Severe forms of dilated cardiomyopathy (DCM) are associated with point mutations in the alternative splicing regulator RBM20 that are frequently located in the arginine/serine-rich domain (RS-domain). Such mutations can cause defective splicing and cytoplasmic mislocalization, which leads to the formation of detrimental cytoplasmic granules. Successful development of personalized therapies requires identifying the direct mechanisms of pathogenic RBM20 variants. Here, we decipher the molecular mechanism of RBM20 mislocalization and its specific role in DCM pathogenesis. We demonstrate that mislocalized RBM20 RS-domain variants retain their splice regulatory activity, which reveals that aberrant cellular localization is the main driver of their pathological phenotype. A genome-wide CRISPR knockout screen combined with image-enabled cell sorting identified Transportin-3 (TNPO3) as the main nuclear importer of RBM20. We show that the direct RBM20-TNPO3 interaction involves the RS-domain, and is disrupted by pathogenic variants. Relocalization of pathogenic RBM20 variants to the nucleus restores alternative splicing and dissolves cytoplasmic granules in cell culture and animal models. These findings provide proof-of-principle for developing therapeutic strategies to restore RBM20’s nuclear localization in RBM20-DCM patients.

严重形式的扩张型心肌病 (DCM) 与选择性剪接调节因子 RBM20 的点突变有关，该突变通常位于精氨酸/富含丝氨酸的结构域（RS 结构域）。这种突变会导致剪接缺陷和细胞质错误定位，从而导致有害细胞质颗粒的形成。个性化疗法的成功开发需要确定致病性 RBM20 变异的直接机制。在这里，我们破译了RBM20错误定位的分子机制及其在DCM发病机制中的具体作用。我们证明错误定位的 RBM20 RS 结构域变体保留了其剪接调节活性，这表明异常的细胞定位是其病理表型的主要驱动因素。全基因组 CRISPR 敲除筛选与图像细胞分选相结合，确定 Transportin-3 (TNPO3) 是 RBM20 的主要核输入蛋白。我们发现 RBM20-TNPO3 的直接相互作用涉及 RS 结构域，并且被致病变异破坏。在细胞培养物和动物模型中，致病性 RBM20 变异重新定位到细胞核可恢复选择性剪接并溶解细胞质颗粒。这些发现为制定恢复 RBM20-DCM 患者中 RBM20 核定位的治疗策略提供了原理验证。