Nanopore sequencing for the 17 modification types in 36 locations inE. coliribosomal RNA enables monitoring of stress-dependent changes

Abstract

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 the signature for each modification, 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-prime-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.

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA

“…12,52 The occupancy of Ψ across the transcriptome is organism, cell-type, and stress-dependent, suggesting that this U isomer has a significant role in cellular processes. 4,12,22,23,37 Determination of these roles will require accurate and quantitative sequencing of RNA, in which nanopore direct RNA sequencing is a candidate method to achieve this goal. 37,53 Our work identified that nanopore signatures for Ψ are highly sequence context dependent and that using a consensus of nanopore current, helicase dwell time, and base miscalling is an approach to sequence this U isomer.…”

“…The E. coli rRNA strands provide a test case for this question because there are 36 well-established modification sites in the 16S and 23S rRNAs with 17 different chemical structures. 4 These RNAs are modified with methyltransferases, isomerases, an oxidase, or a reductase to generate the 17 different structures (see Figure 5a for acronyms and locations of the modifications). The rRNAs were extracted from the stationary phase grown E. coli that have the modifications written at high occupancy based on mass spectrometry analysis.…”

Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications

“…Many different epitranscriptomic modifications have been sequenced with the nanopore system. 14–17 The present discussion will focus on the uridine isomer pseudouridine (Ψ) because the prior work showcases the strengths and challenges of this approach (Fig. 1B).…”

“…27 In nanopore direct RNA sequencing for Ψ, this modification is “miscalled” as a C with the highest frequency, and miscalls to the other bases occur with lower frequencies 16,18–25 Natural U/C sequence variations will present as Ψ in base-call data analyses. These base miscall signatures have allowed sequencing for Ψ directly in rRNA, 17,18,25 mRNA, 20,23,25 tRNA, 14,15 and vRNA 19 that can have lengths >5000 nts, demonstrating the ability for long-read modification-aware sequencing with this method. A challenge to using base-called data for quantitative analysis of Ψ is that the frequency of “miscalls” is sequence-context dependent.…”

“…20,23,28 The sequencer has high overall error for RNA sequencing; 29 therefore, a well-matched control void in the target modification is needed to make comparisons. 12,17,23…”