Mislocalization of pathogenic RBM20 variants in dilated cardiomyopathy is caused by loss-of-interaction with Transportin-3

Abstract

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.

Mechanisms of RBM20 Cardiomyopathy: Insights From Model Systems

“…13,14,27 More recently, Rbm20 P635L mice (analogous to P635L in humans) have been generated. 27,28 In our own studies, analysis of Rbm20 S637A mice revealed marked systolic dysfunction and dilation of the left ventricle in both heterozygous and homozygous variant carriers by 8 weeks of age, consistent with the development of a DCM-like phenotype in these mice. 10 Homozygous Rbm20 S637A mice also exhibited premature mortality with ≈34% of mice dying within 100 days of birth, which mimics the early onset and high mortality associated with pathogenic variants in RBM20 in humans.…”

“…Notably, a second paper published by the Steinmetz group not only identified the RBM20 nuclear import receptor (TNPO3) but also showed that overexpression of this protein was sufficient to at least partially rescue nuclear localization of the P633L variant in hiPSC-CMs and mice (P635L in mice). 28 The observed restoration of variant RBM20 nuclear localization was associated with improved splicing of RBM20 target genes both in vitro and in vivo. 28 This finding is important as it suggests that the variant protein functions similar to WT, at least with respect to splicing, and, thus, strategies aimed at restoring nuclear localization of variant RBM20 may also be viable for the treatment of RBM20 cardiomyopathy.…”

“…28 The observed restoration of variant RBM20 nuclear localization was associated with improved splicing of RBM20 target genes both in vitro and in vivo. 28 This finding is important as it suggests that the variant protein functions similar to WT, at least with respect to splicing, and, thus, strategies aimed at restoring nuclear localization of variant RBM20 may also be viable for the treatment of RBM20 cardiomyopathy. Nevertheless, additional studies, in particular those assessing not only splicing but also cardiac function as an end point, will be necessary to confirm that such strategies are worth pursuing alongside CRISPR-based therapies.…”

“…Moreover, it was demonstrated that overexpression of TNPO3, the recently identified RBM20 nuclear import receptor, in hiPSC-CMs and mice with the P633L variant (P635L in mice) improves nuclear localization of the mutant protein and rescues the splicing of RBM20 target transcripts. 28 Thus, improved function following RBM20 upregulation may be due to partial rescue of splicing defects by increasing the small pool of mutant RBM20 in the nucleus.…”

“…11,12,26 In the years since this discovery, these findings have been confirmed by our laboratory and others in additional rodent models and humaninduced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). 9,10,13-18,27, 28 The finding that RS domain pathogenic variant knock-in animals recapitulate aspects of RBM20 cardiomyopathy not observed in knockout rodents was a significant breakthrough in the field as it not only unambiguously confirmed that these variants are causative in aggressive DCM but also implicated sarcoplasmic RBM20 condensates in the development of this devastating disease. Nevertheless, many questions remain to be answered.…”