Transient interdomain interactions shape the conformational ensemble governing RNA recognition by the tandem RRMs of Sex-lethal
RNA recognition motif (RRM) proteins frequently contain multiple RNA-binding domains connected by flexible linkers, yet the contribution of transient interdomain interactions to RNA recognition remains incompletely understood. Here, we investigated the structural organization of the tandem RRMs of the Drosophila melanogaster splicing regulator Sex-lethal (Sxl) using solution NMR spectroscopy in combination with rational protein engineering, restrained docking and RNA-binding studies. Progressive extension of the native interdomain linker resulted in a gradual decrease in rotational coupling between the two RRMs and continuous chemical shift changes, demonstrating that the RNA-free protein samples a dynamic conformational ensemble rather than behaving as two independently tumbling domains. NMR-guided docking identified a compact arrangement compatible with the experimental data and suggested a transient interface partially overlapping the RNA-binding surfaces. Surprisingly, a mutant designed to weaken this interface produced the opposite effect: instead of increasing interdomain mobility, it exhibited enhanced rotational coupling while remaining natively folded, indicating a redistribution of the conformational ensemble rather than disruption of the domain architecture. Both linker extension and the mutant reduced RNA-binding affinity, and the mutant additionally diminished sequence discrimination, demonstrating that perturbations shifting the conformational equilibrium in either direction compromise RNA recognition. Together, our results demonstrate that RNA recognition by Sxl is governed not by a single apo structure but by a finely balanced conformational ensemble, and that perturbing this equilibrium in either direction compromises high-affinity and sequence-selective RNA binding.