Charting the Structure and Functional Landscape of the SF3B1 Intrinsically Disordered Region

Despite its essential role in pre–mRNA splicing, the structure and functional significance of the intrinsically disordered N-terminal domain (NTD) of SF3B1, a core component of the SF3b spliceosome complex, remain poorly understood. Here, we combine nuclear magnetic resonance (NMR) spectroscopy, small-angle X–ray scattering (SAXS), biochemistry, bioinformatics approaches, with molecular dynamics simulations to characterize the conformational landscape of the SF3B1-NTD and its phosphorylation-dependent remodelling. Specifically, we focus on the 186–341 region (186–341SF3B1), which contains at least four regulatory phosphorylation sites. We reveal that 186–341SF3B1 is highly dynamic, lacking a stable secondary structure, and that phosphorylation of T211, T235, T313, and T328 induces a shift toward more compact states driven by the formation of intramolecular interactions. Additionally, we unveil that 186–341SF3B1 interacts with RNA primarily via the arginine residues flaking one of the conserved tryptophan-centred motifs present within SF3B1–NTD. These interactions are transient and heterogeneous, reflecting a nonspecific yet sequence-dependent recognition mechanism. Collectively, this study provides a comprehensive description of the conformational landscape and functional features of the SF3B1-NTD, and expands the portfolio of its possible interactors, thus broadening our understanding of how intrinsic disorder, post-translational modifications, and sequence-encoded motifs cooperate to regulate pre-mRNA splicing.