Nanopore sequencing for the 17 modification types in 36 locations in E. coli ribosomal RNA enables monitoring of stress-dependent changes

Escherichia coli possess the 16S and 23S rRNA strands that have 36 chemical modification sites with 17 different structures. Direct RNA nanopore sequencing using a protein nanopore sensor and helicase brake, which is also a sensor, was applied to the rRNAs. Nanopore current levels, base calling profile, and helicase dwell times for the modifications relative to non-modified synthetic rRNA controls found signatures for nearly all modifications. Signatures for clustered modifications were determined by selective sequencing of writer knock-out E. coli and sequencing of synthetic RNAs utilizing some custom-synthesized nucleotide triphosphates for their preparation. The knowledge of each modifications signature, apart from 5-methylcytidine, was used to determine how metabolic and cold-shock stress impact rRNA modifications. Metabolic stress resulted in either no change or a decrease, and one site increased in modification occupancy, while cold-shock stress led to either no change or a decrease. In the 16S rRNA, there resides an m4Cm modification at site 1402 that decreased with both stressors. Using helicase dwell time, it was determined that the N4 methyl group is lost during both stressors, and the 2-OMe group remained. In the ribosome, this modification stabilizes binding to the mRNA codon at the P-site resulting in increased translational fidelity that is lost during stress. The E. coli genome has seven rRNA operons (rrn), and earlier studies aligned the nanopore reads to a single operon (rrnA). Here, the reads were aligned to the seven operons to identify operon-specific changes in the 11 pseudouridines. This study demonstrates that direct sequencing for >16 different RNA modifications in a strand is achievable.