Novel L-RNA Aptamer Controls APP Gene Expression in Cells by Targeting RNA G-Quadruplex Structure.

Guanine quadruplex (G4) structure is a four-stranded nucleic acid secondary structure motif with unique chemical properties and important biological roles. Amyloid precursor protein (APP) is an Alzheimer's disease (AD)-related gene, and recently, we reported the formation of RNA G4 (rG4) at the 3'UTR of APP mRNA and demonstrated its repressive role in translation. Herein, we apply rG4-SELEX to develop a novel L-RNA aptamer, L-Apt.8f, which binds to APP 3'UTR D-rG4 strongly with subnanomolar affinity. We structurally characterize the aptamer and find that it contains a thermostable and parallel G4 motif, and mutagenesis analysis identifies the key nucleotides that are involved in the target recognition. We also reveal that the L-Apt.8f-APP D-rG4 interaction is enantiomeric-, magnesium ion-, and potassium ion-dependent. Notably, L-Apt.8f preferentially recognizes APP rG4 over other structural motifs, and it can control the APP reporter gene and native transcript translation in cells. Our work introduces a novel strategy and reports a new L-aptamer candidate to target APP 3'UTR rG4 structure, which laid the foundation for further applying L-RNA as an important class of biomolecule for practical L-aptamer-based targeting and controlling of gene expression in cells.

Alternative splicing modulation by G-quadruplexes.

Alternative splicing is central to metazoan gene regulation, but the regulatory mechanisms are incompletely understood. Here, we show that G-quadruplex (G4) motifs are enriched ~3-fold near splice junctions. The importance of G4s in RNA is emphasised by a higher enrichment for the non-template strand. RNA-seq data from mouse and human neurons reveals an enrichment of G4s at exons that were skipped following depolarisation induced by potassium chloride. We validate the formation of stable RNA G4s for three candidate splice sites by circular dichroism spectroscopy, UV-melting and fluorescence measurements. Moreover, we find that sQTLs are enriched at G4s, and a minigene experiment provides further support for their role in promoting exon inclusion. Analysis of >1,800 high-throughput experiments reveals multiple RNA binding proteins associated with G4s. Finally, exploration of G4 motifs across eleven species shows strong enrichment at splice sites in mammals and birds, suggesting an evolutionary conserved splice regulatory mechanism.

High-throughput characterization of the role of non-B DNA motifs on promoter function.

lternative DNA conformations, termed non-B DNA structures, can affect transcription, but the underlying mechanisms and their functional impact have not been systematically characterized. Here, we used computational genomic analyses coupled with massively parallel reporter assays (MPRAs) to show that certain non-B DNA structures have a substantial effect on gene expression. Genomic analyses found that non-B DNA structures at promoters harbor an excess of germline variants. Analysis of multiple MPRAs, including a promoter library specifically designed to perturb non-B DNA structures, functionally validated that Z-DNA can significantly affect promoter activity. We also observed that biophysical properties of non-B DNA motifs, such as the length of Z-DNA motifs and the orientation of G-quadruplex structures relative to transcriptional direction, have a significant effect on promoter activity. Combined, their higher mutation rate and functional effect on transcription implicate a subset of non-B DNA motifs as major drivers of human gene-expression-associated phenotypes.

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 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.