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

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

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

Fig. 6

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.

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