Abstract 2123 Systematic characterizations of modifications and RNA processing in ribosome assembly via direct RNA sequencing

The ribosome consists of the small and large subunits. Rapid, efficient, and accurate ribosome assembly is essential for cell growth and survival. Ribosomal RNA (rRNA) provides the platform for ribosome assembly. The rRNA molecules are synthesized as one long transcript and later processed. In addition, rRNA molecules are post-transcriptionally modified. Both rRNA processing and the incorporation of RNA modifications modulate translation and ribosome assembly. A complete understanding of ribosome maturation demands the determination of the rRNA processing steps and modification events in all the intermediates and each pathway of ribosome assembly. The large subunit of the bacterial ribosome, which migrates in a sucrose gradient as a particle with a sedimentation coefficient of 50S, consists of two RNA molecules, 5S, which is 120 nucleotide long, and 23S, which is 2907 nucleotide long. Here we investigate the 23S rRNA modifications and 3' end processing of two large subunit intermediates with sedimentation coefficients of 35S and 45S. These intermediates accumulate in cells expressing the helicase inactive R331A DbpA construct and rearrange to form 50S large subunit via independent pathways. We employed Oxford Nanopore direct RNA sequencing, combined with EpiNano to determine the RNA modifications, and Minimap2 alignment to determine the 3' end processing of 23S rRNA. The 35S intermediate is significantly less processed than both the 45S intermediate and 50S large subunit, while the 45S intermediate is less processed than the 50S large subunit. We can detect 16 out of 25 RNA modifications in the 23S rRNA. Our group's previously published Illuimina next generation sequencing, combined with chemical modifications, detected 11 out of 25 23S rRNA modifications in the 35S and 45S intermediates. Hence, this study for the first time describes the extent of five new modifications in 35S and 45S intermediates. The majority of the modifications are incorporated similarly to the 50S wild type in the 35S and 45S. Hence, these modifications are inserted in the cells before the 35S and 45S are accumulated. The adaptation of techniques we have employed here could be used to interrogate RNA modifications and processing steps in other systems and RNA molecules. This work was supported in part by the National Institute of General Medical Sciences grant R01-GM131062, the University of Texas System Rising STARs Program, and the start-up from the Chemistry and Biochemistry Department at the University of Texas at El Paso (to E.K.).