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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.
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Research Article Cardiology

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

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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

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Figure 1

PAR-CLIP in HEK293 cells identifies RBM20 RRE.

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PAR-CLIP in HEK293 cells identifies RBM20 RRE.
(A) Transcriptome-wide di...
(A) Transcriptome-wide distribution of RBM20 PAR-CLIP consensus library clusters. The ratio of PAR-CLIP to control HEK293 RNA-seq percentage coverage in each region is displayed. (B) Binding motif enrichment in RBM20 PAR-CLIP libraries. The log2 enrichments of 5-mers in cluster-centered regions of the 4SU and 6SG libraries are correlated. UCUU core–containing 5-mers were most abundant. (C) Sequence logo for the RBM20 RRE was computed from the top 1000 intronic binding sites of the consensus library using MEME. (D) Positional transition frequency for intronic PAR-CLIP clusters anchored at the UCUU core of the RBM20 RRE (black line) and control CCRs randomly placed in the same intron (blue line). (E) Phosphorimage of native PAGE resolving complexes of immunoprecipitated full-length RBM20 protein with WT and mutated Ryr2 target RNA oligonucleotides.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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