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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.

Publication Types

["Journal Article", "Research Support, N.I.H., Extramural", "Research Support, Non-U.S. Gov't", "Research Support, U.S. Gov't, Non-P.H.S."]

Keywords

[]

MeSH Terms

["Cardiomyopathies", "Cardiomyopathy, Dilated", "DEAD-box RNA Helicases", "DNA Helicases", "Gain of Function Mutation", "Gene Knockdown Techniques", "Humans", "Induced Pluripotent Stem Cells", "Male", "Mutation", "Mutation, Missense", "Poly-ADP-Ribose Binding Proteins", "Proto-Oncogene Proteins", "RNA Helicases", "RNA Recognition Motif Proteins", "RNA Splicing", "RNA-Binding Proteins", "Ribonucleoproteins"]

Funding

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)

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_1_S10.bam RNA-Seq 309.5 MB
8bp_del_Homo_2_S2.bam RNA-Seq 372.0 MB
8bp_del_Homo_2_S2.bam RNA-Seq 372.0 MB
R636S_Het_1_S4.bam RNA-Seq 309.7 MB
R636S_Het_1_S4.bam RNA-Seq 309.7 MB
R636S_Het_2_S5.bam RNA-Seq 312.0 MB
R636S_Het_2_S5.bam RNA-Seq 312.0 MB
R636S_Het_3_S6.bam RNA-Seq 262.5 MB
R636S_Het_3_S6.bam RNA-Seq 262.5 MB
R636S_Homo_S7.bam RNA-Seq 341.0 MB
R636S_Homo_S7.bam RNA-Seq 341.0 MB
WT_GCaMP_1_S1.bam RNA-Seq 355.1 MB
WT_GCaMP_1_S1.bam RNA-Seq 355.1 MB
WT_GCaMP_2_S3.bam RNA-Seq 304.9 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_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_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_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_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_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_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_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
SB_RBM20_WT2_IN_R_S64_L004_R1_001.fastq.gz OTHER 1.1 GB
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
8 data files
FileTypeSize
SRR14713284.lite RNA-Seq 10.7 GB
WTB_Het_R636-S1-r1_R1.fastq.gz RNA-Seq 4.4 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-S1_r1_R1.fastq.gz RNA-Seq 9.9 GB
WTB_WT-S2_r1_R1.fastq.gz RNA-Seq 9.3 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
42 data files
FileTypeSize
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_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_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-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-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-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-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-S20_r3_R1.fastq.gz RNA-Seq 6.8 GB
WTC_Homo_R636-S23_r3_R1.fastq.gz RNA-Seq 6.0 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-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_r3_R1.fastq.gz RNA-Seq 5.1 GB
WTC_WT-S15_r4_R1.fastq.gz RNA-Seq 13.1 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-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-S21_r3_R1.fastq.gz RNA-Seq 9.6 GB
WTC_WT-S2_r2_R1.fastq.gz RNA-Seq 6.4 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
WTC_WT-S3_r2_R1.fastq.gz RNA-Seq 5.7 GB

Potentially Related Datasets (2)

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.

GSE118347 GSE GEO
GEO

Mutant FUS and ELAVL4 (HuD) aberrant crosstalk in Amyotrophic Lateral Sclerosis

PRJNA579336 PRJNA BioProject

Analysis Pipelines (6)

GSE113947 RNA-seq Data Processing 4 steps
RNA-seq geo_data_processing GSE113947
GSE118347 CLIP-seq Data Processing 10 steps
CLIP-seq geo_data_processing GSE118347
GSE175885 RNA-seq Data Processing 5 steps
RNA-seq geo_data_processing GSE175885
GSE175886 RNA-seq Data Processing 4 steps
RNA-seq geo_data_processing GSE175886
GSE176045 RNA-seq Data Processing 4 steps
RNA-seq geo_data_processing GSE176045
GSE176060 eCLIP Data Processing 3 steps
eCLIP geo_data_processing GSE176060