Understanding the Role of NAD-asGlpF in Regulating Glycerol Utilization Genes in E. coli

Living organisms use complex mechanisms to regulate gene activity, with RNA modifications playing a critical role. In eukaryotic cells, messenger RNAs (mRNAs) typically have a methylguanosine (m7G) cap at the 5’ end, essential for gene expression. It was once thought that bacterial RNAs lacked a cap. However, recent findings reveal that some RNAs in both prokaryotic and eukaryotic cells possess a different cap made from nicotinamide adenine dinucleotide (NAD+), a crucial metabolite in energy production. This discovery suggests a novel regulatory mechanism for gene expression through non-traditional RNA capping, though the mechanisms of NAD capping and the functions of NAD-capped RNAs (NAD-RNAs) remain poorly understood.
Our research team has developed methods to identify NAD-RNAs across various genomes, providing insights into their characteristics. Our findings show that some genes produce significant amounts of NAD-RNA, which primarily serve as regulatory molecules rather than templates for protein synthesis. Using highly sensitive techniques, we found that nearly 20% of genes in E. coli produce NAD-RNAs, with around 15% classified as NAD-capped antisense RNAs (NAD-asRNAs), originating from the opposite strand of protein-coding genes. One of the most abundant NAD-asRNAs originates from the glpF gene, part of the glpFK operon, which includes glpF, encoding a glycerol transporter, and glpK, an enzyme that processes glycerol.
We found that NAD-asGlpF (the NAD-asRNA derived from glpF) is more effective in suppressing glpF expression than the uncapped antisense RNA. E. coli overexpressing NAD-asGlpF exhibits poor growth in glycerol-containing media compared to the strain overexpressing the uncapped version. Importantly, glpF mRNA levels remain unchanged, suggesting that NAD-asGlpF inhibits the translation of glpF mRNA. Additionally, we noted differential regulation of glpF and glpK at the protein level in response to glycerol availability. We hypothesize that E. coli utilizes NAD-asGlpF to regulate glycerol uptake and utilization by specifically inhibiting glpF mRNA translation when the transporter is no longer needed.
The function of NAD capping and its mechanism in gene regulation remain unclear. In this project, a collaborative team of scientists specializing in NAD capping and antisense RNA biology will work together to understand how NAD capping modulates gene expression. We aim to use NAD-asGlpF as a model to investigate the regulation of NAD capping and its action in suppressing glpF expression in E. coli. This project could uncover functions and novel mechanisms in gene regulation mediated by this newly discovered RNA modification and offer potential strategies to combat bacterial
pathogens.