Direct nanopore sequencing for the 17 RNA modification types in 36 locations in the E. coli ribosome 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 modification’s 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, resides m⁴C_m1402 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 the 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.