylation modifications in eukaryotic RNA. The chemical derivatization strategy enables the addition of easily ionizable groups to the uridine thiolation and hydroxylation modifications, leading up to a 339-fold increase in detection sensitivities of these modifications by mass spectrometry analysis. The limits of detection of these uridine modifications can be down to 17 amol. With the established method, we discovered and confirmed that a new modification of 5-hydroxyuridine (ho(5)U) was widely present in small RNAs of mammalian cells, expanding the diversity of RNA modifications. The developed method shows superior capability in determining low-abundance RNA modifications and may promote identifying new modifications in RNA, which should be valuable in uncovering the unknown functions of RNA modifications.

Automated summary

The discovery of dynamic and reversible modifications in RNA has expanded their functional repertoires, offering new insight into biological processes. A novel chemical derivatization strategy combined with mass spectrometry analysis has been developed to sensitively and simultaneously determine uridine thiolation and hydroxylation modifications in eukaryotic RNA, enhancing detection sensitivity by up to 339-fold. This method has been used to identify a new modification of 5-hydroxyuridine (ho(5)U) in mammalian cell small RNAs, demonstrating superior capability in detecting low-abundance RNA modifications and potentially leading to the discovery of new modifications in RNA.