Decoding the Escherichia coli epitranscriptome

Modifications of RNA, known as the epitranscriptome, affect mRNA stability, translation, and splicing in eukaryotes and have implications for developmental processes, cancer, and viral infections. In prokaryotes, however, the landscape of the epitranscriptome is still poorly understood. To address this knowledge gap, we used direct RNA sequencing with Nanopore technology to study RNA modifications in the model bacterium Escherichia coli. With a single sequencing reaction, we were able to simultaneously identify and map most of the known modification types in rRNA, tRNA, and mRNA. Subsequently, a multifaceted approach integrating different algorithms for data analysis, deletion mutants, mass spectrometry, qPCR, and in vitro methylation was implemented to evaluate the presence of m5C and m6A in E. coli. Known m5C and m6A sites in rRNA were confirmed, but these modifications could not be localized in the mRNA. Nevertheless, based on the sequencing data, modifications were found to be enriched in the coding regions of genes associated with general metabolism and RNA processing. This study provides a useful resource for experimental and bioinformatic approaches to gain new insights into post-transcriptional regulation in a prokaryotic model. Overall design: Total RNA was extracted from two biological replicates each of wild-type (WT) and ?rlmF?rlmJ mutant E. coli cultured under standard growth conditions and from two biological replicates of WT heat-shocked E. coli to measure RNA modifications throughout the transcriptome. Two replicates of in vitro transcribed RNA were synthesized from WT E. coli DNA template as unmodified controls. Putative modifications were detected after samples were processed with direct RNA sequencing via Nanopore.