Biological relevance and therapeutic potential of G-quadruplex structures in the human noncoding transcriptome

Abstract

Noncoding RNAs are functional transcripts that are not translated into proteins. They represent the largest portion of the human transcriptome and have been shown to regulate gene expression networks in both physiological and pathological cell conditions. Research in this field has made remarkable progress in the comprehension of how aberrations in noncoding RNA drive relevant disease-associated phenotypes; however, the biological role and mechanism of action of several noncoding RNAs still need full understanding. Besides fulfilling its function through sequence-based mechanisms, RNA can form complex secondary and tertiary structures which allow non-canonical interactions with proteins and/or other nucleic acids. In this context, the presence of G-quadruplexes in microRNAs and long noncoding RNAs is increasingly being reported. This evidence suggests a role for RNA G-quadruplexes in controlling microRNA biogenesis and mediating noncoding RNA interaction with biological partners, thus ultimately regulating gene expression. Here, we review the state of the art of G-quadruplexes in the noncoding transcriptome, with their structural and functional characterization. In light of the existence and further possible development of G-quadruplex binders that modulate G-quadruplex conformation and protein interactions, we also discuss the therapeutic potential of G-quadruplexes as targets to interfere with disease-associated noncoding RNAs.

Figure 1.

(A) The canonical miRNA processing pathway. In the nucleus, miRNA genes are initially transcribed into pri-miRNAs, which are then cleaved into pre-miRNAs by Drosha. Pre-miRNAs are exported to the cytoplasm by Exp5 and further processed into a miRNA:miRNA duplex by Dicer. The miRNA:miRNA duplex is unwound and the mature miRNA is then assembled into the RISC complex, where it binds to the target mRNA, thus finally regulating gene expression. (B) rG4s interfere with the biogenesis and function of miRNAs. rG4s found in pri-miRNAs and pre-miRNAs affect miRNA processing by altering Drosha and Dicer binding, respectively, while rG4s in mature miRNAs impede miRNA binding to its target mRNA. (C) rG4s in the 3′-UTR of mRNAs hamper the accessibility to miRNAs and prevent RISC-mediated degradation. Created with BioRender.com.

Figure 2.

The repertoire of lncRNA modes of action. In the cell nucleus, lncRNAs can (A) modify the chromatin state, (B) activate/inhibit transcription of target genes, (C) serve as host genes for the transcription of miRNAs, (D) regulate mRNA alternative splicing. In the cell cytoplasm, lncRNAs can (E) modulate mRNA stability or translation and (F) act as sponges to sequester miRNAs. Some lncRNAs serve as scaffolds for the assembly of different organelles or nuclear condensates, such as (G) paraspeckles. All reported lncRNAs are depicted in purple. RPB: RNA binding protein. Created with BioRender.com.

Figure 3.

RNA can fold into several secondary structural motifs to fulfill its precise biological role in a specific cellular environment. These include hairpin stem or loops, bulges, three-way junctions, internal loops, pseudoknots, rG4s and i-motifs. It is to note that RNA i-motifs have only been observed in vitro (195) to date and their existence in vivo is controversial. However, Zeraati et al. speculate that the antibody fragment (iMab) (196), besides recognizing DNA i-motifs, also binds to RNA i-motifs in cells with high selectivity and affinity. Created with BioRender.com.

Figure 4.

The rG4 structure. (A) Chemical structure and (B) schematic illustration of a G-tetrad (in purple), composed of four Gs linked together through Hoogsteen H-bonds; (C) example of intramolecular parallel rG4. Cations coordinated at the center of the tetrad are represented in gray, while Gs in blue.

Figure 5.

Chemical structures of the reported G4 ligands: PhenDC3, BRACO-19, cPDS, C8,TMPyP4, PDS, Sanguinarine, Jatrorrhizine derivative 2, Jatrorrhizine derivative 7, Pseudopalmatine, NMM, RHPS4, QUMA-1.

Figure 6.

rG4s within lncRNAs influence the interaction between lncRNAs and their targets. (A) Nascent NEAT1 transcripts bind to the NONO proteins through conserved rG4 motifs. The binding of NONO, along with the splicing factor proline- and glutamine-rich (SFPQ), favors the recruitment of additional protein components required for paraspeckle formation and maturation. (B) FLJ39051 interacts with the RNA helicase DHX36 through its rG4 structure and inhibits DHX36 ATP-dependent rG4 unwinding activity, finally promoting motility of colorectal cancer cells. (C) REG1CP promotes cancer cell cycle progression and tumorigenicity by inducing REG3A gene transcription, which occurs as a consequence of the rG4-mediated binding of REG1CP to the helicase FANCJ, which is necessary to unwind the double-stranded DNA and derepress transcriptional inhibition. Created with BioRender.com.