G-Quadruplex structural motifs modulate protein-RNA interactions within the transcriptome

RNA secondary structures, including G-quadruplexes (G4s), have emerged as vital players in protein–RNA interactions. The RNA-binding protein Fused in Sarcoma (FUS), which is strongly implicated in both neurodegenerative disease and cancer, is known to interact with RNA molecules through a variety of GU-rich sequences. However, a definitive consensus motif for FUS–RNA recognition and binding has not yet been determined. Here, we hypothesize that G4 structures, which are inherently G-rich, may play a key role in FUS binding. We examine the role of G4s in FUS–RNA binding by developing an RNA immunoprecipitation sequencing (RIP-seq) protocol under G4-stabilising and non-stabilising conditions. We find that G4s regulate the binding of FUS to target RNAs, providing new information on protein–RNA binding motifs, while reinforcing the importance of RNA secondary structures as pivotal regulators of protein interactions. These insights advance our understanding of FUS-RNA binding dynamics and future potential for identifying new therapeutic targets for neurodegenerative disease and other FUS-related pathologies. RNA G4 secondary structures were annealed in RIP buffer containing 150 mM of K+ or Li+, then bound to recombinant C-terminal FUS protein. FUS-bound RNA was purified and next-generation sequencing performed to identify G4-dependent FUS-RNA binding. Input samples were subject to all steps as FUS-RNA samples except for immunoprecipitation and subsequent washes. Each condition was performed in triplicate.