RNA G-Quadruplex Structures Mediate Gene Regulation in Bacteria.

Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model strain and a major human pathogen, , were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 rG4 sites were found in and , respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as and ), and they were functionally validated in cells by genetic (point mutation and reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species. G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model strain and a major human pathogen, , have 168 and 161 rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria ( and ). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.