Abstract
RBM20 is a major regulator of heart-specific alternative pre-mRNA splicing of TTN encoding a giant sarcomeric protein titin. Mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), yet most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. In the present study, we identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. We demonstrate that the two serine residues in the RSRSP stretch are constitutively phosphorylated and mutations in the stretch disturb nuclear localization of RBM20. Rbm20S637A knock-in mouse mimicking an S635A mutation reported in a familial case showed a remarkable effect on titin isoform expression like in a patient carrying the mutation. These results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20S637A mouse as a good model for in vivo study.
Highlights
We investigated functional alterations caused by the dilated cardiomyopathy (DCM)-associated RNA binding motif protein 20 (RBM20) mutations including three other missense variants that were reported to be found in other DCM patients
When conserved serine residues in the RSRSP stretch (Ser[637] and/or Ser[639], corresponding to Ser[635] and Ser[637], respectively, in human RBM20) were replaced with alanine, the splicing regulation activity was significantly affected (Fig. 1B,C, lanes 3–5). These results indicated that the TtnE50-E51E218-exon 219 (E219)-EGFP/mCherry reporter could recapitulate RBM20-mediated regulation of the Ttn pre-mRNA processing and could be utilized to validate functional alterations caused by mutations in the RBM20 protein
With the tools we developed in this study, we evaluated the functional relevance of mutations in the coding region of RBM20 we found and/or reported in DCM patients[19]
Summary
RBM20 has later been identified as an alternative splicing regulator of a gene responsible for expression of an aberrantly giant isoform of a sarcomeric protein titin in a spontaneously occurring rat strain[6]. Some missense and nonsense mutations outside of the hotspot were identified in such screenings[19,20,23,24], yet neither family study nor functional study was performed and significance of such mutations remains to be elucidated This situation is unusual considering that most of missense mutations were mapped to the RRM domains in our previous genetic screenings for loss- or reduction-of-function mutants for splicing factors[25,26,27]. We investigated functional alterations caused by the DCM-associated RBM20 mutations including three other missense variants that were reported to be found in other DCM patients
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