Degradation of splicing factor SRSF3 contributes to progressive liver disease
Deepak Kumar, … , Olivia Osborn, Nicholas J.G. Webster
Deepak Kumar, … , Olivia Osborn, Nicholas J.G. Webster
Published August 8, 2019
Citation Information: J Clin Invest. 2019;129(10):4477-4491. https://doi.org/10.1172/JCI127374.
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Research Article Endocrinology Hepatology

Degradation of splicing factor SRSF3 contributes to progressive liver disease

Abstract

Serine-rich splicing factor 3 (SRSF3) plays a critical role in liver function and its loss promotes chronic liver damage and regeneration. As a consequence, genetic deletion of SRSF3 in hepatocytes caused progressive liver disease and ultimately led to hepatocellular carcinoma. Here we show that SRSF3 is decreased in human liver samples with nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), or cirrhosis that was associated with alterations in RNA splicing of known SRSF3 target genes. Hepatic SRSF3 expression was similarly decreased and RNA splicing dysregulated in mouse models of NAFLD and NASH. We showed that palmitic acid–induced oxidative stress caused conjugation of the ubiquitin-like NEDD8 protein to SRSF3 and proteasome-mediated degradation. SRSF3 was selectively neddylated at lysine 11 and mutation of this residue (SRSF3-K11R) was sufficient to prevent both SRSF3 degradation and alterations in RNA splicing. Lastly, prevention of SRSF3 degradation in vivo partially protected mice from hepatic steatosis, fibrosis, and inflammation. These results highlight a neddylation-dependent mechanism regulating gene expression in the liver that is disrupted in early metabolic liver disease and may contribute to the progression to NASH, cirrhosis, and ultimately hepatocellular carcinoma.

Authors

Deepak Kumar, Manasi Das, Consuelo Sauceda, Lesley G. Ellies, Karina Kuo, Purva Parwal, Mehak Kaur, Lily Jih, Gautam K. Bandyopadhyay, Douglas Burton, Rohit Loomba, Olivia Osborn, Nicholas J.G. Webster

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Figure 7

Degradation-resistant SRSF3-K11R attenuated expression of genes involved in lipid metabolism, fibrosis, and inflammation, and impaired SRSF3 target gene splicing in RNA from hepatocytes of mice on a NASH diet.

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Degradation-resistant SRSF3-K11R attenuated expression of genes involved...
(A) Expression of lipid storage, synthesis, and transport genes by qPCR. Cell death–inducing DFFA-like effector A (Cidea), cell death–inducing DFFA-like effector C/fat-specific protein 27 (Cidec), fatty acid synthase (Fasn), and fatty acid translocase (Cd36) RNA expression in hepatocytes is shown. (B) Expression of fibrosis genes by qPCR. Collagen 1a (Col1a1), fibronectin (Fn1), tissue inhibitor of metalloprotease (Timp1), and smooth muscle actin (Acta2) RNA expression in hepatocytes is shown. (C) Expression of inflammatory genes by qPCR. Macrophage F4/80 gene (Emr1), Kupffer cell C-type lectin domain family 4 member F (Clec4f), tumor necrosis factor α (Tnfa), and interleukin 6 (Il6) RNA expression in hepatocytes is shown. Lean mice shown in white, GFP-expressing mice in red, SRSF3-WT–expressing mice in blue, and SRSF3-K11R–expressing mice in cyan. (D) Analysis of RNA splicing in infected livers. Primary hepatocytes were generated from lean mice or mice on a NASH diet infected with AAV8-GFP, AAV8-SRSF3-WT, or AAV8-SRSF3-K11R. Splicing of SRSF3 target genes was assessed by RT-PCR as in Figure 2. Representative gels are shown for splicing of the insulin receptor exon 11 (Insr), fibronectin EDA exon 33 (Fn1), STE20-like kinase exon 13 (Slk), and myosin 1b exon 23 (Myo1b) (n = 4/group). Graphs show the percentage inclusion of the spliced exon. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Tukey’s post hoc testing.