Analyzing RNA posttranscriptional modifications to decipher the epitranscriptomic code

Abstract

The discovery of RNA silencing has revealed that non‐protein‐coding sequences (ncRNAs) can cover essential roles in regulatory networks and their malfunction may result in severe consequences on human health. These findings have prompted a general reassessment of the significance of RNA as a key player in cellular processes. This reassessment, however, will not be complete without a greater understanding of the distribution and function of the over 170 variants of the canonical ribonucleotides, which contribute to the breathtaking structural diversity of natural RNA. This review surveys the analytical approaches employed for the identification, characterization, and detection of RNA posttranscriptional modifications (rPTMs). The merits of analyzing individual units after exhaustive hydrolysis of the initial biopolymer are outlined together with those of identifying their position in the sequence of parent strands. Approaches based on next generation sequencing and mass spectrometry technologies are covered in depth to provide a comprehensive view of their respective merits. Deciphering the epitranscriptomic code will require not only mapping the location of rPTMs in the various classes of RNAs, but also assessing the variations of expression levels under different experimental conditions. The fact that no individual platform is currently capable of meeting all such demands implies that it will be essential to capitalize on complementary approaches to obtain the desired information. For this reason, the review strived to cover the broadest possible range of techniques to provide readers with the fundamental elements necessary to make informed choices and design the most effective possible strategy to accomplish the task at hand.

SWAMNA: a comprehensive platform for analysis of nucleic acid modifications

“…Liquid chromatography with tandem mass spectrometry (LC-MS/MS) enables simultaneous characterization and quantification of different modifications. 7 The ability to identify modifications directly based on their distinct mass and fragmentation patterns distinguishes MS-based techniques from other analytical approaches. LC-MS/MS analysis of nucleic acid modifications has often been performed at the nucleoside level.…”

Characterization of mRNA therapeutics

“…IMS‐MS has been successfully deployed in the analysis heavily modified RNA, such as tRNA (Lauman et al, 2023; Rose et al, 2015), and allowed for the development of analytical platforms that can explore the relationship between RNA modifications and biological significance (Rose et al, 2015). Further applications of IMS‐MS in the analysis of ribonucleotide components has been recently reviewed in detail (Deng et al, 2022). Overall, the ability to identify complex mononucleotide mixtures may be a valuable tool in the analysis of digested therapeutic RNAs.…”

Identification of glycosylated nucleosides in small synthetic glyco‐RNAs

“…These functions can be affected by posttranscriptional modifications of the RNAs including methylation, hydroxylation and acetylation. [1] Post-transcriptional modifications can occur at various locations on a nucleotide. For example, methylation can occur at 2, 7 and 2'-O for guanosine.…”

“…RNAs can have various functions such messenger RNA (mRNA) that forms the coding template for protein synthesis, transfer RNA (tRNA) involved in translating messenger RNA into protein, and other non‐coding RNAs that regulate gene expression among other roles. These functions can be affected by post‐transcriptional modifications of the RNAs including methylation, hydroxylation and acetylation [1] . Post‐transcriptional modifications can occur at various locations on a nucleotide.…”