RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing
Henrike Maatz, … , Markus Landthaler, Norbert Hubner
Henrike Maatz, … , Markus Landthaler, Norbert Hubner
Published June 24, 2014
Citation Information: J Clin Invest. 2014;124(8):3419-3430. https://doi.org/10.1172/JCI74523.
View: Text | PDF
Research Article Cardiology

RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing

Abstract

Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3′ and 5′ splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure.

Authors

Henrike Maatz, Marvin Jens, Martin Liss, Sebastian Schafer, Matthias Heinig, Marieluise Kirchner, Eleonora Adami, Carola Rintisch, Vita Dauksaite, Michael H. Radke, Matthias Selbach, Paul J.R. Barton, Stuart A. Cook, Nikolaus Rajewsky, Michael Gotthardt, Markus Landthaler, Norbert Hubner

×

Figure 3

RBM20 binds to intronic splicing silencers upstream and downstream of repressed exons.

Options: View larger image (or click on image) Download as PowerPoint
RBM20 binds to intronic splicing silencers upstream and downstream of re...
(A–C) ΔPSIs are shown as black lines (WT compared with homozygous rats). Elevated versus decreased ΔPSIs show inclusion or exclusion of exons, respectively. Red ticks indicate mapped RBM20 cluster positions. (A) Ttn is predominantly spliced in the elastic region. Mapped RBM20-binding sites coincide with differential splicing. (B) Camk2d exons 14–16 are mutually exclusive and regulated by RBM20. Mapped RBM20-binding sites are located upstream of exon 14 and downstream of exon 15. (C) Ldb3 undergoes an exon switch affecting exon 4 versus exons 5–7. Mapped binding sites are upstream of exon 5 and downstream of exon 8. (D) Mean CLIP density near RBM20 activated (red) and repressed exons (blue). Dotted line indicates density around exons not regulated by RBM20. Gray shows 90% CIs. (E) Right panel: specific activity of RBM20 on Ttn RNA from the PEVK region in a splice-reporter assay. Rbm20 expression leads to exclusion of the firefly luciferase–containing (Fluc) exon. Ratio of Fluc to Renilla luciferase (Rluc) activity (downstream exon) reflects splice activity. Reporter activity depends on mutation of 2 UCUU elements in cluster CID016226, but not outside the cluster. Mutations are indicated as X. White boxes only show native reporter. Controls: PEVK-construct cotransfected with empty vector (–), Rbm20 cotransfected with a Ttn M-band region construct not affected by RBM20 (M). ***P < 0.001 compared with native reporter. Left: EMSA evaluating RBM20 binding to Ttn PEVK-derived RNAs. White arrows indicate input RNA; black arrows indicate RNA-protein complex.