An RBM20 E-rich domain mutation induces atypical Ttn splicing alterations despite preserved nuclear localization

RBM20-related cardiomyopathy has been defined by mutations largely within the RSRSP stretch, impairing nuclear localization of RNA-binding motif protein 20 (RBM20) leading to aberrant cytoplasmic granule formation. However, some disease-associated mutations have also been identified in its E-rich region in unrelated families with dilated cardiomyopathy (DCM), suggesting a distinct pathogenic mechanism. In this study, we generated an Rbm20V894A knock-in mouse model to examine the effects of an E-rich region mutation on RBM20-dependent splicing regulation and cardiac functions. Endogenous RBM20V894A was phosphorylated on the RSRSP stretch and predominantly localized to nuclear speckles in cardiomyocytes. Nevertheless, splicing abnormalities were observed in established RBM20 target genes, Camk2d, Ldb3, and Ttn. Long-read amplicon sequencing of the Ttn transcripts across the RBM20-regulated region revealed loss of the major wild-type N2BA isoform together with emergence of atypical isoforms lacking the N2A region in the heart of Rbm20V894A homozygotes. Despite these molecular abnormalities, Rbm20V894A/V894A mice did not develop an apparent DCM-like phenotype under standard conditions. These findings indicate that the myopathy-related missense mutation in the E-rich domain in RBM20 caused unique splicing abnormalities, which may not be sufficient to cause cardiomyopathies in the mouse model.