Direct RNA sequencing of the Escherichia coli epitranscriptome uncovers alterations under heat stress

Modifications of RNA, known as the epitranscriptome, affect gene expression, translation, and splicing in eukaryotes, with implications for developmental processes, cancer, and viral infections. In prokaryotes, regulation at the level of the epitranscriptome is still poorly understood. Here, we used nanopore direct RNA sequencing of Escherichia coli to study RNA modifications and their changes under heat stress. With a single sequencing reaction, we detected most known modification types in ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). RNA sequencing was complemented by a multifaceted approach that included mass spectrometry, deletion mutants, single-nucleotide polymerase chain reaction, and in vitro methylation. Known 5-methylcytidine (m5C) and N6-methyladenosine (m6A) sites in the rRNA were confirmed, but these types of modifications could not be localized in the mRNA. In response to heat stress, levels of m5C, m6A, and N6,N6-dimethyladenosine increased in the 16S rRNA. Sequencing and mass spectrometry data demonstrated a decrease in tRNA modification abundance in the anticodon loop at 45°C. In general, mRNA modifications at 37°C were enriched in the coding regions of genes associated with general metabolism and RNA processing, which shifted to genes involved in cell wall synthesis and membrane transport under heat stress. This study provides new insights into the complexity of post-transcriptional regulation in bacteria.