Properties and biological impact of RNA G-quadruplexes: from order to turmoil and back

Abstract

Guanine-quadruplexes (G4s) are non-canonical four-stranded structures that can be formed in guanine (G) rich nucleic acid sequences. A great number of G-rich sequences capable of forming G4 structures have been described based on in vitro analysis, and evidence supporting their formation in live cells continues to accumulate. While formation of DNA G4s (dG4s) within chromatin in vivo has been supported by different chemical, imaging and genomic approaches, formation of RNA G4s (rG4s) in vivo remains a matter of discussion. Recent data support the dynamic nature of G4 formation in the transcriptome. Such dynamic fluctuation of rG4 folding-unfolding underpins the biological significance of these structures in the regulation of RNA metabolism. Moreover, rG4-mediated functions may ultimately be connected to mechanisms underlying disease pathologies and, potentially, provide novel options for therapeutics. In this framework, we will review the landscape of rG4s within the transcriptome, focus on their potential impact on biological processes, and consider an emerging connection of these functions in human health and disease.

Figure 1.

RNA G-quadruplex architecture, complexity and formation. (A) G-quartet, a square planar arrangement of 4 Gs stabilized by a cation, is a structural unit of G-quadruplexes. (B) The presence of structurally diverse RNA G-quadruplexes in various RNA species reflects their roles in various aspects of RNA metabolism. (C) Most commonly used cations in G4 studies, and their G4 stabilizing order. (D) Various types of commonly reported rG4s based on different number of G-quartet stacking. (E, F) Proposed complex rG4 structures in pG4 repeat containing sequences like TERRA and C9ORF72 intronic RNAs.

Figure 2.

Models of rG4 types based on structural predictions. (A, B) A three-tier parallel rG4 modeled for 5′-GGGAGGGGCGGGUCUGGG, lateral and top view, respectively. (C, D) An anti-parallel rG4, modeled for 5′-UUAGGGUUAGGGUUAGGGUUAGGGUUA based on analogous dG4 PDB: 2mbj (208). (E, F) A proposed hybrid rG4, modeled for 5′-UAGGGUUAGGGUUAGGGUUAGGG based on analogous dG4 PDB: 2jsm (209). (G, H) A tetramolecular rG4, modeled for 5′-GGGGGUGUAGCUCAGUGGUAGAGCGCGUGC based on the sequence of 5′-tiRNA^Ala (details in Figure 6E). The nucleotides are colored as follows: Gs in syn conformation in quartets are yellow, Gs in anti-conformation in quartets are green, the remaining nucleotides in loops are magenta. K⁺ ions are shown in black. Gs in quartets are depicted by ‘ball and stick’ model. The models were created using the method described in ‘RNA G-quadruplex-topological diversity and dynamics’. The rG4 models shown in C, D and E, F have the same number of tetrads and loops, but their topology is different.

Figure 3.

Recent developments in cellular rG4 mapping tools that have been used in tandem with high throughput sequencing technologies. (A) rG4 sequencing. (B) SHAPE probing in combination with other validation methods can be used to map the presence of rG4s. (C) iCLIP-RNA-seq can be used to map potential rG4BP binding regions within the transcriptome. (D) Differential DMS reactivity of N7 of Gs can be a valuable tool in mapping in cella rG4 formation. (E) G4 antibodies can detect rG4s in fixed cells. (F) An rG4 specific small molecule with intrinsic fluorescence properties. (G) Hemin-bound rG4 can show peroxidase like activity which can be exploited to biotinylate rG4s for their potential in cella detection.

Figure 4.

RNA G-quadruplex binding proteins are key in regulating rG4 dynamics in vivo. (A) Different possibilities of rG4–rG4BP interactions—unfolded RNA folds into an rG4 with the help of rG4BP (1,2) or the later can interact with pre-folded rG4, potentially resulting in one of the following consequences—stabilization of the rG4 or melting of rG4 to transit into an alternate structure (3) or a non-structured form (4), or recruitment of other binding factors to further stabilize the rG4 (5). (B, C) Classification of reported proteins composition based on their loosely defined domain and motif compositions (supplementary file available for the analyzed proteins). (D) Crystal structure of the complex between the FMRP RGG box (RGGGGR peptide) and sc1 RNA quadruplex-duplex junction, peptide is in green color and RNA is in gray color with G-quartets and the mixed tetrad in orange. (E) Hydrogen bonding pattern between peptide and nucleic acids (complex from D). (F) NMR solution structure of G4 binding segment of DHX36 with human telomeric parallel dG4, with detailed intermolecular interactions between peptide positively charged side-chains of K8, R10 and K19 and the DNA phosphate backbone. DHX36, red; guanines, cyan; thymines, orange; DNA backbone, gray; O4′ atoms, yellow. D–F are reproduced with permission from the National Academy of Sciences of the United States of America (114,118).

Figure 5.

rG4s are implicated in different layers of gene regulation. (A) Proposed transcriptional regulation model via a DNA:RNA hybrid G4 formation in the R-loops. (B) The RNA Helicase DDX1 Converts rG4 into R-Loops to Promote IgH Class Switch Recombination. (C) rG4s regulate mRNA maturation. (D) rG4s regulate mRNA transport. (E, F) rG4s regulate mRNA stability. (G) rG4s modulate mRNA translation.

Figure 6.

rG4s influence ncRNA biology. (A) TRF2 interacts with both TERRA rG4s and telomere end-situated dG4s to influence telomere biology. (B) rG4s are present in different expansion segments of human rRNA (ES7 and ES27 are indicated). (C) rG4s modulate miRNA biogenesis and their target recognition. (D) rG4s affect piRNA biogenesis and their target recognition. (E) rG4s formed by 5′tiRNA^ala in response to cellular stress inhibit cap-dependent mRNA translation.