Selective Neddylation facilitates proteasome‐mediated degradation of Serine Rich Splicing Factor 3 (SRSF3) in non‐alcoholic fatty liver disease

Background Hepatocellular Carcinoma (HCC) is the most common primary liver cancer. Over 80% of patients with HCC develop liver cirrhosis. Metabolic disturbances such as non‐alcoholic fatty liver disease (NAFLD) and its more severe form non‐alcoholic steatosis (NASH) are becoming more common in the obese population and both are risk factors for cirrhosis. Serine‐rich proteins are RNA‐binding proteins that contain an amino‐terminal RNA recognition motif (RRM) and have a C‐terminal domain enriched in arginine/serine dipeptides (RS domain). Previously, our group has demonstrated that liver‐specific SRSF3 deletion causes a multi‐step dysregulation of liver morphology and hepatocyte function, ultimately leading to hepatocellular carcinoma. Although studies have assessed the mechanism of SRSF3 degradation and its effect on cell metabolism. No investigations have been carried out to study the effect of SRSF3 degradation in liver diseases such as NAFLD, NASH or cirrhosis that predispose to HCC. The work presented here addresses the mechanism of SRSF3 degradation and its impact on liver disease such as NASH. Methods To assess the degradation of SRSF3 in NAFLD, mice were maintained on a 60% high‐fat diet (HFD) or western diet (NASH diet) and SRSF3 protein expression in liver was studied. In addition, we also tested the effect of oxidative stress on SRSF3. To understand the molecular mechanism of SRSF3 degradation, we performed experiments with cycloheximide and the proteasome inhibitor MG132. Further, we assessed different SRSF3 lysine residues to determine the SRSF3 lysine site modification. We also performed immunoblot and RT‐PCR of SRSF3 and its target genes to define functions of Lys11. Results Immunoblot data showed loss of SRSF3 in HFD and NASH hepatocyte cells. Immunohistochemistry showed a stronger staining for SRSF3 in the nucleus of liver samples from normal chow‐fed mice compared to HFD or NASH fed mice, indicating loss of SRSF3. Fibronectin 1 (Fn1) and insulin receptor (INSR) are direct target for SRSF3. Immunohistochemistry and RT‐PCR results showed higher expression of Fn1 and greater inclusion of exon 33 of the profibrogenic EDA exon in the Fn1 transcript in hepatocytes and liver of HFD and NASH mice. Increased expression of the INSR‐A isoform was evident in HFD and NASH hepatocytes. Further, we found that palmitic acid and H 2 O 2 cause loss of SRSF3 in HepG2 and primary hepatocyte cells. Co‐IP data suggested that SRSF3 is selectively neddylated at Lys11. More importantly, SRSF3 degradation is mediated following Neddylation of the protein and subsequent degradation via a proteasome complex degradation pathway. We observed that, Lys11 impaired the palmitic acid induced FN1‐EDA inclusion and other EMT genes. Conclusion Lipid accumulation causes loss of SRSF3 in HFD and NASH liver. Further, neddylation may be a primary mechanism of SRSF3 degradation following modification at Lys11. However, the degradation profile of SRSF3 in a large number of well‐characterized pathological samples needs to be determined. A detailed understanding of the mechanisms involved in SRSF3 degradation might allow the identification of novel therapeutic targets and may aid in the treatment of hepatocellular carcinoma. Support or Funding Information VMRF This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .