Essential Roles and Risks of G-Quadruplex Regulation: Recognition Targets of ALS-Linked TDP-43 and FUS

Abstract

A non-canonical DNA/RNA structure, G-quadruplex (G4), is a unique structure formed by two or more guanine quartets, which associate through Hoogsteen hydrogen bonding leading to form a square planar arrangement. A set of RNA-binding proteins specifically recognize G4 structures and play certain unique physiological roles. These G4-binding proteins form ribonucleoprotein (RNP) through a physicochemical phenomenon called liquid-liquid phase separation (LLPS). G4-containing RNP granules are identified in both prokaryotes and eukaryotes, but extensive studies have been performed in eukaryotes. We have been involved in analyses of the roles of G4-containing RNAs recognized by two G4-RNA-binding proteins, TDP-43 and FUS, which both are the amyotrophic lateral sclerosis (ALS) causative gene products. These RNA-binding proteins play the essential roles in both G4 recognition and LLPS, but they also carry the risk of agglutination. The biological significance of G4-binding proteins is controlled through unique 3D structure of G4, of which the risk of conformational stability is influenced by environmental conditions such as monovalent metals and guanine oxidation.

Keywords: FUS (fused in sarcoma); G-quadruplex (G4); RNP granule; TDP-43 (43 kDa TAR DNA-binding protein); amyotrophic lateral sclerosis (ALS); liquid-liquid phase separation (LLPS).

FIGURE 1

General roles of DNA/RNA G4s in prokaryote and eukaryote. G4 has also been suggested to be involved in prokaryotic stress responses through replication, transcription, and translation. Dysregulation of telomere regulation, transcription, translation and replication by G4 are suggested to be involved in the pathogenic mechanism of human cancer. Mutations in G4 containing RNAs and G4 binding proteins involved in RNA transport and LLPS have been found in patients with neurodegenerative diseases such as ALS.

FIGURE 2

ALS-linked G4 binding proteins. (A), Binding specificity of TDP-43 and FUS. TDP-43 can bind only for the parallel-stranded G4-DNA/RNAs, however, FUS binds to parallel, hybrid, and even G-rich hairpin forms (Ishiguro et al., 2021). (B), Different G4 binding modes for TDP-43 and FUS. TDP-43 protects and stabilizes the G4 structure, while FUS destabilizes the G4 conformation and recreates it into a “terminal associated” configuration. In both cases, the G4-protein interaction was inhibited by introducing amino acid substitutions from ALS patients (Ishiguro et al., 2021). In some FUS mutations have been found that promotion of the phase transition of liquid to solid phase of the droplet formed by LLPS (Ishiguro et al., 2021). (C), Concerned risks for G4s and G4 binding proteins. Low-complexity IDRs have considered to be tended to be aggregated (van der Lee et al., 2014; Taylor et al., 2016). 8OG produced by oxidation of guanine interferes with the normal formation of G4. Li+ is the same monocation as K+ and Na+, but it is known that it may not contribute to the structural stabilization of G4.