The RNA Stem–Loop to G-Quadruplex Equilibrium Controls Mature MicroRNA Production inside the Cell

Abstract

Biological role for existence of overlapping structures in RNA is possible yet remains very less explored. G-rich tracts of RNA form G-quadruplexes while GC-rich sequences prefer stem-loop structures. Equilibrium between alternate structures within RNA may occur and influence its functionality. We tested equilibrium between G-quadruplex and stem-loop structure in RNA and its effect on biological processes using pre-miRNA as a model system. Dicer enzyme recognizes canonical stem-loop structures in pre-miRNA to produce mature miRNAs. Deviation from stemloop leads to deregulated mature miRNA levels, providing readout of existence of alternate structure per se G-quadruplex mediated structural interference in miRNA maturation. In vitro analysis using beacon and Dicer cleavage assays indicated that mature miRNA levels depend on relative amounts of K + and Mg 2+ ions suggesting an ion-dependent structural shift. Further in cellulo studies with and without TmPyP 4 (RNA G-quadruplex destabilizer) demonstrated that miRNA biogenesis is modulated by G-quadruplex-stem-loop equilibrium in a subset of premiRNAs. Our combined analysis thus provides evidence for formation of non-canonical Gquadruplexes in competition with canonical stem-loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner.

RNA G-Quadruplexes in Biology: Principles and Molecular Mechanisms

“…One of the duplex strands as a mature miR is effectively loaded into the RISC complex (RNA-induced silencing complex) where, in the complex with the protein Argonaute (Ago), regulates mRNA stability and translation. About 130 miR genes (∼ 5% of all miR genes) are predicted to contain G4s in their promoter regions 168 and some G4 are found within miR transcripts 169; 170 . Since Dicer effectively recognizes canonical hairpin structures in pre-miRs, alternative structures such as G4s in pre-miRs should inhibit their maturation into miRs.…”

“…In contrast, conditions that favor G4 formation significantly inhibited pre-miR-92b processing in vitro . In another study, authors compared processing of the naturally occurring (pre-let-7e) and artificial synthetic (quad-pre-miR-27a) pre-miRs containing G4 structures into mature miRs 170 . In agreement with the previous work, G4s inhibit Dicer-mediated conversion of pre-miRs into mature forms.…”

RNA G-quadruplexes: emerging mechanisms in disease

“…Indeed, a number of studies provided evidence of the formation of G4s in competition with stem-loop structures (10,38–41,124) or with alternate G4 conformations (125). These structural transitions may play a role in telomere homeostasis (38), translational regulation (39,40) and miRNA biogenesis (10,41) as well as seed binding site accessibility (125), and can be modulated by ions (10,41,124), G4 ligands (41) and trans -acting factors, such as ncRNAs (39). Noteworthy, transcriptome-wide analysis of in vitro G4 formation supports the notion that hairpin-G4 transitions may be prevalent in human transcripts and play a role in gene expression regulation (31).…”

“…Other examples suggest that the interplay between RNA and DNA can involve the formation of hybrid G4 structures that recruit G-tracts from both the DNA and RNA molecules (Figure 2C) to modulate transcription regulation (35) or telomere homeostasis (36,37). RNA G4s can also exist in equilibrium with hairpin structures (Figure 2D) and play a role in telomere homeostasis (38) or gene expression mechanisms (10,39–41). Finally, sequences with the potential to form G4s might play an active role in the formation or dissolution of stable RNA/DNA hybrids (R-loops) (42–45) (Figure 2E).…”

G Quadruplex in Plants: A Ubiquitous Regulatory Element and Its Biological Relevance

“…In human genome, there are ∼16% of pre-mi-RNA that contains putative GQFS and can adopt these secondary structures to modulate canonical stem-loop structure of mi-RNA to adopt G quadruplex structure and thus impeded dicer mediated cleavage of mi-RNA (Mirihana Arachchilage et al, 2015). In addition, the equilibrium between the G quadruplex structure and stem loop structure influence the miRNA functionality as dicer enzyme recognize canonical stem loop structure in pre mi-RNA to produce mature miRNA and thus in turn formation of G quadruplex affect miRNA maturation [The RNA Stem–Loop to G Quadruplex Equilibrium Controls Mature MicroRNA Production inside the Cell (Pandey et al, 2015)]. In silico transcriptome wide analyses have identified significant number of G quadruplex motifs in human long non-coding RNA (lncRNA).…”