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Abstract

Dilated cardiomyopathy (DCM) is amongst the most common causes of heart failure. However, targeted therapy approaches to treat DCM patients are still largely absent in clinics. Mutations in the heart-specific alternative splicing (AS) regulator RBM20 are usually associated with a particularly severe form of the disease. All identified pathogenic RBM20 mutations result in loss of RBM20’s splicing function. In addition, mutations in the protein’s RS-rich region cause its cytoplasmic mislocalization which worsens the disease phenotype. To date, it has been unclear why these mutations lead to cytoplasmic mislocalization, and whether relocalizing these variants to the nucleus may be beneficial. In my PhD, I combined the novel image-enabled cell sorting technology (ICS, Schraivogel et al., Science, 2022) with RNA-sequencing, CRISPR screens and other functional studies to decipher the mechanism of pathological RS-domain RBM20 variants.

I found that, unlike other known RS-domain mutations, a newly identified P633L leads to a mixed mislocalization pattern, with a significant fraction of the protein localizing to the nucleus. Using the ICS technology together with RNA-sequencing, I showed that nuclear-localised RBM20-P633L functions similar to the wild type (WT) in iPSC-derived cardiomyocytes (iPSC-CMs), displaying functional AS activity. Forcing nuclear localization of RBM20 harbouring a more severely mislocalizing mutation (R634Q) by fusing it with the SV40 NLS sequence, rescued splicing of RBM20 targets. In addition, protein interactors were mainly unchanged for nuclear-localised RBM20-WT, –P633L, or -R634Q in HeLa cells. These results indicate that RS-domain mutations do not affect RBM20 splicing activity but rather its nuclear import.

To identify factors responsible for RBM20 nuclear import in an unbiased way, I performed two genome-wide CRISPR-based screens with ICS-measured RBM20-WT or -R634Q localization as readouts in HeLa cells. I identified TNPO3 to be the main nuclear importer of RBM20. I showed that loss of TNPO3 resulted in cytoplasmic mislocalization of RBM20-WT protein, similar to what is observed in the presence of RS-domain mutations. Together with Dr. Marta Rodríguez-Martínez, I then used biochemistry and mass spectrometry to show that RBM20 interacts with TNPO3 and that their interaction is disrupted by RS-domain mutations. Notably, since TNPO3 is still able to bind RS-domain mutated RBM20 to some extent, I hypothesised that it may serve as an anchor for developing therapeutic strategies targeted to stabilize this interaction and restore the nuclear import.

To test this hypothesis, I first delivered TNPO3 to iPSC-CMs with RS-domain RBM20 variants, which restored their nuclear localization and proportionally restored splicing of RBM20 targets. Next, I tested AAv9-Tnpo3 delivery in mouse models mimicking patients’ mutations P633L and R634Q. I found that Tnpo3 overexpression in vivo restored alternative splicing of RBM20 targets, proportionally restoring cardiac ejection fraction, without causing systemic changes in gene expression or alternative splicing. These results demonstrate that, enhancing nuclear import of RBM20 variants either by up-regulating TNPO3, or by other means, can serve as a promising therapeutic strategy.

Altogether, I identified the molecular mechanism responsible of RBM20 nuclear transport and deciphered the cause of RBM20 mislocalization in DCM. In addition, I showed that restoring nuclear localization of RBM20 variants presents a viable strategy to rescue both disease phenotypes: dissolve detrimental cytoplasmic granules and restore splicing deficiency.