Spliceosome component Usp39 contributes to hepatic lipid homeostasis through the regulation of autophagy

Abstract

Regulation of alternative splicing (AS) enables a single transcript to yield multiple isoforms that increase transcriptome and proteome diversity. Here, we report that spliceosome component Usp39 plays a role in the regulation of hepatocyte lipid homeostasis. We demonstrate that Usp39 expression is downregulated in hepatic tissues of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) subjects. Hepatocyte-specific Usp39 deletion in mice leads to increased lipid accumulation, spontaneous steatosis and impaired autophagy. Combined analysis of RNA immunoprecipitation (RIP-seq) and bulk RNA sequencing (RNA-seq) data reveals that Usp39 regulates AS of several autophagy-related genes. In particular, deletion of Usp39 results in alternative 5' splice site selection of exon 6 in Heat shock transcription factor 1 (Hsf1) and consequently its reduced expression. Importantly, overexpression of Hsf1 could attenuate lipid accumulation caused by Usp39 deficiency. Taken together, our findings indicate that Usp39-mediated AS is required for sustaining autophagy and lipid homeostasis in the liver.

Fig. 1. Hepatic Usp39 expression is decreased in NAFLD and NASH.

a, b Heatmap showed the expression of core U4/U6. U5 tri-snRNP components in the mouse liver models of NAFLD (n = 6) and NASH (n = 8) compared with controls using publicly available RNA-seq data (GSE165855 and (GSE154892). The quantification data was shown in Supplementary Fig. S1a, b. c Interaction network analysis of 16 U4/U6. U5 tri-snRNP components using RNA-seq data (GSE165855) and (GSE154892) visualized with Cytoscape. U4/U6. U5 complex related splicing factors were classified into U5 snRNP (orange), U4/U6 snRNP (green), and tri-snRNP (blue) according to Spliceosome DB. d Correlation analysis of U4/U6. U5 splicing factors expression and NALFD activity score based on RNA-seq data of a human NAFLD cohort of 271 patients. e USP39 mRNA expression was compared between high (score ≥ 3) (n = 196) and low (score <3) (n = 75) NALFD activity score group. The bounds of the box were the upper and lower quartile with the median value in the center. The whiskers indicated the minima and maxima. The range from 10–90 percentile. f USP39 mRNA expression was compared between stage 0–1 (n = 78) and stage 2–4 (n = 192) fibrosis stage group. g, h Immunoblotting was performed to measure Usp39 protein in the livers of HFD-fed (n = 7 per group) and MCD-fed (n = 6 per group) male mice and chow-fed mice. i The USP39 protein level was measured in human healthy (n = 5) and NASH (n = 4) livers. j Immunohistochemical (IHC) staining of Usp39 in the liver sections of MCD-fed and HFD-fed male mice compared to those of the chow-fed mice (n = 12 per group). Scale bars, 50 µm. IHC image were quantified by pathologists. Resultes were shown in Fig. S1i. All immunoblotting band intensities were quantified by Image J. The p-value was obtained by Pearson R Correlation (d) and others performed unpaired two-sided Student’s t-test, and the results are presented as the mean ± S.D. CD, control diet. MW, molecular weight. Source data are provided as a Source data file.

Fig. 2. Hepatic Usp39 knockout impairs liver homeostasis and function.

a Schematic illustrating the generation of the Usp39 conditional knockout mice. The strategy was to insert loxp sites to flank exons 2 of the mouse Usp39 gene. Usp39^fl/fl mice were crossed with albumin-Cre mice to generate Usp39-HKO mice. b Immunoblotting was performed to determine Usp39 knockout efficiency using liver tissues and primary hepatocytes (n = 3 independent experiments). Band intensities were quantified by Image J. c Body weight of male mice was recorded from 1 to 16 weeks after birth (n = 5 per group). d Representative images of 5-week-old control and Usp39-HKO male mice. e–g Body weight, liver weight, and liver/body weight ratio were compared between control and Usp39-HKO mice (n = 10 per group). h H&E staining (n = 6), IHC staining of Pcna (n = 15) and Ki67 (n = 10) in the liver sections of 5-week-old male mice. Red arrows indicate Ki67-positive nuclei. Scale bars, 50 µm. i The Pcna-positive cells count of 5-week-old male mice liver sections (n = 15). j The Ki67-positive cells count of 5-week-old control (n = 11) and Usp39-HKO (n = 10) male mice liver sections. k–m Relative mRNA expression Afp (n = 4), H19 (n = 4), and Alb (n = 3) in the liver of 5-week-old male mice. n Serum ALT, AST, LDH and albumin levels were measured in 5-week-old male mice fasted for 16 h (n  =  20 per group). o IHC staining of F4/80, Sirius Red staining in the liver sections of 5- and 10-week-old male mice (n = 5 per group). Scale bars, 50 µm. p qPCR was performed to analyze mRNA expression indicated genes in livers of 5-week-old male mice (n = 4 per group). Images are representative of at least three independent experiments. The p-value was obtained by unpaired two-sided Student’s t-test, and the results are presented as the mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.  NS, stands for non-significant. Alb, albumin. Afp, α-Fetoprotein. MW, molecular weight. Source data are provided as a Source data file.

Fig. 3. Spontaneous steatosis in hepatocyte-specific Usp39 knockout mice.

a Representative liver and spleen images from 12-month-old male mice fasted for 16 h. b H&E staining and Sirius Red staining of liver sections in 12-month-old male mice (n = 3 per group). Scale bars, 50 µm. c Hepatic TG levels were analyzed in 12-month-old male mice fasted for 16 h (n = 8 per group). d qPCR was performed to analyze mRNA expression of indicated genes in livers of 12-month-old male mice (n = 3). e Representative liver images from control and Usp39-HKO male mice fed the HFD for 12 weeks. f H&E (left), Oil Red O (middle), and Sirius Red staining (right) of liver sections from 16-week-old male mice fed the HFD for 12 weeks (n = 3). Scale bars, 50 µm. g Hepatic TG levels were analyzed in16-week-old male mice fed the HFD for 12 weeks (n = 3). h qPCR was performed to analyze mRNA expression of indicated genes in livers of control and Usp39-HKO male mice fed the HFD for 12 weeks (n = 3). i Heatmap showing lipidomic data in the livers of 5-week-old male mice fasted 16 h (n = 6). j Volcano plot of the differential lipids between the livers (n = 6 per group). k Pathway enrichment analysis using Ingenuity Pathway Analysis (IPA) of the lipidomic and RNA-seq data. l Enrichment level of the subcategories of liver steatosis shown in (k). Gray squares indicate the activate patterns of the subcategories are not available in reference. m Heatmap showing differential hepatic steatosis-related genes in Usp39-HKO mice compared to control mice (n = 4 per group). n IPA analysis of hepatic steatosis-related genes using RNA-seq data in control and Usp39-HKO mice. Correlation of differential genes with hepatic steatosis were shown with arrows (activation), and dashes (inhibition). Images are representative of at least three independent experiments. The p-value was obtained by IPA (k–l) and others performed unpaired two-sided Student’s t-test, and the results are presented as the mean ± S.D. NS, stands for non-significant. Source data are provided as a Source data file.

Fig. 4. Autophagy is impaired in hepatocytes lacking Usp39.

a Pathway enrichment analysis of differentially expressed genes using RNA-seq data for control and Usp39-HKO male mice. b qPCR analysis of autophagy-related genes in the livers (n = 3 per group). c qPCR was performed to analyze expression of autophagy-related genes in Usp39 knockdown and control AML12 cells (n = 3). d Immunoblotting was performed to analyze protein levels of autophagy-related genes in 5-week-old control and Usp39-HKO male mice (n = 3 per group) fasted for 16 h. e, f Immunoblotting was performed to analyze protein levels of autophagy-related genes in Usp39 knockdown and control AML12 cells (n = 3) (e) and primary cells (n = 4) (f) supplemented with 0.4 mM oleic acid for 6 h. Immunoblotting band intensities were quantified by Image J, which were shown in Fig. S5g–i. g Electron micrographs in the livers of control (n = 11) and Usp39-HKO (n = 10) male mice showing autophagic vesicles with quantification. Scale bars, 2 µm. h Confocal image of the liver sections in control and Usp39-HKO mice showing co-localization of Plin2 (green) and Lamp2 (red). Scale bars, 10 µm. i Confocal images of primary hepatocytes transfected with Ad-EGFP-LC3 and control vectors followed by serum deprivation to 2 h, showing EGFP-LC3 puncta/cell (n  =  56 cells for control, n  =  64 cells for HKO). Scale bars, 10 µm. j Co-localization of EGFP-LC3 puncta and BODIPY C₁₂ (white arrows) in primary hepatocytes transfected with Ad-EGFP-LC3 and control vectors for 72 h, cells were then cultured in medium supplemented with 0.4 mM oleic acid (n  =  37 cells for control, n  =  39 cells for HKO). Scale bars, 10 µm. Immunofluorescence staining and co-localization was quantified by Image J. Images are representative of at least three independent experiments. The p-value was obtained by IPA (a) and others performed unpaired two-sided Student’s t-test, and the results are presented as the mean ± S.D. NS, stands for non-significant. OA, Oleic acid. MW, molecular weight. Source data are provided as a Source data file.

Fig. 5. Usp39 deletion leads to altered splicing of autophagy-related genes in hepatocytes.

a Pie chart showing the distribution of AS events in the RNA-seq data in the livers of 5-week-old control and Usp39-HKO male mice (n = 4 per group) fasted 16 h. A total of 1937 splicing events from 1472 genes were identified. SE, skipped exons; RI, retained introns; A5SS, alternative 5’ splice site; A3SS, alternative 3’ splice site; MXE, mutually exclusive exons (p < 0.05, |delInclevel| > 0.1). b Heatmap of autophagy-related genes using RNA-seq data of control and Usp39-HKO male mice (n = 4 per group). c Volcano plot showing differential AS events in the RNA-seq data of livers from Usp39-HKO male mice compared to controls. Negative log10 transformed p-values and the delInclevel values were analyzed with rMATS (n = 4 per group). d The distribution of Usp39 binding peaks (35, 980), corresponding to 5002 genes, were identified from RIP-seq data with Piranha software (-p 0.001 -z 100). e HOMER de novo motif analysis of Usp39 binding peaks based on the RIP-seq data showing top-two Usp39 binding motifs. Binding motifs were ranked by p-value, and the statistical results including the percentage of targets and, the background are shown. f Venn diagram of 611 overlap genes from 5002 Usp39 binding genes (RIP-seq) and 1472 differentially expressed genes (RNA-seq). g Biological process enrichment analysis of the 611 genes from (f). Accumulative hypergeometric q-values and gene ratios were calculated. h–k Sashimi plots of AS and binding peaks for indicated genes using RNA-seq and RIP-seq data. The light blue region highlights the AS sites and the Usp39 binding region. l PCR was performed to analyze AS events in indicated genes using splice-variant specific primers in the liver tissues of 5-week-old control and Usp39-HKO male mice. Images are representative of at least three independent experiments. The results are presented as the mean ± S.D. NS, stands for non-significant. RIP, RNA immunoprecipitation sequencing. AS, alternative splicing. Source data are provided as a Source data file.

Fig. 6. Usp39 deletion leads to mis-splicing and fast degradation of Hsf1 in hepatocytes.

a AS pattern and Usp39 binding sites on Hsf1 were visualized with IGV using RNA-seq and RIP-seq data. AS sites and the Usp39 binding region was highlighted. b Schematic structure of two Hsf1 isoforms. Hsf1-FL is the canonical isoform, and Hsf1-NMD is the altered splicing (A5SS) of exon 6. c RT-PCR was performed to validate AS events of Hsf1 in the livers of 5-week-old male mice. Percent spliced in (PSI) was quantified (n = 4). d Analysis of the splicing of the Hsf1 by minigene in 293 T cells. e Relative expression of Hsf1-FL and Hsf1-NMD was analyzed by qPCR in liver tissues (n = 4). f Hsf1-NMD was measured by qPCR in Upf1 knockdown and control AML12 cells treated with 10 μg/mL actinomycin D. g RIP-qPCR was performed to validate the binding of Usp39 on the Hsf1 transcript in AML12 cells (n = 4). U6 snRNA and Ptbp1 were used as the positive and negative controls. h RNA pull-down showing the interaction between Usp39 protein and Hsf1 pre-mRNA. PC: positive control, NC: negative control. Protein was quantified with Input. i–k Immunoblotting was performed to measure the levels of indicated genes in Usp39-HKO livers compared with controls under fed (i), fasted (j) and HFD (k) conditions (n = 3). l, m Hsf1 level was analyzed by immunoblotting in the livers of MCD-fed (n = 6) and HFD-fed (n = 7) mice compared to chow-fed mice. n HSF1 was measured by immunoblotting in the livers of individuals with NASH (n = 4) compared with those without NASH (n = 5). Immunoblotting bands intensities were quantified by Image J (Fig. S9a-c, g-i). Images are representative of at least three independent experiments. P-value was obtained by unpaired two-sided Student’s t-test and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test for (f), and the results are presented as the mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001. NS, stands for non-significant. CD, control diet. MW, molecular weight. Source data are provided as a Source data file.

Fig. 7. Hsf1 promotes autophagy and alleviates hepatic steatosis caused by Usp39 deletion.

a, b AML12 cells transfected with indicated vectors. Cells were stained with BODIPY and imaged using confocal microscopy. Lipid droplet numbers were quantified of 30 BODIPY-stained cells in each group (n = 3 independent experiments) by Image J. Scale bar, 10 µm. c Co-localization between Plin2 (green) and Lamp2 (red) was analyzed by immunofluorescence staining in AML12 cells transfected with indicated vectors, co-localization signal was quantified by Image J. Scale bars, 10 µm. d Immunoblotting analysis of Usp39, Hsf1 and autophagy-related genes in AML12 cell transfected with indicated vectors in complete or serum deprived medium (n = 3, 4 per group, from three independent experiments). e Oil Red O staining of liver sections of Usp39-floxed mice injected with AAV-empty, AAV-TBG-Cre, or Ad-Hsf1. The mice were sacrificed after fasted for 16 h. Scale bars, 50 µm. f Immunofluorescence experiments showed co-localization of Plin2 (green) and Lamp2 (red). Scale bar, 10 µm. g Immunoblotting analysis of protein levels of indicated genes in the livers from Usp39-floxed mice injected with AAV- empty, AAV-TBG-Cre, or Ad-Hsf1. The mice were sacrificed after fasted for 16 h (n = 3, 4, 6 per group, from three independent experiments). Immunoblotting band intensities were quantified by Image J, which were shown in Fig. S10d, j. Co-localization was quantified by Image J. Images are representative of at least three independent experiments. P-value was obtained by unpaired two-sided Student’s t-test and the results are presented as the mean ± S.D. NS, stands for non-significant. AD, Adenovirus. AAV, Adeno-associated virus. TBG, thyroxine-binding globulin. MW, molecular weight. Source data are provided as a Source data file.