Gracia Mazuca, L. A. | Search bioRxiv

bioRxiv2026

Direct RNA Sequencing Reveals Stress-Dependent and Pathway-Specific rRNA Modification Reprogramming During 50S Biogenesis

Ribosomal RNA (rRNA) modification and processing are essential steps in ribosome assembly. Using Oxford Nanopore direct RNA sequencing, we simultaneously detect and quantify eight classes of 23S rRNA modifications in the mature 50S large subunit (LSU) from Escherichia coli cells expressing either wild-type DbpA or the helicase-inactive R331A DbpA variant, as well as in two LSU assembly intermediates, 35S and 45S, which accumulate along distinct maturation pathways in R331A DbpA expressing cells. In addition, we analyze 3'-end processing of 23S and 5S rRNAs across these particles. Many 23S rRNA modifications are incorporated at similar levels in LSU assembly intermediates and mature 50S subunits from both wild-type and R331A DbpA expressing cells, indicating that these modifications are incorporated prior to intermediate accumulation and are not preferentially reprogrammed under R331A DbpA induced assembly stress. In contrast, a subset of three modifications exhibits altered incorporation patterns. N2-methyladenosine 2507 incorporation is reduced in the 50S LSU from R331A DbpA expressing cells compared with the cells expressing wild-type DbpA, whereas pseudouridine ({Psi}) 2508 is increased. In addition, {Psi} 2608 is reduced in the 50S subunit from R331A DbpA expressing cells compared with the 35S and 45S intermediates from the same cells and the 50S subunit from wild-type cells. Because the 35S and 45S pathways account for only [~]40% of ribosome assembly in R331A DbpA expressing cells, these findings demonstrate that {Psi}2608 incorporation is selectively reprogrammed across alternative in vivo assembly routes, revealing an additional regulatory layer in ribosome biogenesis.

biochemistry↗

bioRxiv2025

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 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.

biochemistry↗