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 3

Reduction in SRSF3 protein in response to excess lipid and oxidative stress.

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Reduction in SRSF3 protein in response to excess lipid and oxidative str...
Human HepG2 cells were exposed to (A) 500 μM PA, (B) 500 μM PA in the presence of the antioxidant N-acetyl-cysteine (NAC) to scavenge ROS, (C) 500 μM H2O2, and (D) 500 μM H2O2 in the presence of NAC over time, and extracts were immunoblotted for SRSF3. Graphs in A and C show quantification of SRSF3 protein normalized to β-actin (mean ± SEM, n = 3/group). **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. time 0 by 1-way ANOVA. (E) HepG2 cells were exposed to 500 μM PA or vehicle in the presence of 50 μg/mL cycloheximide for 0–8 hours. Cell extracts were immunoblotted for SRSF3 and results quantified by densitometry. Normalized SRSF3 expression results were fit to a 1-phase exponential decay to calculate the protein half-life (t1/2). The R2 values of the curve fits are given. The 2 curves were significantly different (P = 0.0004). (F) HepG2 cells were exposed to 500 μM PA or vehicle in the presence of 50 μg/mL cycloheximide for 0–8 hours. Cells were separated into nuclear and cytoplasmic fractions and immunoblotted for SRSF3 and normalized to lamin B or β-actin. Nuclear SRSF3 levels again fit a 1-phase exponential decay but there was no significant difference between treatments, so the single-curve fit is shown. Cytoplasmic SRSF3 did not fit an exponential decay, but the data were fit by a linear equation. The 2 treatments had significantly different linear fits. (G) Nuclear and cytoplasmic proteins were isolated from HepG2 cells treated with PA, leptomycin B (LMB), or both (LMB + PA) over time and immunoblotted for SRSF3 and lamin B or β-actin. Graph shows normalized nuclear SRSF3 levels in the 3 groups over time. Cytoplasmic levels of SRSF3 were very low in leptomycin B–treated groups. Results are presented as mean ± SEM (n = 3/group). *P < 0.05, **P < 0.01 vs. cotreatment by 2-way ANOVA.