RNA stem-loop to G-quadruplex equilibrium controls mature miRNA production inside the cell

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 stem-loop 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 Mg2+ ions suggesting an ion-dependent structural shift. Further in cellulo studies with and without TmPyP4 (RNA G-quadruplex destabilizer) demonstrated that miRNA biogenesis is modulated by G-quadruplex-stem-loop equilibrium in a subset of pre-miRNAs. Our combined analysis thus provides evidence for formation of non-canonical G-quadruplexes in competition with canonical stem-loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner

Author Correction to: Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage

An amendment to this paper has been published and can be accessed via the original article.</p

Role of G-quadruplex located at 5&#x2032; end of mRNAs

Background: Secondary structures in 5&#x2032; UTR of mRNAs play a critical role in regulating protein synthesis. Though studies have indicated the role of secondary structure G-quadruplex in translational regulation, position-specific effect of G-quadruplex in naturally occurring mRNAs is still not understood. As a pre-initiation complex recognises 5&#x2032; cap of the mRNA and scans along the untranslated region (UTR) before initiating translation, the presence of G-quadruplex in 5&#x2032; region may have a significant contribution in regulating translation. Here, we investigate the role of G-quadruplex located at the 5&#x2032; end of an mRNA. Methods: Biophysical characterisation of putative G-quadruplexes was performed using UV and CD spectroscopy. Functional implication of G-quadruplex in the context of their location was assessed in cellulo using qRT-PCR and dual luciferase assay system. Results: PG4 sequences in 5&#x2032; UTR of AKT interacting protein (AKTIP), cathepsin B (CTSB) and forkhead box E3 (FOXE3) mRNAs form G-quadruplex whereas it is unable to form G-quadruplex in apolipoprotein A-I binding protein (APOA1BP). Our results demonstrated diverse roles of G-quadruplex located at 5&#x2032; end of mRNAs. Though G-quadruplex in AKTIP and CTSB mRNA act as inhibitory modules, it activates translation in FOXE3 mRNA. Conclusions: Our works suggests that G-quadruplex present at the 5&#x2032; terminal of an mRNA behaves differently in a different gene context. It can activate or inhibit gene expression

The tale of RNA G-quadruplex

G-quadruplexes are non-canonical secondary structures found in guanine rich regions of DNA and RNA. Reports have indicated the wide occurrence of RNA G-quadruplexes across the transcriptome in various regions of mRNAs and non-coding RNAs. RNA G-quadruplexes have been implicated in playing an important role in translational regulation, mRNA processing events and maintenance of chromosomal end integrity. In this review, we summarize the structural and functional aspects of RNA G-quadruplexes with emphasis on recent progress to understand the protein/trans factors binding these motifs. With the revelation of the importance of these secondary structures as regulatory modules in biology, we have also evaluated the various advancements towards targeting these structures and the challenges associated with them. Apart from this, numerous potential applications of this secondary motif have also been discussed

Effect of loops and G-quartets on the stability of RNA G-quadruplexes

The loop length, loop composition, salt concentration, and number of G-quartets are major determinants of G-quadruplex stability. We examined the effect of each of these factors on the thermal stability and folding topology of a library of RNA quadruplexes. The thermal stability of G2 and G3 RNA quadruplexes was investigated upon varying the loop length (from 1-1-1 to 15-15-15) and salt concentration (from 1 to 100 mM KCl), while the effect of loop composition was explored using 18 naturally occurring potential RNA quadruplexes predicted in untranslated regions (UTRs). We found loop length and quadruplex stability to be inversely related for G2 RNA quadruplexes and G3 RNA quadruplexes with shorter loops. However, melting temperature saturates for G3 RNA quadruplexes with longer loops. RNA G-quadruplexes with longer loops (G3 15-15-15) displayed Tm values significantly higher than the physiological temperature. This study thus highlights the need to modify the consensus motif presently used by quadruplex prediction tools. An increase in the loop size from 7 bases to 15 bases in the consensus motif will add to its predictive value for the discovery of potential RNA quadruplexes across transcriptomes

Effect of Loops and G‑Quartets on the Stability of RNA G‑Quadruplexes

The loop length, loop composition, salt concentration, and number of G-quartets are major determinants of G-quadruplex stability. We examined the effect of each of these factors on the thermal stability and folding topology of a library of RNA quadruplexes. The thermal stability of G2 and G3 RNA quadruplexes was investigated upon varying the loop length (from 1-1-1 to 15-15-15) and salt concentration (from 1 to 100 mM KCl), while the effect of loop composition was explored using 18 naturally occurring potential RNA quadruplexes predicted in untranslated regions (UTRs). We found loop length and quadruplex stability to be inversely related for G2 RNA quadruplexes and G3 RNA quadruplexes with shorter loops. However, melting temperature saturates for G3 RNA quadruplexes with longer loops. RNA G-quadruplexes with longer loops (G3 15-15-15) displayed <i>T</i>_m values significantly higher than the physiological temperature. This study thus highlights the need to modify the consensus motif presently used by quadruplex prediction tools. An increase in the loop size from 7 bases to 15 bases in the consensus motif will add to its predictive value for the discovery of potential RNA quadruplexes across transcriptomes

A Novel G-Quadruplex Binding Protein in Yeast—Slx9

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo