ABSTRACT: RNA post-transcriptional modifications are ubiquitous across all organisms and serve as fundamental regulators of cellular homeostasis, growth, and stress adaptation. Techniques for the simultaneous detection of multiple RNA modifications in a high-throughput, single-nucleotide resolution manner are largely lacking in the field. Given the crucial role of RNA modifications in cellular metabolism, developing such techniques is of paramount importance. We used the Escherichia coli ribosome as a model system to develop novel techniques for RNA post-transcriptional modification, leveraging its extensive and diverse array of modifications to enable the simultaneous interrogation of multiple modification types. For modification detection, we performed reverse transcriptase reactions in the presence of Mn²⁺ and quantified the reverse transcriptase deletions and misincorporations at modification positions using Illumina next-generation sequencing. In the absence of chemical treatment, we simultaneously detected the following modifications in ribosomal RNA: 1-methylguanosine (m¹G), 2-methylguanosine (m²G), 3-methylpseudouridine, N⁶,N⁶-dimethyladenosine, and 3-methyluridine. Furthermore, subjecting the RNA samples to 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate followed by alkaline conditions allowed us to simultaneously detect pseudouridine, 7-methylguanosine (m⁷G), 5-hydroxycytosine (OH⁵C), 2-methyladenosine, and dihydrouridine (D). Finally, subjecting the rRNA samples to KMnO₄ followed by alkaline conditions allowed us to simultaneously detect m⁷G, OH⁵C, and D. Our findings reveal that m¹G, m²G, m⁷G, and D are incorporated before the accumulation of the 27S, 35S, and 45S in cells, suggesting that the enzymes responsible for these modifications complete their functions before these three intermediates are populated.
