P-328 - NAD+-capped RNA: A new layer of regulation in bacterial virulence

RNA capping is well known to play crucial roles in RNA stability, splicing, and translation initiation in eukaryotes. It was long believed that RNA capping was absent in prokaryotes until the recent discovery of nicotinamide adenine dinucleotide (NAD+) as an ancient RNA cap across domains of life. However, how NAD+-RNA capping regulates gene expression in bacteria is poorly understood. Here, we determined the NAD+-capped transcriptome of Pseudomonas syringae during infection. Using captureSeq, we found hundreds of genes whose transcripts can be NAD+-capped. Interestingly, genes encoding type III secretion machinery and secreted effectors were among the most highly NAD+-capped RNAs. To explore the functional significance of this regulation, we further investigated hrpA, which encodes harpin, the building block of the type III pili. NAD+ is incorporated in RNA by replacing ATP at the transcription start site (TSS). By generating mutants at the hrpA TSS, we revealed a strong correlation between the level of NAD+-capped hrpA transcripts and HrpA protein accumulation, indicating that NAD+-capping of mRNA may enhance protein translation during infection and subsequently promoting bacterial virulence. This research provides novel insights into NAD+-RNA capping as a regulatory mechanism in bacterial pathogens and has broader implications for the impact of RNA modifications on bacterial physiology and pathogenesis.