Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization

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.

Structural basis of UCUU RNA motif recognition by splicing factor RBM20

“…The C-terminal zinc finger is essential for RBM20 function , however so far neither of the two zinc fingers have been shown to bind RNA. The RS domain is required for nuclear localization (Murayama et al, 2018) and likely mediates protein-protein interactions (Maatz et al, 2014). We have shown that inclusion of the unphosphorylated RS domain does not directly affect affinity to AUCUUA RNA ( Table 1).…”

“…Most of the RBM20 mutations implicated in cardiomyopathy are localized within the RS region, and these mutants mainly disrupt nuclear localization (Murayama et al, 2018). To see if the addition of the RS region could in general affect RNA binding, we prepared a construct with the C-terminus extended to residue 649.…”

“…Nevertheless, few RBM20 disease mutations have so far been found within the RRM domain: there is a single example of a sporadic V535I mutation (V537I in mice) that has only limited effect on RNA binding (Table 1), and the proximal I536T mutation that may be secondary to mRNA processing defects in LDB3 (Yamamoto et al, 2019). In contrast, the RS domain remains the hotspot of RBM20 disease mutations, and mutations such as S637A can act as a dominant negative toward titin splicing (Murayama et al, 2018;Kimura, 2016). It may be that functional mutants of RBM20 that can still bind to native pre-mRNA binding sites are more deleterious that RBM20 mutant proteins that simply affect RNA-binding specificity.…”

“…Although not corresponding to a folded domain, a region Cterminal to the RRM domain is enriched in arginine and serine residues (RS), and most of the disease mutations map to this small segment (Brauch et al, 2009) (Li et al, 2010;Watanabe et al, 2018). Improper phosphorylation of key serine residues in this RS domain prevents proper nuclear localization of RBM20 mutants (Murayama et al, 2018).…”

RBM20 mutations in left ventricular non‐compaction cardiomyopathy

“…[22][23][24] Loss of RBM20 function leads to mis-splicing of target genes in humans and rodent models. [22][23][24][25] RBM20 is composed of two zink finger domains, one RNA-recognition motif (RRM)-type RNA binding domain and an arginine-/serine-(RS)-rich region. In 2019, two mutational hot spot regions in exons 9 and 11 of RBM20 have been recognized.…”