Quantitative Analysis of RNA Modifications and Processing Dynamics in the Course of Large Ribosomal Subunit Assembly

Most 23S rRNA modifications are incorporated into the 35S and 45S intermediates at levels comparable to those in the 50S LSU from both wild-type and R331A DbpA-expressing cells, indicating that modification enzymes complete their functions before 35S and 45S accumulation. However, three enzymes exhibit differential modification patterns. RlmN incorporates N²-methyladenosine (m²A) at position 2507 more extensively in the 50S LSU from wild-type DbpA-expressing cells than in the 35S, 45S, and 50S LSU from R331A DbpA-expressing cells.

Conversely, RluC incorporates pseudouridine (Ψ) at position 2508 at lower levels in the 50S LSU from wild-type DbpA-expressing cells than in the 35S, 45S, and 50S LSU from R331A DbpA-expressing cells. The RluF enzyme incorporates Ψ at position 2508 at a lower level in the 50S LSU from R331A DbpA-expressing cells than in the 35S and 45S intermediates and the 50S LSU from wild-type DbpA-expressing cells. Since the pathways leading to 35S and 45S account for only ~50% of total ribosome assembly in R331A DbpA-expressing cells, these findings reveal that RluF activity is reduced in a subset of maturation pathways.

The 3′ end of 23S rRNA is less mature in the early-stage LSU intermediate, 35S, and more mature in the late-stage LSU intermediate, 45S. Interestingly, the 3′ end of 5S rRNA is less processed in the 45S than in the 35S, suggesting that 5S rRNA in the 45S intermediate matures during the final steps of large subunit ribosome assembly. A complete understanding of ribosome assembly demands a precise characterization of ribosome assembly pathways and processes across diverse cellular conditions.

This study reveals critical insights into rRNA modifications and processing dynamics in bacterial ribosomes under stress.