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Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies.

Aidan M Fenix, Yuichiro Miyaoka, Alessandro Bertero, Steven M Blue, Matthew J Spindler, Kenneth K B Tan, Juan A Perez-Bermejo, Amanda H Chan, Steven J Mayerl, Trieu D Nguyen, Caitlin R Russell, Paweena P Lizarraga, Annie Truong, Po-Lin So, Aishwarya Kulkarni, Kashish Chetal, Shashank Sathe, Nathan J Sniadecki, Gene W Yeo, Charles E Murry, Bruce R Conklin, Nathan Salomonis

Nature communications · 2021 · Vol. 12 (1) · pp. 6324

PubMed ↗ DOI ↗ PMC full text ↗

Abstract

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

Linked datasets 5

GSE175885 GSE · via ncbi_elink
GEO ↗

RNA-Seq of isogenic human iPS cell-derived cardiomyocytes with RBM20 mutations created by genome editing (RiboMinus RNA-Seq)

Homo sapiens

16 data files
FileTypeSize
8bp_del_Homo_1_S10.bam RNA-Seq 309.5 MB
8bp_del_Homo_2_S2.bam RNA-Seq 372.0 MB
R636S_Het_1_S4.bam RNA-Seq 309.7 MB
R636S_Het_2_S5.bam RNA-Seq 312.0 MB
R636S_Het_3_S6.bam RNA-Seq 262.5 MB
R636S_Homo_S7.bam RNA-Seq 341.0 MB
SRR14699656_GSM5350776 RNA-Seq 355.0 MB
SRR14699657_GSM5350777 RNA-Seq 304.0 MB
SRR14699658_GSM5350778 RNA-Seq 309.0 MB
SRR14699659_GSM5350779 RNA-Seq 312.0 MB
SRR14699660_GSM5350780 RNA-Seq 262.0 MB
SRR14699661_GSM5350781 RNA-Seq 341.0 MB
SRR14699662_GSM5350782 RNA-Seq 309.0 MB
SRR14699663_GSM5350783 RNA-Seq 372.0 MB
WT_GCaMP_1_S1.bam RNA-Seq 355.1 MB
WT_GCaMP_2_S3.bam RNA-Seq 304.9 MB
GSE175886 GSE · via ncbi_elink
GEO ↗

RNA-Seq of isogenic human iPS cell-derived cardiomyocytes with RBM20 mutations created by genome editing

Homo sapiens

GSE176060 GSE · via ncbi_elink
GEO ↗

RNA-Seq of isogenic human iPS cell-derived cardiomyocytes with RBM20 mutations created by genome editing (eCLIP)

Homo sapiens

16 data files
FileTypeSize
SB_RBM20_Homo1_CLIP_R_S53_L003_R1_001.fastq.gz OTHER 955.4 MB
SB_RBM20_Homo1_IN_R_S52_L003_R1_001.fastq.gz OTHER 1008.4 MB
SB_RBM20_Homo2_CLIP_R_S67_L004_R1_001.fastq.gz OTHER 969.1 MB
SB_RBM20_Homo2_IN_R_S66_L004_R1_001.fastq.gz OTHER 1009.9 MB
SB_RBM20_WT1_CLIP_R_S51_L003_R1_001.fastq.gz OTHER 1.0 GB
SB_RBM20_WT1_IN_R_S50_L003_R1_001.fastq.gz OTHER 1.0 GB
SB_RBM20_WT2_CLIP_R_S65_L004_R1_001.fastq.gz OTHER 1.0 GB
SB_RBM20_WT2_IN_R_S64_L004_R1_001.fastq.gz OTHER 1.1 GB
SRR14724399_GSM5354304 OTHER 1.0 GB
SRR14724400_GSM5354305 OTHER 1.0 GB
SRR14724401_GSM5354306 OTHER 1.1 GB
SRR14724402_GSM5354307 OTHER 1.0 GB
SRR14724403_GSM5354308 OTHER 1008.0 MB
SRR14724404_GSM5354309 OTHER 955.0 MB
SRR14724405_GSM5354310 OTHER 1009.0 MB
SRR14724406_GSM5354311 OTHER 969.0 MB
GSE176045 GSE · via ncbi_elink
GEO ↗

RNA-Seq of isogenic human iPS cell-derived cardiomyocytes with RBM20 mutations created by genome editing (WTB RNA-Seq)

Homo sapiens

9 data files
FileTypeSize
SRR14713281_GSM5353720 RNA-Seq 9.9 GB
SRR14713282_GSM5353721 RNA-Seq 9.3 GB
SRR14713283_GSM5353722 RNA-Seq 4.4 GB
SRR14713284_GSM5353723 RNA-Seq 10.7 GB
SRR14713284.lite RNA-Seq 10.7 GB
WTB_Het_R636-S1-r1_R1.fastq.gz RNA-Seq 4.4 GB
WTB_Het_R636-S2_r1_R1.fastq.gz RNA-Seq 10.7 GB
WTB_WT-S1_r1_R1.fastq.gz RNA-Seq 9.9 GB
WTB_WT-S2_r1_R1.fastq.gz RNA-Seq 9.3 GB
GSE113947 GSE · via ncbi_elink
GEO ↗

RNA-Seq of isogenic human iPS cell-derived cardiomyocytes with RBM20 mutations created by genome editing [RNA-Seq]

Homo sapiens

48 data files
FileTypeSize
SRR7092055_GSM3124390 RNA-Seq 12.0 GB
SRR7092056_GSM3124391 RNA-Seq 6.8 GB
SRR7092057_GSM3124392 RNA-Seq 6.8 GB
SRR7092058_GSM3124393 RNA-Seq 14.6 GB
SRR7092059_GSM3124394 RNA-Seq 7.8 GB
SRR7092060_GSM3124395 RNA-Seq 10.5 GB
SRR7092061_GSM3124396 RNA-Seq 9.3 GB
SRR7092062_GSM3124397 RNA-Seq 3.5 GB
SRR7092063_GSM3124398 RNA-Seq 6.0 GB
SRR7092064_GSM3124399 RNA-Seq 7.8 GB
SRR7092065_GSM3124400 RNA-Seq 5.7 GB
SRR7092066_GSM3124401 RNA-Seq 6.8 GB
SRR7092067_GSM3124402 RNA-Seq 6.0 GB
SRR7092068_GSM3124403 RNA-Seq 7.4 GB
SRR7092069_GSM3124404 RNA-Seq 6.4 GB
SRR7092070_GSM3124405 RNA-Seq 5.7 GB
SRR7092071_GSM3124406 RNA-Seq 11.3 GB
SRR7092072_GSM3124407 RNA-Seq 5.1 GB
SRR7092073_GSM3124408 RNA-Seq 13.1 GB
SRR7092074_GSM3124409 RNA-Seq 4.8 GB
SRR7092075_GSM3124410 RNA-Seq 9.6 GB
WTC_638_Del_8_1-S6_r4_R1.fastq.gz RNA-Seq 12.0 GB
WTC_638_Del_8_1-S6_r4_R2.fastq.gz RNA-Seq 12.0 GB
WTC_638_Del_8_2-S7_r4_R1.fastq.gz RNA-Seq 6.8 GB
WTC_638_Del_8_3-S8_r4_R1.fastq.gz RNA-Seq 6.8 GB
WTC_638_Del_8_3-S8_r4_R1.fastq.gz RNA-Seq 6.8 GB
WTC_638_Del_8_4_S9_r4_R1.fastq.gz RNA-Seq 14.6 GB
WTC_Het_R636-S16_r3_R1.fastq.gz RNA-Seq 3.5 GB
WTC_Het_R636-S19_r3_R1.fastq.gz RNA-Seq 6.0 GB
WTC_Het_R636-S1_r2_R1.fastq.gz RNA-Seq 7.8 GB
WTC_Het_R636-S1_r2_R1.fastq.gz RNA-Seq 7.8 GB
WTC_Het_R636-S22_r3_R1.fastq.gz RNA-Seq 7.8 GB
WTC_Het_R636-S2_r2_R1.fastq.gz RNA-Seq 10.5 GB
WTC_Het_R636-S3_r2_R1.fastq.gz RNA-Seq 9.3 GB
WTC_Het_R636-S3_r2_R1.fastq.gz RNA-Seq 9.3 GB
WTC_Homo_R636-S17_r3_R1.fastq.gz RNA-Seq 5.7 GB
WTC_Homo_R636-S17_r3_R1.fastq.gz RNA-Seq 5.7 GB
WTC_Homo_R636-S20_r3_R1.fastq.gz RNA-Seq 6.8 GB
WTC_Homo_R636-S23_r3_R1.fastq.gz RNA-Seq 6.0 GB
WTC_WT-S14_r4_R1.fastq.gz RNA-Seq 11.3 GB
WTC_WT-S15_r3_R1.fastq.gz RNA-Seq 5.1 GB
WTC_WT-S15_r4_R1.fastq.gz RNA-Seq 13.1 GB
WTC_WT-S18_r3_R1.fastq.gz RNA-Seq 4.8 GB
WTC_WT-S1_r2_R1.fastq.gz RNA-Seq 7.4 GB
WTC_WT-S1_r2_R1.fastq.gz RNA-Seq 7.4 GB
WTC_WT-S21_r3_R1.fastq.gz RNA-Seq 9.6 GB
WTC_WT-S2_r2_R1.fastq.gz RNA-Seq 6.4 GB
WTC_WT-S3_r2_R1.fastq.gz RNA-Seq 5.7 GB

Potentially related datasets 3

These accessions were text-mined from the PMC full text. They may be referenced for comparison, cited from other studies, or otherwise mentioned without being primary data for this paper.

Accession Source Title
GSE118347 GSE · GEO GEO ↗ Mutant FUS and ELAVL4 (HuD) aberrant crosstalk in Amyotrophic Lateral Sclerosis
PRJNA579336 PRJNA · BioProject No title in the index
GSE175886 GSE · GEO GEO ↗ RNA-Seq of isogenic human iPS cell-derived cardiomyocytes with RBM20 mutations created by genome editing

Analysis pipelines 6

Pipeline Assay Steps Dataset
GSE113947 RNA-seq Data Processing geo_data_processing RNA-seq 4 GSE113947
GSE118347 CLIP-seq Data Processing geo_data_processing CLIP-seq 10 GSE118347
GSE175885 RNA-seq Data Processing geo_data_processing RNA-seq 5 GSE175885
GSE175886 RNA-seq Data Processing geo_data_processing RNA-seq 4 GSE175886
GSE176045 RNA-seq Data Processing geo_data_processing RNA-seq 4 GSE176045
GSE176060 eCLIP Data Processing geo_data_processing eCLIP 3 GSE176060
Funding 14 grants
Grant Funder
R01 HG004659 NHGRI NIH HHS (United States)
P01 HL089707 NHLBI NIH HHS (United States)
U01 HL099997 NHLBI NIH HHS (United States)
U01 ES032673 NIEHS NIH HHS (United States)
R01 HL128368 NHLBI NIH HHS (United States)
U54 DK107979 NIDDK NIH HHS (United States)
R01 HL141570 NHLBI NIH HHS (United States)
R01 CA226802 NCI NIH HHS (United States)
F32 HL156361 NHLBI NIH HHS (United States)
R01 HL128362 NHLBI NIH HHS (United States)
R01 HL149734 NHLBI NIH HHS (United States)
R01 HL146868 NHLBI NIH HHS (United States)
U54 HG007005 NHGRI NIH HHS (United States)
R01 HL130533 NHLBI NIH HHS (United States)