G-Quadruplexes in Nuclear Biomolecular Condensates

G-quadruplexes (G4s) have long been implicated in the regulation of chromatin packaging and gene expression. These processes require or are accelerated by the separation of related proteins into liquid condensates on DNA/RNA matrices. While cytoplasmic G4s are acknowledged scaffolds of potentially pathogenic condensates, the possible contribution of G4s to phase transitions in the nucleus has only recently come to light. In this review, we summarize the growing evidence for the G4-dependent assembly of biomolecular condensates at telomeres and transcription initiation sites, as well as nucleoli, speckles, and paraspeckles. The limitations of the underlying assays and the remaining open questions are outlined. We also discuss the molecular basis for the apparent permissive role of G4s in the in vitro condensate assembly based on the interactome data. To highlight the prospects and risks of G4-targeting therapies with respect to the phase transitions, we also touch upon the reported effects of G4-stabilizing small molecules on nuclear biomolecular condensates. (Genes (Basel). 2023 May; 14(5): 1076.)

G-quadruplexes (G4s) are planar arrangements of Hoogsteen bonded guanine tetrads. They can be formed through the association of several nucleic acid strands that harbor G2+ tracts or an intramolecular tetrahelical folding of a single strand. Within genomic DNA, intramolecular G4 folding is favored by a low nucleosome density and negative supercoiling. Chromatin immunoprecipitation with the quadruplex-specific antibodies BG4 and 1H6 or an artificial protein probe PG4 revealed the G4 association with DNA damage hotspots and an abundance in the regulatory genomic regions, including promotors and 5′-UTRs, telomeres, and boundaries of topologically associating domains, etc. Such a distribution pointed to a G4 relevance for reparation, transcription, genome integrity maintenance, and chromatin remodeling, which has been analyzed comprehensively from the biological perspective.

RNA G4s have recently emerged as prospective drivers for various phase transitions in the cytoplasm. The evidence for their contribution to the assembly of stress granules and toxic aggregates associated with neurodegenerative diseases has reinforced the interest in RNA G4 ligands as drug candidates. 

Biomacromolecular condensates are assembled spontaneously through the liquid–liquid phase separation (LLPS) of biopolymers, typically nucleic acids and proteins with low-complexity domains (LCDs) or intrinsically disordered regions (IDRs). Such proteins and nucleic acids are prone to weak multivalent homo/heterotypical interactions, which outcompete the water–biopolymer interactions within a certain polymer concentration range, favoring solution demixing into polymer-depleted and condensed liquid phases. The resulting increase in the local macromolecule concentration can promote the assembly of multicomponent complexes, facilitate the enzyme–substrate recognition, and increase the reaction rates. At the same time, the LLPS-mediated compartmentalization enables the reversible isolation of excessive or toxic biopolymers from the bulk cellular media. Assuming the G4s trigger or assist in the phase separation, G4-(de)stabilizing endogenous or exogenous ligands (potential drugs) may have profound effects on the proteostasis. This possibility should be taken into account in the development of G4-targeted therapeutics.

Despite the technical limitations, several lines of research have converged to support the importance of G4s in nuclear condensates. Some of the major results and interpretations are summarized below.

  • G4s promote the LLPS of heterochromatin-associated proteins in artificial systems, but the biological relevance of these findings awaits verification.
  • G4s promote the LLPS of RNA-binding proteins in the pseudo-cellular environment. These findings are in line with the studies of cytoplasmic condensates and may be relevant to the assembly of nuclear RNA processing factor-rich condensates, namely nucleoli, speckles, and paraspeckles.
  • The integrity and/or functions of speckles/paraspeckles are disrupted by G4 mutations and G4-stabilizing ligands. The shelterin integrity and function are also disrupted by G4 ligands. The effects of these ligands are attributed to their interference with G4 protein interactions.
  • The colocalization with Pol II clusters, TFs, and chromatin loop boundaries supports the idea that G4s assist in the transcription initiation. However, conclusive evidence is lacking. A comparison of transcription burst rates at G4-rich and non-G4 SEs could probably clarify this matter.

The proposed mechanisms for the G4-mediated LLPS can be classified as follows.

  • The nucleobase exposure in the G4 outer tetrads and the adjoining ssDNA regions for transient π–π interactions with aromatic amino acid-rich proteins and cation–π interactions with Arg-rich ones.
  • The exposure of a protein IDR/LCD for transient interactions with other macromolecules following a G4-binding-induced conformational transition.
  • The accumulation of multiple IDR/LCD-containing proteins at the G4 repeats through the G4 recognition by the structured domains of these proteins or their partners.
  • The assembly of a transient nucleic acid “net” through the formation of G4–G4 kissing complexes, intermolecular G4 folding, or chromatin looping mediated by G4-binding proteins.