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.
Introduction
Serine-rich splicing factor 3 (SRSF3) is the smallest member of the SR protein family (1, 2) that functions to promote RNA splicing by recruiting components of the spliceosome at constitutive and alternatively spliced exons (3, 4). SRSF3 has also been ascribed a number of cellular functions including controlling cellular proliferation, as it is regulated during G1/S by the E2F transcription factor (5) and controls the G2/M transition of immortal rat fibroblasts (6). These findings have led to the suggestion that SRSF3 is a proto-oncogene and, indeed, it is overexpressed in some tumors (7). Previously, we demonstrated that SRSF3 regulates splicing of the insulin receptor gene (INSR) (8) and plays an important role in hepatocyte differentiation and hepatic glucose and lipid metabolism (9). Furthermore, we have shown that hepatocyte-specific deletion of the SRSF3 gene (SRSF3H-KO) promotes hepatocellular carcinoma (HCC) in aged SRSF3H-KO mice (10, 11), and we and others observed that SRSF3 is reduced in samples of human HCC (10, 11).
The global increase in obesity over the past few decades has caused a corresponding increase in nonalcoholic fatty liver disease (NAFLD) and its more severe form, nonalcoholic steatohepatitis (NASH) (12–16). Both NAFLD and NASH are associated with liver insulin resistance, nonalcoholic cirrhosis, and the development of HCC (17, 18). HCC is currently ranked as the fifth most common cancer worldwide (19, 20) and has a high mortality. It usually arises after years of liver disease and inflammation (19) either due to chronic hepatitis B or C virus (HBV/HCV) infection (21), or alcoholic and nonalcoholic cirrhosis.
The finding that loss of SRSF3 triggers metabolic changes, hepatic fibrosis, and increased liver inflammation (10, 11) that are all features of early liver disease led us to propose that SRSF3 expression might be sensitive to hepatic stress. Indeed, studies have shown that SRSF3 is ubiquitinated under both normal and stressed conditions (22–24). Additionally, it has been reported that SRSF3 is conjugated to the NEDD8 protein, a process termed neddylation, under arsenite-induced stress that is important for stress granule assembly (25). Therefore, we investigated whether SRSF3 is lost in early liver disease. We found that SRSF3 protein expression is lost in early liver disease, including NAFLD, NASH, and cirrhosis, in both humans and mice. We report here that SRSF3 is degraded in the proteasome in response to lipid-induced oxidative stress via neddylation on lysine 11 and that prevention of SRSF3 loss inhibited many of the deleterious changes observed previously. These results identify a neddylation-dependent pathway regulating liver health and may provide novel therapeutic targets that may aid in the treatment of liver diseases.
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