Post-transcriptional Modifications of the Large Ribosome Subunit Assembly Intermediates in E. coli Expressing Helicase-Inactive DbpA Variant

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 absent in the field, and 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 detection, leveraging its extensive and diverse array of modifications. For modification detection, we performed reverse transcriptase reactions in the presence of Mn ² ⁺ and quantified the reverse transcriptase deletions and misincorporations at modification positions using Illumina next-generation sequencing. We simultaneously detected the following modifications in ribosomal RNA (rRNA): 1-methylguanosine (m ¹ G), 2-methylguanosine (m ² G), 3-methylpseudouridine, N ⁶ ,N ⁶ -dimethyladenosine, and 3-methyluridine, without chemical treatment. Furthermore, subjecting the rRNA samples to 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho- p -toluenesulfonate followed by alkaline conditions allowed us to simultaneously detect pseudouridine, 7-methylguanosine (m ⁷ G), 5-hydroxycytidine (OH ⁵ C), 2-methyladenosine, and dihydrouridine (D). Finally, subjecting the rRNA samples to KMnO ₄ followed by alkaline conditions allowed us to simultaneously detect m ⁷ G, OH ⁵ C, and D. Our results reveal that m ¹ G, m ² G, m ⁷ G, and D are incorporated prior to the accumulation of the 27S, 35S, and 45S large subunit intermediates in cells expressing the helicase-inactive R331A DbpA construct. These intermediates belong to three distinct stages and pathways of large subunit ribosome assembly. Therefore, our results identify the time points in three pathways at which m ¹ G, m ² G, m ⁷ G, and D are incorporated into the large ribosome subunit and provide a framework for broader studies on RNA modification dynamics.