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 5

Mutation of lysine 11 prevented SRSF3 protein degradation.

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Mutation of lysine 11 prevented SRSF3 protein degradation. (A) Amino aci...
(A) Amino acid sequence of SRSF3 protein. The RNA recognition motif (RRM) is shown in blue, and the arginine- and serine-rich (RS) domain in red. Lysine residues are bold face and underlined. (B) HepG2 cells were transfected with empty vector (Control), wild-type Flag-SRSF3 (WT), or a series of single lysine-to-arginine mutants (K11R, K23R, K85R), double lysine mutants (K11/23R, K23/85R), or a triple lysine mutant (3KR) as indicated. Cells were treated with cycloheximide (CHX) for 1 and 4 hours, and then lysates were immunoblotted using the anti-Flag antibody. Graphs below show quantification of Flag-SRSF3 levels from transfected HepG2 cells. Results are presented as mean ± SEM (n = 3/group). *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. time 0 by 1-way ANOVA. Colors indicate the different mutants. (C) HepG2 cells were transfected with Flag-SRSF3 or single lysine mutants (K11R, K23R, K85R) as indicated and treated with 500 μM PA for 12 hours. Cell lysates were immunoprecipitated with anti-Flag antibodies and then immunoblotted for NEDD8. Input indicates input control, Co-IP indicates the coimmunoprecipitated complex. Monomeric NEDD8 and the NEDD8–Flag-SRSF3 conjugate are indicated by arrows. (D) Analysis of PA-induced SRSF3 degradation. Primary hepatocytes (top panels) and HepG2 cells (bottom panels) were transfected with Flag-SRSF3-WT or Flag-SRSF3-K11R. Twenty-four hours later, primary hepatocytes or HepG2 cells were treated with 250 μM or 500 μM PA, respectively, for 12 hours. Cells were then lysed and immunoblotted with the anti-Flag antibody. (E) Analysis of PA-induced changes in RNA splicing. Primary hepatocytes (left panels) and HepG2 cells (right panels) were transfected with Flag-SRSF3-WT or Flag-SRSF3-K11R. Twenty-four hours later, cells were treated with 250 μM or 500 μM PA, respectively, for 12 hours. Exon 33 (EDA exon) incorporation in the Fn1 mRNA, Slk–exon 13 skipping, and Myo1b–exon 23 inclusion was analyzed by RT-PCR.