Quantitative mapping of pseudouridines in bacterial RNA

RNA pseudouridylation is one of the most prevalent post-transcriptional modifications, occurring universally across all organisms. Although pseudouridines have been extensively studied in bacterial tRNAs and rRNAs, their presence and role in bacterial mRNA remain poorly characterized. Here, we used a bisulfite-based deep sequencing approach to provide a comprehensive and quantitative measurement of bacterial pseudouridines using E. coli, to provide proof of concept. We identified 1,954 high-confidence sites in 1,331 transcripts, which is 29 times above previous estimates and representing almost 30% of the transcriptome. Furthermore, pseudouridines were significantly associated with mRNA stability and enriched in transcripts associated with secondary metabolite production and adaptation to diverse environments. Finally, we mapped pseudouridines in oral microbiome samples of human subjects, demonstrating the broad applicability of our approach in complex microbiomes. This way, we observe that, although uridines are required for modification, mRNAs from GC-rich bacteria harbored more pseudouridine sites than AT-rich genomes in our dataset. Altogether, our work highlights the advantages of mapping bacterial pseudouridines and provides a tool to study posttranscription regulation in microbial communities.