RNA G-Quadruplex Structures Mediate Gene Regulation in Bacteria

Abstract

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 in vitro 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 Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, 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 hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux 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. IMPORTANCE 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 E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.

Specific suppression of D-RNA G-quadruplex–protein interaction with an L-RNA aptamer

“…cPDS is a synthetic small molecule derived from parent molecule PDS, with demonstrated molecular specificity toward rG4 compared to dG4 ( 56 ). QUMA-1 is developed initially to track the folding of rG4s in live human cells, and has been recently applied to rG4s in other systems ( 57 , 58 ). As the cPDS and QUMA-1 ligands are small in size (Figure 7D and G ) and thus unobservable gel-shift in EMSA, we have carried out the comparison using MST assay (Figure 7E ,F, H–I).…”

G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum

“…In several model organisms, the rG4 content of the transcriptome has recently been investigated by ‘rG4-seq’ 18–20 , a technique in which the transcriptome is extracted from a cell and reverse-transcribed in two parallel conditions, one allowing rG4s to fold and the other preventing them from folding. Folded rG4s tend to stall the reverse transcriptase, leading to rG4-specific truncations that can be detected by comparing the two resultant cDNA libraries.…”

Exploring G and C-quadruplex structures as potential targets against the severe acute respiratory syndrome coronavirus 2

“…They have been found to be regulatory elements in the human genome implicated in key functions such as telomere maintenance and genome transcription regulation, replication and repair (17). G4 structures have also been identified in fungi (18)(19)(20)(21), bacteria (22)(23)(24)(25)(26) and parasites (27)(28)(29)(30)(31)(32). Their occurrence are known in many viruses that afflict humans as well.…”