Abstract 16079: Transcriptome-Wide Identification of Targets and Binding Sites of the Dilated Cardiomyopathy Associated RNA-Binding Motif Protein 20 by PAR-CLIP
Background Mutations in RNA-binding Motif Protein 20 (RBM20) were recently described in families with early onset dilated cardiomyopathy (DCM). In silico predictions indicated that RBM20 is an RNA-binding protein (RBP). Most reported mutations either flank or are located within the predicted RNA recognition motif. To gain insights into the role of RNA-binding in RBM20-related pathogenesis, we identified targets and binding sites, on the transcriptome level, using our recently published PAR-CLIP protocol.
Methods We generated HEK293 cells that constitutively synthesize FLAG/HA-tagged RBM20 wild-type protein. We performed PAR-CLIP which results in the generation of a cDNA library that is subjected to Illumina sequencing. Bioinformatic analyses of this library were performed using a customized annotation pipeline, Gibbs sampling, and R/Bioconductor.
Results RBM20 is localized to the nucleus. Through PAR-CLIP, we obtained 38,563,857 reads, of which 4,659,447 (12 %) were mRNA. Overlapping mRNAs with more than 5 reads and greater than 20 nt in length were clustered, resulting in 80,747 mRNA clusters, of which 15677 (19 %) represented high confidence clusters (HCTs). Sixty percent of binding sites were located within introns, the remainder were located in exons; exonic target sequences were mostly (70%) located within 3’ UTRs, 25% within coding sequences. To identify RNA-binding motifs, we used a Gibbs algorithm, and identified UCUU as the most consistent and highly significant binding motif. It occured at least once in 12235 out of the 15677 clusters (53036 out of all 80,747). Within the HCTs, 5 DCM-causing genes (TMPO, RBM20, EYA4, VCL, TPM1, ACTN2) are targeted by RBM20. With less restrictive parameters, this extended to 23 genes, including those that encode structural proteins such as DSG2, FKTN, or the ion channel RYR2, that are related to distinct types of myopathies, cardiomyopathies, or primary electric disorders.
Conclusions RBM20 is an RBP that targets transcripts from genes that are known to cause DCM. Here we present RBM20 targets and binding sites on a transcriptome-wide level at nucleotide resolution. This study will help us to elucidate RBM20-related pathogenesis and may lead to the identification of additional myopathy-linked genes.
- © 2011 by American Heart Association, Inc.