SRp55 Regulates a Splicing Network That Controls Human Pancreatic β-Cell Function and Survival

Abstract

Progressive failure of insulin-producing β-cells is the central event leading to diabetes, but the signaling networks controlling β-cell fate remain poorly understood. Here we show that SRp55, a splicing factor regulated by the diabetes susceptibility gene GLIS3, has a major role in maintaining the function and survival of human β-cells. RNA sequencing analysis revealed that SRp55 regulates the splicing of genes involved in cell survival and death, insulin secretion, and c-Jun N-terminal kinase (JNK) signaling. In particular, SRp55-mediated splicing changes modulate the function of the proapoptotic proteins BIM and BAX, JNK signaling, and endoplasmic reticulum stress, explaining why SRp55 depletion triggers β-cell apoptosis. Furthermore, SRp55 depletion inhibits β-cell mitochondrial function, explaining the observed decrease in insulin release. These data unveil a novel layer of regulation of human β-cell function and survival, namely alternative splicing modulated by key splicing regulators such as SRp55, that may cross talk with candidate genes for diabetes.

Figure 1

SRp55 is highly expressed in human pancreatic β-cells, and its depletion leads to increased β-cell apoptosis. A: Fluorescence microscopy of insulin and SRp55 in human islets (left panel) and in the human EndoC-βH1 cell line (right panel) shows staining of SRp55 (red), insulin (green), and nuclei (blue). B: mRNA expression of SRp55 in human islets, EndoC-βH1 cells, and a panel of normal human tissues was measured by quantitative RT-PCR (qRT-PCR) and normalized by the housekeeping gene β-actin. C and D: Human islets were transfected with siCTL or specific siRNA against SRp55 (siSR#1 and siSR#2) for 48 h. SRp55 KD levels were assessed by qRT-PCR (C), and apoptosis was evaluated by Hoechst/propidium iodide (PI) staining (D). E–I: EndoC-βH1 cells were transfected with control or specific siRNA against SRp55 for 48 h. SRp55 KD levels were assessed by qRT-PCR (E) and by Western blotting (F). Apoptosis of EndoC-βH1 cells after SRp55 KD was evaluated by Hoechst/PI staining (G) and by cleaved caspase 3 immunofluorescence (H and I). Scale bars = 1 µm. Results are the mean ± SEM of three to nine independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. siCTL (paired t test).

Figure 2

RNA sequencing of EndoC-βH1 cells after SRp55 depletion. A: Pairwise comparison of control vs. SRp55 KD EndoC-βH1 cassette exons shown as a volcano plot. AS events presenting a difference of ΔPSI >5% and an FDR ≤0.01% were considered modified, as indicated by the dotted lines. B: Numbers and proportions of the different AS events modified after SRp55 silencing, as identified by rMATS analysis. C: Ingenuity pathway analysis of genes showing differential AS (enhanced or inhibited) after SRp55 depletion. D: Heat maps showing genes enriched with Gene Ontology terms involved in cell survival and β-cell function. PSI values are represented by gradient colors and shown for each individual control and SRp55 KD sample. Red represents a higher PSI; blue represents a lower PSI. Results are based on five RNA sequencing samples.

Figure 3

Confirmation of SRp55-regulated splicing events shown through representative RT-PCR validations (A–L). cDNA were amplified by RT-PCR using primers located in the upstream and downstream exons of the modified splicing event. PCR fragments were analyzed by automated electrophoresis using a bioanalyzer machine and quantified by comparison with a loading control. For each gene, representative gel images showing different splice variants affected by SRp55 KD and the corresponding inclusion-to-exclusion ratios are shown. The structure of each isoform is indicated by exons (blocks) and introns (solid lines). Alternatively spliced regions are indicated in red, green, or blue. Results are the mean ± SEM of three to eight independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. siCTL (paired t test).

Figure 4

SRp55 depletion impairs insulin secretion and mitochondrial respiration. A: Insulin secretion in EndoC-βH1 cells was evaluated by ELISA after 1 h of stimulation with 1 mmol/L glucose, 20 mmol/L glucose, or 20 mmol/L glucose plus forskolin. A, B, and D–H: Black bars indicate transfection with control siRNA and white bars indicate transfection with siRNA against SRp55. B: Insulin content after SRp55 KD was evaluated by ELISA. C–G: Analysis of mitochondrial respiration parameters in EndoC-βH1 cells using a Seahorse oximeter. C: Oxygen consumption rate (OCR) profiles of control and SRp55 KD cells at basal conditions (1 mmol/L glucose) and after sequential treatment with glucose (20 mmol/L), oligomycin (5 μmol/L), FCCP (4 μmol/L), and rotenone plus antimycin A (1 μmol/L each). Injection of different compounds is indicated by arrows. D: Basal respiration (1 mmol/L glucose), calculated by subtracting nonmitochondrial respiration from the last measurement before 20 mmol/L glucose injection. E: Response to high glucose, calculated by subtracting the last basal respiration measurement from the last measurement after injection of 20 mmol/L glucose. F: ATP production, calculated by subtracting the minimum measurement after oligomycin injection from the last measurement after glucose injection. G: Maximal respiration, calculated by subtracting nonmitochondrial respiration from the maximum measurement after FCCP injection. H: mRNA expression of transcription factors regulating β-cell identity and phenotype. In the top panels, RNA sequencing expression values are shown in RPKM, and in the bottom panels, confirmation is indicated by quantitative RT-PCR (qRT-PCR) normalized by the housekeeping gene β-actin. Results are the mean ± SEM of three to nine experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. siCTL (ANOVA followed by the Bonferroni post hoc test [A] or the paired t test [B and D–H]).

Figure 5

SRp55 controls the expression of a constitutively active isoform of the apoptotic inducer BAX, contributing to increased β-cell apoptosis. A: Schematic representation of BAX isoforms α and β, and RNA sequencing reads in control and SRp55 KD cells mapping to the distal part of the gene. Boxes represent exons; gray represents untranslated regions; black represents coding regions; and solid lines represent introns. B: Model of activation of apoptosis by BAX α and BAX β isoforms proposed by Fu et al. (33). Upon apoptotic signaling, BH3-only molecules such as BIM activate BAX α to promote its translocation and oligomerization to the mitochondrial outer membrane, leading to cytochrome c release and apoptosis activation. On the other hand, BAX β spontaneously targets, oligomerizes, and permeabilizes mitochondria, behaving as a constitutively active isoform. C–G: Double KD of SRp55 and BAX β in EndoC-βH1 cells (C–F) and in human islets (G). Cells were transfected with siCTL, siSR#2, siBaxβ, or siSR#2 plus siBaxβ for 48 h. C: Fluorescence microscopy analysis of BAX and the mitochondrial marker ATP synthase in EndoC-βH1 cells, showing that SRp55 KD leads to increased translocation of BAX to the mitochondria, a phenomenon prevented by BAX β silencing. Scale bar = 1 µm. mRNA expression of SRp55 (D) and BAX β (E) was measured by quantitative RT-PCR and normalized by the housekeeping gene β-actin. mRNA expression values were normalized by the highest value of each experiment, considered as 1. Proportion of apoptotic cells in EndoC-βH1 cells (F) and in dispersed human islets (G). Results are the mean ± SEM of four or five independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. siCTL; ##P < 0.01 and ###P < 0.001 as indicated by bars (ANOVA followed by the Bonferroni post hoc test).

Figure 6

SRp55 modifies the splicing of JNK signaling cascade genes, leading to JNK1 hyperactivation and β-cell apoptosis. A: Schematic representation of the JNK signaling pathway. Proteins showing AS detected by RNA sequencing after SRp55 KD are shown in blue. Proteins exhibiting overphosphorylation upon SRp55 depletion are shown in red. B: Representative Western blotting and densitometric measurements of total and phosphorylated forms of MKK7, JNK1, and c-JUN in EndoC-βH1 cells under control conditions and after SRp55 KD. C–F: Double KD of SRp55 and JNK1 in EndoC-βH1 cells (C–E) and in human islets (F). Cells were transfected with siCTL, siSR#2, siJNK1, or siSR#2 plus siJNK1 for 48 h. mRNA expression of SRp55 (C) and JNK1 (D) was measured by quantitative RT-PCR and normalized by the housekeeping gene β-actin. mRNA expression values were normalized by the highest value of each experiment, considered as 1. E and F: Proportion of apoptotic cells in EndoC-βH1 cells (E) and in dispersed human islets (F). G–K: Specific KD of three SRp55-regulated spliced variants of the JNK cascade. EndoC-βH1 cells were transfected with siCTL, siSR#2, or specific siRNA targeting cassette exons of MAP3K7 (exon 12, siMAP3K7e12), JNK1 (exon 3, siJNK1e3), and JNK2 (exon 2, siJNK2e2). G–I: Representative RT-PCR validations showing increased exon skipping in MAP3K7 (G), JNK1 (H), and JNK2 (I). J and K: Percentage of apoptotic cells (J) and JNK phosphorylation (K) after SRp55 KD or skipping of MAP3K7, JNK1, and JNK2 cassette exons. Results are the mean ± SEM of four or five independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. siCTL; ##P < 0.01 and ###P < 0.001 as indicated by bars (paired t test [B, G–K] or ANOVA followed by the Bonferroni post hoc test [C–F]).

Figure 7

SRp55 KD–induced ER stress contributes to β-cell death. A: Heat map showing AS and gene expression changes in genes involved in the ER-associated protein degradation process (top panel) and markers of the unfolded protein response (bottom panel). Red represents higher expression and blue, lower expression. B–E: Representative Western blotting (B) and densitometric measurements of total and phosphorylated forms of IRE1α (C), PERK (D), and eIF2α (E). mRNA expression of BIP (F), XBP1 spliced (G), and CHOP (H) after SRp55 KD was measured by quantitative RT-PCR (qRT-PCR) and normalized by the housekeeping gene β-actin. I–K: Double KD of SRp55 and IRE1α in EndoC-βH1 cells. Cells were transfected with siCTL, siSR#2, siIRE1α, or siSR#2 plus siIRE1α for 48 h. mRNA expression of SRp55 (I) and IRE1α (J) was measured by qRT-PCR and normalized by the housekeeping gene β-actin. mRNA expression values were normalized by the highest value of each experiment, considered as 1. K: The proportion of apoptotic cells was evaluated by Hoechst/propidium iodide staining. Results are the mean ± SEM of four to nine independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. siCTL; #P < 0.05 and ###P < 0.001 as indicated by bars (paired t test [C–H] or ANOVA followed by the Bonferroni post hoc test [I–K]).