Direct RNA Sequencing Reveals Stress-Dependent and Pathway-Specific rRNA Modification Reprogramming during 50S...

Ribosomal RNA (rRNA) modification and processing are essential steps in the ribosome assembly. Using Oxford Nanopore direct RNA sequencing, we simultaneously detect and quantify N²-methyladenosine (m²A), pseudouridine (Ψ), 2'-O-methyluridine, 1-methylguanosine, 7-methylguanosine, dihydrouridine, 3-methylpseudouridine, and 5-methyluridine modification in 23S rRNA of the mature 50S large subunit (LSU) from Escherichia coli cells expressing either wild-type DbpA or the helicase-inactive R331A DbpA variant and in two LSU assembly intermediates, 35S and 45S, which accumulate along distinct maturation pathways in R331A DbpA-expressing cells. Furthermore, we analyze the 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. A subset of three modifications exhibits altered incorporation patterns. m²A 2507 incorporation is reduced in the 50S LSU from R331A DbpA-expressing cells compared with the cells expressing wild-type DbpA, whereas 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 Ψ 2608 incorporation is selectively reprogrammed across alternative in vivo assembly routes, revealing an additional regulatory layer in ribosome biogenesis.