Characterizations of G-Quadruplex RNA-Protein Interactions in Living Cells

RNA guanine quadruplexes (rG4s) are noncanonical nucleic acid structures that contribute to diverse cellular functions and disease mechanisms. Defining the proteins that interact with rG4s (rG4IPs) is essential for elucidating their biological roles. Here, we build on the RNA-protein interaction detection (RaPID) platform to develop G4-RaPID, a tailored chemoproteomic strategy for the unbiased profiling of rG4IPs in living cells. Using G4-RaPID, we identified 105 candidate rG4IPs that were commonly enriched across three distinct rG4 sequences. Biochemical analyses confirmed that recombinant hnRNPA0, CHD4, and IGF2BP1 proteins directly bind rG4 structures in vitro. In addition, CLIP-seq experiments revealed significant enrichment of hnRNPA0 binding at endogenous rG4 loci. Luciferase reporter assays further demonstrated that hnRNPA0 engages the rG4 in the 5' UTR of NRAS mRNA to negatively regulate its translation. Together, these results establish G4-RaPID as a robust approach for mapping rG4-protein interactions in living cells and document hnRNPA0-rG4 recognition as a regulatory mechanism controlling NRAS mRNA translation.