Molecular basis of titin exon exclusion by RBM20 and the novel titin splice regulator PTB4

Abstract

RNA-binding motif protein 20 (RBM20) is a cardiac splice regulator that adapts cardiac filling via its diverse substrates—including the sarcomeric protein titin. The molecular basis and regulation of RBM20-dependent exon exclusion are largely unknown. In tissue culture experiments, we show that the combination of RNA recognition motif (RRM) and C-terminus is necessary and sufficient for RBM20 activity, indicating an important function of the ZnF2 domain in splicing repression. Using splice reporter and in vitro binding assays targeting titin exons 241–243, we identified a minimal genomic segment that is necessary for RBM20-mediated splicing repression of the alternative exon. Here, RBM20 binds the cluster containing most RBM20 binding motifs through its RRM domain and represses the upstream and downstream introns. For subsequent exon exclusion, specific regions upstream, downstream and within the alternative exon 242 are required. Regulation of exon exclusion involves PTB4 as a novel titin splice regulator, which counteracts RBM20 repressor activity in HEK293 cells. Together, these mechanistic insights into the regulation and action of RBM20 and PTB4 provide a basis for the future development of RBM20 modulators that adapt titin elasticity in cardiac disease.

Compliant Titin Isoform Content Is Reduced in Left Ventricles of Sedentary Versus Active Rats

“…An increased RBM20 content is consistent with a reduced N2BA:N2B ratio, as additional exons of titin mRNA are spliced out to form the shorter, stiffer, N2B protein isoform (Guo et al, 2012(Guo et al, , 2013Methawasin et al, 2016;. RBM20's activity may be inhibited by the Polypyrimidine Tract Binding protein 4 (PTB4) (Dauksaite and Gotthardt, 2018). Thus, multiple mechanisms to modify titin isoform content have been proposed.…”

The Emerging Role of the RBM20 and PTBP1 Ribonucleoproteins in Heart Development and Cardiovascular Diseases

“…The balance between splicing activation and repression mediated by splicing factors, as well as the combinatorial effect of RNA-binding competition or promotion are expected to play a crucial role in directing regulated alternative splicing events in tissues differentiation. A study that investigated the molecular bases of TTN exon exclusion regulated by RBM20, using a splicing reporter and in vitro binding assay, demonstrated that the PTBP1 isoform PTB4 regulates titin splicing [31]. PTB4 may counteract the RBM20 splicing repressor activity, binding the same consensus motif on the 5 splice site (5 SS), located downstream of the TTN 242 alternative exon (Figure 2c).…”

“…PTB4 may counteract the RBM20 splicing repressor activity, binding the same consensus motif on the 5 splice site (5 SS), located downstream of the TTN 242 alternative exon (Figure 2c). Binding of both PTB4 and RBM20 to the downstream intron may differentially interfere with U1 snRNP, favoring the inclusion of the alternative exon [31].…”

“…Remarkably, RBM20 regulates alternative splicing of titin (TTN), one of the major disease-causing genes in cardiac muscle. Mutations or altered expression of RBM20 can lead to the shift in the expression pattern of titin transcript variants, which are associated with cardiac diseases, including DCM [31].…”

“…Based on recent studies, novel evidences have highlighted the role of the well-known splicing regulator polypyrimidine-tract binding protein 1 (PTBP1) in the regulation of alternatively spliced variants that are critical for the heart functionality [31,32]. In this review, we summarize the functional evidences of the role of RBM20 in the regulation of TTN and additional genes involved in heart function and cardiac diseases development.…”