Fig 2: slm2 knockdown results in heart failure in zebrafishA. Conservation of the SLM2 proteins in humans, rats, and zebrafish. The KH domain which mediates RNA interaction and the tyrosine (Y) residue which mediates splicing are labeled. B. PCR analysis of zebrafish injected with splice morpholino targeting slm2 mRNA (MO-slm2) or control MO (MO-Ctrl) at 1–2-cell-stage embryos. The 320-bp product indicates the wild-type slm2 and the 120-bp product indicates loss of function by exon skipping. C. The effectiveness of MO-slm2 validated by Sanger sequencing of the PCR products shown in (B). D. Representative images of zebrafish injected with MO-slm2 and MO-Ctrl, respectively. Scale bar, 500 µm. E. Magnification of the hearts of zebrafish shown in (D). F. Quantitative analysis of the phenotypes of zebrafish after MO-slm2 or MO-Ctrl injection. G. Ventricular fractional shortening of zebrafish after MO-slm2 or MO-Ctrl injection. H. Atrial fractional shortening of zebrafish after MO-slm2 or MO-Ctrl injection. I. Heart rate of zebrafish after MO-slm2 or MO-Ctrl injection. *, P < 0.05 (Student’s t-test). A, atrium; V, ventricle; HF, heart failure; WT, wild-type.

Fig 3: SLM2 binding to RNAs in the failing human heartA. Circos plot showing 172 SLM2-bound RNAs which exhibit significant changes in alternative splicing (indicated by PSI). SLM2 was immunoprecipitated from myocardial tissue lysates of the left ventricle from DCM patients (n = 3, orange) and controls (n = 4, blue), respectively, and bound RNAs were deep sequenced. Chr. 5 and Chr. Y were excluded due to lack of significant associations. B. GO analysis of the disease relevance of the 172 SLM2-bound RNAs showing differential splicing between DCM and control hearts (P < 0.05 indicating false discovery rate by Benjamini). C. Enrichment of alternatively spliced RNAs by SLM2 RIP from DCM hearts followed by sequencing. Data are presented as mean ± SD (n = 3 DCM samples). D. Alternative splicing of RNAs bound to SLM2 in hearts of DCM patients (n = 3) compared to controls (n = 4). Student’s t-test (*, P < 0.05). E. Total gene expression of the selected SLM2-bound RNA candidates in the hearts of DCM patients (n = 33) and controls (n = 24). Student’s t-test. PSI, percent spliced in; GO, Gene Ontology; RIP, RNA immunoprecipitation.

Fig 4: SLM2 promotes intron retention and prevents exon exclusion in TTN mRNA opposing the role of RBM20A. Correlation coefficients of TTN exon expression to RNA expression of RBM20 (blue) and SLM2 (yellow) in LV biopsies analyzed by RNA-seq in DCM patients (n = 56). B. Binding of SLM2 to TTN exons 131–332 verified by RIP followed by RT-PCR. n = 3. C. RT-PCR showing TTN mRNA splicing after transfection of the TTN131–133 minigene along with SLM2 or RBM20. D. Quantification of TTN mRNA splice ratios as shown in (C). n = 3. E. RT-PCR showing TTN mRNA splicing after transfection of the TTN241–243 minigene along with SLM2 or RBM20. F. Quantification of TTN mRNA splice ratios as shown in (E). n = 3. G. RT-PCR showing TTN mRNA splicing after transfection of the TTN241–243 minigene along with increasing RBM20 and decreasing SLM2. *, P < 0.05 (Student’s t-test). LV, left-ventricular.

Fig 5: Regulation and enrichment of mRBPs in failing human heartsA. Regulation of 421 mRPB-coding genes in the heart of patients diagnosed with DCM (n = 33) compared to controls (n = 24) (Student’s t-test). Size of the bubble indicates a HSS (arbitrary unit) in 53 different human tissues from a total of 11,467 human tissue samples. B.SLM2 mRNA expression levels in heart tissues of patients (DCM and ICM; each n = 3) and controls (n = 5). The SLM2 mRNA expression levels were normalized to the mRNA expression level of RPLP0, which served as the reference gene. C. Representative IB image depicting the protein expression levels of SLM2 in heart tissues of DCM patients and controls. a-actin served as a loading control. D. Densitometric quantification of SLM2 protein expression in heart tissues of DCM patients (n = 4) and controls (n = 3). a-actin served as a loading control and was used for normalization. *, P < 0.05 (Student’s t-test). mRPB, mRNA-binding protein; DCM, dilated cardiomyopathy; ICM, ischemic cardiomyopathy; HSS, heart-specificity score; SLM2, Sam68-like mammalian protein 2; IB, immunoblot.