Mutational mapping of sequence variants within the arginine- and serine-rich (RS) domain of RBM20

Abstract Background/Purpose Mutations in the cardiac splicing factor RBM20, are associated with severe and early onset forms of dilated cardiomyopathy (DCM). DCM-associated mutations predominate in the RS domain within a RSRSP sequence motif (aa 634-638). This sequence motif serves as a nuclear localization signal, which is recognized by the nuclear import receptor transportin 3. Consequently, mutations within the RSRSP stretch are characterized by mislocalization of RBM20 to the cytoplasm and formation of ribonucleoprotein granules. The RSRSP motif is highly conserved across different species. However, the conservation of this motif is not exclusive, as a sequence downstream of the RSRSP stretch (aa 640-645, SRSLSP-Stretch) within the RS-domain is also highly conserved. To date, the impact of the SRSLSP-Stretch on RBM20-localization and RBM20-dependent splicing is not clear. Therefore, we performed mutational mapping at codons 634, 635, 636, 640, 642, 644 and 645, to gain a more profound understanding of the function of the RS domain. Methods The subcellular localization of RBM20 variants was analyzed in HEK293 cells after transient transfection with RBM20-pEYFP plasmids with confocal microscopy. Splicing analyses of RBM20 target genes TTN and RYR2 were conducted using human myocardium from patients carrying the variants p.R634W or p.R641W, using quantitative real-time PCR. Additionally, the subcellular localization of RBM20 in patient myocardium was investigated using paraffin-embedded tissue sections. Results As anticipated, variants within the RSRSP stretch exhibited a cytoplasmic localization of RBM20 (e.g., p.R634W). For RBM20-p.R634W an aberrant splicing of TTN, comparable to other pathogenic RBM20-variants, was observed in patient myocardium. In contrast, variants within the SRSLSP-stretch exhibited predominantly a nuclear localization. Interestingly, different amino acid substitutions at position p.R641 led to partial mislocalization of RBM20 and formation of RNP granules (e.g. p.R641W). Of note, despite the partial RBM20-mislocalization no missplicing of the RBM20 target genes TTN and RYR2 was detected in human myocardium with the p.R641W variant. Conclusion Pathogenic RBM20 variants correlate with clinically aggressive forms of DCM. Therefore, the classification of variants according to the ACMG criteria and the understanding of the pathomechanisms is of significant consequence. Notably, in silico predictions, such as the AlphaMissense score, can be utilized as a PP3 criteria for ACMG classification. Nevertheless, the findings of our study indicate that in silico predictions are still not reliable to predict the molecular phenotype of the mutation, thereby underscoring the significance of functional data for variant classification. Additionally, we show that although the RSRSP stretch, as well as the SRSLSP-stretch, are highly conserved within the RS-domain they differ in their impact on subcellular localization and splicing function of RBM20.