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 <i>Escherichia coli</i> 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 (Ψ) 2508 is increased. In addition, Ψ 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 Ψ2608 incorporation is selectively reprogrammed across alternative <i>in vivo</i> assembly routes, revealing an additional regulatory layer in ribosome biogenesis.