Molecular insights into DDX3X-androgen receptor mRNA regulation via non-canonical G-quadruplex in castration-resistant prostate cancer.

Prostate cancer (PC) is one of the most common malignancies in men, and the emergence of androgen receptor-low/negative castration-resistant PC (ARL/- CRPC) following androgen receptor signaling inhibitor (ARSI) therapy remains a critical clinical challenge. The RNA-binding protein DEAD-box helicase 3 X-linked (DDX3X) has been implicated in the translational regulation of androgen receptor (AR) mRNA; however, the underlying binding mechanisms are not well defined. Here, we show that DDX3X colocalizes with AR mRNA in ARL/- CRPC cells and selectively recognizes non-canonical RNA G-quadruplex (rG4) motifs within the sequence of AR mRNA. RNA immunoprecipitation sequencing (RIP-seq) revealed enrichment of DDX3X-AR mRNA interactions in ARL/- CRPC cells. Fluorescence imaging confirmed the colocalization of DDX3X and AR mRNA within cytoplasmic granules, and biochemical assays confirmed the ability of selected AR mRNA fragments to form rG4 structures bound by DDX3X. Proteomic profiling of DDX3X-Ras GTPase-activating protein-binding protein 1 (G3BP1) complexes identified several RNA-binding proteins, including IGF2BP1, PUM2, and UBAP2, which may act as candidate cofactors. Together, these findings shed light on the interaction between AR mRNA and DDX3X and identify putative protein partners, offering insights into future therapeutic strategies.