Inactivation of SRSF2 in MyoD+ progenitors resulted in skeletal muscle dysfunction and respiratory failure.

(A) Macroscopic appearance of WT and SRSF2-KO pups immediately after birth (P1). Scale bars, 5 mm. (B) Comparison of body weight between WT and SRSF2-KO pups (n=10). (C) Survival curve of WT and SRSF2-KO pups (n=10). (D) Sinking test for lungs excised from P1 WT and SRSF2-KO mice and placed in a 5 ml Ep tube. (E) Representative HE staining of lung sections at P1 (n=3). Scale bars, 50 μm. (F) Representative HE staining on longitudinal sections of hindlimbs (top panels), and transverse tongue sections at P1 (bottom panels) (n=3). Scale bars, 10 μm. (G) Representative HE staining of transverse-sections of diaphragms at P1 (n=3). Scale bars, 50 μm. Comparison of diaphragm thickness is displayed on the right. (H) Representative confocal images of desmin staining in transverse-sections and longitudinal sections of hindlimbs from WT and SRSF2-KO at P1 (n=3). Scale bars, 10 μm. (I) Representative confocal images of Myh3 staining in intercostal muscle sections of WT and SRSF2-KO at P1 (n=3). Scale bars, 10 μm. Enlarged views shown on the upper right corners.

RNA-seq analysis and disrupted sarcomere structures in the KO skeletal muscle.

(A) RNAs-seq was conducted on P1 diaphragm samples. Based on statistical significance (P value <0.05) and fold change, over 160 upregulated genes and 244 downregulated genes were identified. (B) Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) was performed, focusing on their cellular component and molecular function categories. (C) qPCR analysis on a selection of genes based on RNA-seq data, using P1 diaphragm samples (n=6). (D) Representative confocal images of phalloidin staining in longitudinal sections of P1 diaphragm muscle of WT and SRSF2-KO mice. Scale bars, 10 μm. (E) Transmission electron microscopy images of the diaphragm in newborn WT and SRSF2-KO mice. Sarcomere is indicated by the green box, Z-disc by the blue arrow, and Triad by the red arrow. Scale bars, 20 μm.

SRSF2-deficinent MyoD-derived cells migrated into non-skeletal muscle regions and displayed significant differentiation defects.

(A) A schematic diagram of SRSF2-KO/tdT mice (SRSF2f/f;MyoDCre;tdT) was generated through the breeding SRSF2f/f mice with MyoDCre mice and Rosa26-tdTomato mice. SRSF2f/w;MyoDcre;tdT mice served as the control group. (B) Representative confocal images of tdT (red) staining on sagittal sections from E14.5 control and KO/tdT embryos. Scale bar is 500 μm. The red boxes indicated the enlarged images on the right, with a scale bar of 100 μm. (C) Representative confocal images of MHC (green) staining on sagittal sections from E14.5 control and KO/tdT embryos. Scale bars, 500 μm. White arrows indicated non-skeletal muscle regions. The enlarged images were shown on the right, with a scale bar of 100 μm. (D) Representative confocal images of tdT (red) staining in hindlimb sections from E15.5 mice. Scale bars, 100 μm. White arrows indicated non-skeletal muscle regions. (E) Representative confocal images of tdT (red) staining in hindlimb sections from P1 mice. Scale bars, 100 μm. White arrows indicated non-skeletal muscle regions. (F) Representative confocal images of tdT staining in transverse sections of intercostal muscle isolated from P1 mice. Scale bars, 200μm. Enlarged images were shown on the left with a scale bar of 50μm. (G) Representative confocal images of tdT (red) and MHC staining on longitudinal diaphragm sections from P1 mice. Scale bars, 20μm. White arrows indicate undifferentiated cells or tdT-labeled cells without MHC staining. (H) Representative confocal images of tdT (red) and Laminin (green) immunostaining in transverse diaphragm sections from P1 mice. Scale bars, 20μm. (I) Representative confocal images of tdT (red) and Laminin (green) immunostaining in hindlimb sections from P1 mice. Scale bars, 50μm. White arrows highlight cavities within tdT/Lamin labeled cells.

snRNA-seq analysis identifies distinct cell populations within P1 diaphragms.

(A) Diagram of single-cell preparation from P1 diaphragms and processing using the 10X Genomics platform for single-cell RNA-sequencing analysis. (B) t-SNE plot showing 8 distinct cell populations identified in P1 WT and SRSF2-KO groups. Note that clusters expressing same marker genes were grouped into the same population. (C) t-SNE plot showing the expression of representative marker genes within distinct cell populations. (D)Heatmap showing the top 10 most differently expressed genes among 8 distinct cell populations. (E) Proportion comparison of cell populations between WT and SRSF2-KO groups. (F) The levels of SRSF2 are reduced in the SRSF2-KO skeletal muscle (SKM) cells compared to WT cells

Loss of SRSF2 results in precocious differentiation of myoblasts.

(A) The t-SNE plot reveals six distinct subclusters from WT and SRSF2-KO SKM cells. (B) Violin plot showing expression levels of the marker genes in the subclusters. (C) Pseudotime-ordered analysis of SKM subclusters, shown as arrangement of subclusters along the two branches of the pseudotime (top), and their pseudotime order (bottom). (D) Heatmap showing expression dynamics of marker genes along the pseudotime branches. Representative marker genes in the four expression modules along the pseudotime shown on the left to the heatmap. (E) Separate t-SNE subcluster projections of WT (top) and SRSF2-KO (bottom) SKM cells. (F) Proportion comparison of WT and SRSF2-KO SKM subclusters. (G) Plots showing the relative levels of marker genes of WT (left) and SRSF2-KO (right) along the differentiation and quiescence branches of the pseudotime, respectively.

Decreased levels of ECM and cytoskeleton, along with mitosis abnormalities and differentiation defects, were observed in the KO group.

(A) GO analysis of DEGs within four subclusters of SKM cells. (B) Violin plots showing relative expression levels of ECM components between WT and KO subclusters. (C) Violin plots showing relative expression levels of cytoskeleton-related genes between WT and KO subclusters. (D) Violin plots showing relative expression levels of mitosis-related genes between WT and KO sC2 cells. (E) Violin plots showing relative expression levels of differentiation-related genes between WT and KO subclusters.

SRSF2 regulates Aurka expression to control mitotic processes and differentiation of C2C12 myoblasts.

(A) UCSC genome browser screenshots illustrate SRSF2 ChIP-seq signals on the Aurka gene. A pair of primers is highlighted by red arrows, indicating their relative positions from the transcription start sites (TSS) within the Aurka promoter region on the top. (B) ChIP-qPCR analysis quantifies SRSF2-bound Aurka promoter regions in C2C12 myoblasts, with relative enrichment folds normalized to the IgG group. (C) Western blot analysis for indicated proteins in C2C12 cells transiently transfected with siRNAs against SRSF2 (siSRSF2-#1, siSRSF2-#2) for 48 hours (left) or against Aurka (siAurka-#1, siAurka-#2) for 48 hours (right). (D) qPCR analysis of spindle formation-related genes in C2C12 cells following SRSF2 knockdown for 48 hours. (E) qPCR analysis of spindle formation-related genes in C2C12 cells with Aurka knockdown for 48 hours. (F) Immunostaining of phalloidin(red) and DAPI (blue) after SRSF2 or Aurka knockdown. Scale bars, 20μm. (G) Representative confocal images of MHC (green), Myog (red), and DAPI (blue) immunostaining in C2C12 cells. Scale bars, 50 μm. C2C12 cells were transfected with indicated siRNAs, then induced into differentiation for 3 days. Qualification of MHC+ cells were shown on the right graphs. (H) Western blot analysis was performed using whole cell extracts isolated from C2C12 cells transfected with indicated siRNAs (lanes 1-6), or with the indicated siRNAs along with either vector plasmid or Aurka plasmid, respectively (lanes 7-10). (I) Representative confocal images of MHC (green), Myog (red), and DAPI (blue) immunostaining in C2C12 cells. Scale bars, 50 μm. C2C12 cells were co-transfected with the indicated siRNAs along with either vector plasmid or Aurka plasmid, respectively, then induced into differentiation for 3 days. Qualification of MHC+ cells were shown on the right graphs.

Alternative splicing events affected by SRSF2 during skeletal muscle development.

(A) A table displaying genes alternatively spliced by SRSF2 during skeletal muscle development. (B) RT-PCR analysis of AS events observed in Bin1, Dmpk, Fhl1 and Ldb3 genes due to the absence of SRSF2, using total RNA isolated from three independent WT and KO samples. Alternative exons are highlighted in red with their respective exon number. Note that there were three 3’ alternative sites within exon14 of Dmpk. SRSF2 deficiency resulted in selection of the proximal 3’ site, while the presence of SRSF1 leads to the inclusion of the distal 3’ site. (C) RT-PCR analysis of AS events observed in Nrap, Pdlim3, Pdlim7 and Trim54 genes in three independent WT and KO skeletal muscle samples. (D) RT-PCR analysis of AS events observed in Aamdc, Dap3, Dnm1l, Ppp3ca and Ppp3cb genes in three independent WT and KO skeletal muscle samples. (E) RT-PCR analysis of AS events observed in indicated targets following knockdown of SRSF2 in C2C12 cells for 48 hours. (F) Diagram of Bin1 splice variants present in both normal and diseased tissues. Note that green, red or gray exons represent alternative exons, while slight blue exons are constitutive. (G) RT-PCR analysis of Bin1 exon 11 inclusion in three independent WT and KO skeletal muscle samples. (H) RT-PCR analysis was conducted to assess the levels of Bin1 exon 17-containing and exon 17-lacking variants after transfection with siBin1-#1 or siBin1-#2 in C2C12 cells for 48 hours. The black arrows indicate primer positions. (I) Representative confocal images of MHC (green), Myog (red), and DAPI (blue) immunostaining in C2C12 cells. Scale bars, 50 μm. C2C12 cells were transfected with siBin1 for 48 hours, then induced into differentiation for 3 days. Quantification of MHC+ cells were shown on the right graphs.