Mutational mapping of sequence variants within the arginine- and serine-rich domain of RNA binding motif protein 20

Mutations in the cardiac splicing factor RNA binding motif protein 20 (RBM20) are associated with severe forms of dilated cardiomyopathy. Mutations predominate within the highly conserved RSRSP-stretch and are characterized by a cytoplasmic mislocalization of RBM20. However, a sequence downstream of the RSRSP-stretch, the SRSLSP-stretch, is also phylogenetically highly conserved. The impact of the SRSLSP-stretch is unclear. In our study we investigated subcellular localization of RBM20 variants in HEK293 cells after transient transfection. We showed that variants within the RSRSP-stretch lead to significant cytoplasmic mislocalization. Aberrant TTN-splicing was observed in the explanted myocardium of an RBM20-p.R634W carrier. Within the SRSLSP-stretch, variants at amino acid position 641 are associated with partial mislocalization of RBM20. Molecular modelling of RBM20 and transportin-3 interaction by AlphaFold-3 suggests that the RSRSP stretch and arginine at position 641 are involved in transportin-3 interaction. In summary, our study supports the functional relevance of the RSRSP-stretch for the RBM20-TNPO3 interaction, whereas variants within the adjacent SRSLSP stretch have a minor impact on the nuclear localization. Furthermore, the data underscore the limitations of in silico-based pathogenicity predictions of RBM20 variants.