Abstract Body: Objective: Mutations in the nuclear localization signal (NLS) of RNA-binding motif protein 20 (RBM20) cause aggressive dilated cardiomyopathy (DCM) by impairing nuclear import and promoting cytoplasmic RBM20 granule formation. While these granules are emerging as key drivers of RBM20-associated DCM, the nuclear import machinery regulating RBM20 localization remains undefined. Here, we sought to identify the molecular regulator of RBM20 nuclear import to enable targeted therapeutic strategies.
Methods: To identify nuclear transport receptors (NTRs) for RBM20, we performed proximity labeling proteomics using TurboID-fused RBM20 NLS-mutant constructs in H9c2 cells. Biotinylated proteins were analyzed by mass spectrometry and validated by Western blot. Interactions between RBM20 and candidate NTRs were assessed by immunocytochemical and co-immunoprecipitation (co-IP). Functional roles of candidate NTRs were interrogated using pharmacologic inhibition or shRNA-mediated silencing, followed by analysis of RBM20 localization and target gene splicing. Kpnb1 conditional knockout mice were used for in vivo validation.
Results: Proximity labeling proteomics identified karyopherin β1 (KPNB1) as a candidate RBM20 nuclear import receptor. Pulldown assays and co-IP confirmed a direct interaction between RBM20 and KPNB1, with robust co-localization observed in H9c2 cells and hiPSC-derived cardiomyocytes expressing WT or NLS-mutant RBM20. Functionally, KPNB1 loss via knockdown or pharmacologic inhibition drove RBM20 cytoplasmic mislocalization, induced granule formation, and recapitulated splicing defects characteristic of RBM20 dysfunction. Importantly, Kpnb1 deficiency in vivo led to perinuclear RBM20 accumulation and impaired splicing, establishing KPNB1 as an essential and previously unrecognized regulator of RBM20 nuclear import and function.
Conclusion: KPNB1 is a central mediator of RBM20 nuclear import that restrains pathological granule formation. Disruption of this pathway drives RBM20 mislocalization and granule-mediated cardiomyopathy. Targeting KPNB1-dependent nuclear import represents a novel therapeutic strategy to restore RBM20 function in DCM.