Advantages and Challenges of G‑Quadruplexes in Regulating Alternative Splicing

Alternative splicing (AS) is a fundamental process that dramatically expands the proteomic diversity of eukaryotes. The precise regulation of AS is governed by the complex interplay between cis-regulatory elements in the pre-mRNA and trans-acting protein factors. Recently, RNA secondary structures have emerged as critical players in the regulatory landscape. Among these, G-quadruplexes (G4s), which are stable four-stranded structures formed by guanine-rich (G-rich) sequences, have been identified as potent regulators of splicing outcomes. This review synthesizes the current understanding of the role of RNA G4s in AS, focusing on three interconnected pillars: (1) the characteristic sequence features and genomic distribution of G4-forming motifs near splice sites revealing a significant strand-specific enrichment that suggests a conserved regulatory function; (2) the multifaceted interactions between G4 structures and RNA-binding proteins which can act as splicing enhancers or repressors by binding to single-stranded G-tracts or structured G4s; and (3) the profound impact of G4-stabilizing or -destabilizing small molecules, which can be harnessed to deliberately re-engineer splicing patterns in genes linked to cancer, neurodegeneration, and other diseases. By integrating findings from genomic analyses, biochemical studies, and chemical biology approaches, this review aims to expound G4 structures as dynamic, druggable nodes in the splicing regulatory network, offering novel mechanistic insights and promising therapeutic avenues.