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.
Keywords: NGS; RNA posttranscriptional modifications; RNA-seq; epitranscriptomics; high-throughput sequencing; mononucleotide variants; nanopore sequencing; natural RNA; reverse transcriptase; single-molecule analysis; strand cleavage; tandem MS sequencing.
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