Direct Nanopore Sequencing for the 17 RNA Modification Types in 36 Locations in the E. coli Ribosome Enables Monitoring of Stress-Dependent Changes

The bacterium Escherichia coli possesses16S and 23S rRNA strands that have 36 chemical modification siteswith 17 different structures. Nanopore direct RNA sequencing usinga 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 unmodifiedsynthetic rRNA controls found signatures for nearly all modifications.Signatures for clustered modifications were determined by selectivesequencing of writer knockout E. coli and sequencing of synthetic RNAs utilizing some custom-synthesizednucleotide triphosphates for their preparation. The knowledge of eachmodification's signature, apart from 5-methylcytidine, wasused to determine how metabolic and cold-shock stress impact rRNAmodifications. Metabolic stress resulted in either no change or adecrease, and one site increased in modification occupancy, whilecold-shock stress led to either no change or a decrease. The doublemodification m(4)C(m)1402 resides in 16S rRNA, andit decreased with both stressors. Using the helicase dwell time, itwas determined that the N (4) methyl groupis lost during both stressors, and the 2 & PRIME;-OMe group remained.In the ribosome, this modification stabilizes binding to the mRNAcodon at the P-site resulting in increased translational fidelitythat is lost during stress. The E. coli genome has seven rRNA operons (rrn), and the earlierstudies aligned the nanopore reads to a single operon (rrnA). Here, the reads were aligned to all seven operons to identifyoperon-specific changes in the 11 pseudouridines. This study demonstratesthat direct sequencing for >16 different RNA modifications in astrandis achievable.

Automated summary

This study successfully identified the signatures of almost all modifications in the 16S and 23S rRNA strands of Escherichia coli using nanopore direct RNA sequencing. Furthermore, the study also revealed the impact of metabolic and cold-shock stress on rRNA modifications.