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

In the absence of chemical treatment, we simultaneously detected the following modifications in ribosomal RNA: 1-methylguanosine (m¹G), 2-methylguanosine (m²G), 3-methylpseudouridine, N⁶,N⁶-dimethyladenosine, and 3-methyluridine. Furthermore, subjecting the RNA 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-hydroxycytosine (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 findings reveal that m¹G, m²G, m⁷G, and D are incorporated before the accumulation of the 27S, 35S, and 45S in cells, suggesting that the enzymes responsible for these modifications complete their functions before these three intermediates are populated.