Phosphorylation-dependent remodeling of the XIAP IRES by hnRNPA1

The heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a ubiquitously expressed RNA-binding protein with essential roles in splicing, mRNA stability, and translation. Its activity as an internal ribosome entry site (IRES) trans-acting factor (ITAF) is particularly relevant in stress adaptation and cancer, where dysregulated IRES-mediated translation promotes cell survival and therapy resistance. In small-cell lung cancer (SCLC), FGF-2 signalling activates S6K2-dependent phosphorylation of hnRNPA1 at serines 4 and 6, selectively enhancing expression of the anti-apoptotic XIAP and Bcl-xL protein. Here, we combine quantitative binding assays, X-ray crystallography, NMR spectroscopy, and multi-microsecond molecular dynamics (MD) simulations to define how phosphorylation modulates hnRNPA1–XIAP IRES interactions. We show that phosphorylation confers RNA- and sequence-specific recognition, with binding resolving into two cooperative interactions of distinct affinities at the RRM1 and RRM2 domains. This behaviour is consistent with phosphorylation-enhanced RNA melting activity that exposes otherwise inaccessible motifs. Structural and spectroscopic analyses reveal that phosphorylation does not induce structural rearrangements but perturbs the conformational ensemble of the intrinsically disordered (IDR) N-terminal tail, reshaping transient intramolecular contacts with the RRM domains. Our findings reveal that fine-tuning of IDR conformational dynamics is a key component of RRM-mediated RNA recognition, coupling post-translational regulation of RNA-binding proteins to translational control. *Sayan Das & Louise Dunnett contributed equally.