RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function

Figure 1.. Restricted expression of RNA-binding proteins Rbpms2a (RNA-binding protein with multiple splicing [variants] 2a) and Rbpms2b to the zebrafish myocardium. A, Workflow used to identify 1848 protein-coding RNAs enriched in ZsYellow-positive (ZsY+) cardiopharyngeal progenitors or cardiomyocytes of the anterior lateral plate mesoderm in comparison to ZsYellow-negative (ZsY−) cells from the same 14–16 somites stage (ss) Tg(nkx2.5:ZsYellow) embryos. B–G, Representative brightfield images of 16 ss (B and E), 28 hours postfertilization (hpf; C and F), and 48 hpf (D,G) embryos processed for in situ hybridization with riboprobes that detect rbpms2a (B–D) or rbpms2b (E–G). n=10/group. H–K′, Representative confocal projections of hearts in 24 hpf (H and H′), 33 hpf (I and I′), 48 hpf (J and J′), and 72 hpf (K and K′) zebrafish embryos coimmunostained with antibodies that detect Rbpms2 (magenta) or cardiac troponin T (CT3 antibody; green). Single (H, I, J, and K) and merged double channel (H′, I′, J′, and K′) images are shown. n=10/group. L–L′′′, Single optical section through the ventricular wall of a 48 hpf embryo coimmunostained with antibodies that detect Rbpms2 (magenta) or cardiac…

Figure 2.. rbpms2a and rbpms2b are required redundantly for ventricular function in zebrafish. A and B, Schematic diagrams of the zebrafish Rbpms2a and Rbpms2b proteins (top) and the predicted protein products of the rbpms2achb3 and rbpms2bchb4 mutant alleles (bottom). The asterisks (*) mark the locations of premature stop codons within the RRMs (RNA-recognition motifs) caused by frame-shifting deletions. The white box in (A) shows the location of divergent amino acids before the stop codon. C and D, Representative brightfield images of control-sibling (C; CTRL) and rbpms2-null (rbpms2achb3/chb3; rbpms2bchb4/chb4; D) embryos at 72 hours postfertilization (hpf). Arrowhead highlights pericardial edema in the mutant. n=50/group. Scale bars=250μm. E, Western blot showing Rbpms2 levels in 48 hpf wild-type (WT), CTRL, and rbpms2-null embryos (top) relative to the loading control alpha-tubulin (bottom). F and G, Representative confocal projections of hearts in 48 hpf CTRL (F) and rbpms2-null (G) embryos immunostained with an anti-Rbpms2 antibody (magenta) and counterstained with DAPI (4'‚6-diamidino-2-phenylindole; blue). The dotted lines delineate the myocardium as ascertained from the Rbpms2…

Figure 3.. Identification of differential alternative splicing events (ASEs) in rbpms2-null embryos. A, Schematic diagrams of the categories of ASEs investigated by the RNA-sequencing Multivariate Analysis of Transcript Splicing algorithm including skipped exon (SE), alternative 3′ splice site (A3SS), alternative 5′ splice site (A5SS), mutually exclusive exon (MXE), and retained intron (RI). B, Volcano plot showing the inclusion (Inc) level differences (InclevelCTRL – Inclevelrbpms2-null) and false discovery rates (FDRs) for differential ASEs between 33 hours postfertilization (hpf) control-sibling (CTRL; n=4, 10 pooled embryos per replicate) and rbpms2-null animals (n=5) filtered by 0 uncalled replicates, FDR<0.1, and |Incleveldifference|>0.1. The ASEs shown in bold were validated with splicing-sensitive quantitative PCR (qPCR) or in situ hybridization. C. Pie chart showing the proportions of differential ASEs in each category in rbpms2-null animals. Also shown are the percentages of exons in the skipped exon category that are differentially included or excluded in rbpms2 mutants. D, Bar graph showing the relative total transcript levels in 33 hpf CTRL and rbpms2-null animals that are…

Figure 4.. Myofibrillar disarray and calcium handling defects in rbpms2-null ventricular cardiomyocytes. A and B′, Representative confocal projections of ventricles in 48 hours postfertilization (hpf) control-sibling (CTRL) or rbpms2-null embryos immunostained with an anti-TroponinT antibody (CT3 [cardiac troponin T]) to visualize thin filaments. Regions demarcated in (A) and (B) are enlarged in (A′) and (B′). C and D′′, Confocal projections of ventricles in 48 hpf CTRL and rbpms2-null Tg(α-actn3b-EGFP) animals coimmunostained with antibodies that detect tropomyosin (CH1; green) to visualize thin filaments or GFP (magenta) to visualize Z-disks. E and F, Confocal projections of ventricles in 48 hpf CTRL and rbpms2-null embryos immunostained with an antibody that detects myosin heavy chain (MF20) to visualize thick filaments. For A–F, n=10 animals/group. Scale bars=10 μm. G–N, Color maps (G, H, K, and L) and dot plots (I, J, M, and N) of calcium transient duration (G–J) and calcium release amplitude (K–N) in the ventricle (G–I; K–M) and atrium (G, H, J, K, L, and N) of 48 hours postfertilization control-sibling (CTRL; G, I, J, K, M, and N) and rbpms2-null (H–J; L–N) hearts. Color code depicts…

Figure 5.. Human RBPMS2- (RNA-binding protein with multiple splicing [variants] 2)-null cardiomyocytes exhibit myofibrillar disarray and calcium handling defects similar to those in zebrafish rbpms2-null cardiomyocytes. A, Schematic diagrams of human RBPMS2 (top) and the predicted protein product of the ΔRBPMS2 null allele created with CRISPR/Cas9-mediated genome editing (bottom). The asterisk shows the location of a premature stop codon within the RRM (RNA-recognition motifs) caused by a frame-shifting 2 base pair deletion. The white box shows the location of divergent amino acids before the stop codon. B and C, Bar graphs showing the relative expression levels of RBPMS2 in wild-type control (CTRL) and ΔRBPMS2 human induced pluripotent stem cells (hiPSCs; B) and cardiomyocytes (hiPSC-CMs; C) after 15 days of directed differentiation. n=6 biological replicates/group, each with three technical replicates. Error bars show one standard deviation. Statistical significance was determined by an unpaired, 2-tailed Student's t test assuming equal variances. Significant P values are shown. D–K, Representative confocal projections of CTRL (D–G) and ΔRBPMS2 (H–K) hiPSC-CMs immunostained with…

Figure 6.. Conservation of RBPMS2 (RNA-binding protein with multiple splicing [variants] 2)-regulated splicing events in zebrafish and human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). A, Volcano plot showing the inclusion (Inc) level differences (InclevelCTRL–InclevelΔRBPMS2) and false discovery rates (FDRs) for differential alternative splicing events (ASEs) between wild-type control (CTRL; n=4 from independent differentiations) and ΔRBPMS2 (n=5) filtered by 0 uncalled replicates, FDR<0.05 (capped at 10-16), and |Incleveldifference|>0.1. B, Pie chart showing the proportions of differential ASEs in each category in ΔRBPMS2 hiPSC-CMs. Also shown are the percentages of exons in the skipped exon category that are differentially included or excluded in ΔRBPMS2 hiPSC-CMs. C, Schematic diagram (left, top), table (left, bottom), and stacked bar chart (right) showing gene- and exon-level comparisons of the splicing defects in RBPMS2-deficient zebrafish (GRCz11[genome reference consortium zebrafish build 11]) and hiPSC-CMs (Hg38 [genome reference consortium human build 38]) made by ExOrthist software using the zebrafish ASEs as the query group. Of the 166 differential ASEs, 6…