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

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 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 Mn2 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 (m1G), 2-methylguanosine (m2G), 3-methylpseudouridine, N6,N6-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 (m7G), 5-hydroxycytidine (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 results reveal that m1G, m2G, m7G, 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 m1G, m2G, m7G, and D are incorporated into the large ribosome subunit and provide a framework for broader studies on RNA modification dynamics.