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 October 1, 2019; First 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 4

Neddylation of SRSF3 in response to palmitic acid.

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Neddylation of SRSF3 in response to palmitic acid. HepG2 cells (A) and p...
HepG2 cells (A) and primary hepatocytes (B) were treated with 10 μM MG132 (MG) for 2 hours. Cell lysates were immunoprecipitated (IP) with anti-NEDD8 antibodies and then immunoblotted (IB) for SRSF3. Input indicates input control, Co-IP indicates the coimmunoprecipitated proteins. The unmodified SRSF3 and NEDD8-SRSF3 conjugate are indicated by arrows. (C) HepG2 cells were transfected with Flag-tagged SRSF3 and then treated with 10 μM MG132; cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted for NEDD8. Monomeric NEDD8 and the NEDD8–Flag-SRSF3 conjugate are indicated by arrows. (D) HepG2 cells were transfected with Flag-tagged SRSF3 and then treated with 10 μM MG132 and 500 μM palmitic acid (PA); cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted for NEDD8. (E) HepG2 cells were transfected with Flag-tagged SRSF3 and then treated with the NEDD8-activating enzyme 1 inhibitor 3 mM MLN4924 (MLN49) for 1 hour and then PA for 12 hours, and cell lysates immunoprecipitated with an anti-Flag antibody and then immunoblotted for NEDD8. The NEDD8–Flag-SRSF3 conjugate is indicated by an arrow in D and E. (F) HepG2 cells were cotreated with the antioxidant N-acetyl-cysteine (NAC) and PA for 12 hours; cell lysates were immunoprecipitated with anti-NEDD8 antibody and then immunoblotted for SRSF3. NEDD8-SRSF3 is indicated by an arrow. (G) Primary hepatocytes or HepG2 cells were treated with 3 mM MLN49 for 1 hour and then PA for 12 hours, and cell lysates were immunoblotted for SRSF3. (H) Primary hepatocytes or HepG2 cells were treated with 3 mM MLN49 for 1 hour and then PA for 12 hours; splicing of the Fn1-EDA exon 33, Myo1b–exon 23, and Slk–exon 13 was analyzed by RT-PCR. (I) HepG2 cells were treated with 3 mM MLN49 and 1 mM MLN7243 (MLN72, ubiquitin-activating enzyme 1 inhibitor) for 1 hour and then PA for 12 hours; cell lysates were immunoblotted for SRSF3. Results are presented as mean ± SEM (n = 3). *P < 0.05 by 1-way ANOVA.