RG/RGG boxes are common binding motifs in RNA-G-quadruplex-interacting proteins

G-quadruplexes (rG4s) are recognized as key structures involved in RNA biology and are linked to neurodegenerative disease and cancer. However, detailed knowledge of rG4s and their binding partners is lacking. Here, a systematic, unbiased affinity proteomics approach identified 80 potential high-confidence interactors of the rG4 structure in the 5’UTR of the NRAS oncogene. Using epitope-tagged protein expression, we validated a subset of interactions including DDX3X, DDX5, DDX17, DHX9, DHX36, FXR1, FXR2, GRSF1 and NSUN5. More than half of the identified rG4 interactors were enriched in glycine-arginine (GAR) domains, and for DDX3X and DDX17, we show that the GAR domain is required for rG4 interaction. Transcriptome-wide binding analysis using DDX3X iCLIP revealed a striking association with rG4s in 5’UTRs that was lacking in the GAR-domain mutant. Overall, our work highlights a hitherto unrecognized complexity in rG4 structure-protein interactions that should be considered for the understanding of rG4 function. 6 libraries for Flp-In T-Rex 293 cell line iCLIP experiment: 3 for mutant-DDX3X and 3 for WT-DDX3X; 11 libraries for Flp-In T-Rex 293 cell line RNA-seq experiment: 4 for WT-DDX3X, 4 for mutant-DDX3X and 3 for no expression (negative).