Non-Alcoholic Steatohepatitis (NASH) is emerging as one of the most common liver disease in the American population. It is a metabolic disorder in which fat accumulation within the liver (steatosis) is associated with inflammation, hepatic injury and cirrhosis without significant consumption of alcohol. Despite affecting 2-5% of the American population, there are currently no effective therapeutic treatments for NASH. Current knowledge of this disease is limited because early stages (simple steatosis) are asymptomatic and difficult to detect. Furthermore, development of effective therapeutics against NASH pathology has been slow due to lack of a feasible and robust model system. We have discovered that hepatocyte-specific ablation of SRSF1 (SRSF1 HKO), a splicing regulatory protein, triggers severe and early onset of NASH phenotype. SRSF1 plays direct roles in both constitutive and alternative splicing and has recently been shown to also regulate translation and non-sense mediated decay of specific transcripts. Although the structural and functional roles of SRSF1 in splicing are extensively characterized, its role in tissue physiology is not well understood. The overall objective of this proposal is to determine the pathogenic mechanism(s) by which loss of SRSF1 results in NASH.
Aim 1. We will first determine the underlying molecular irregularities promoting liver damage in our mouse model. Using an in vivo viral mediated SRSF1 HKO model, I will identify which of the activated mechanisms are primary versus secondary responses to loss of SRSF1 activity. We also have preliminary data which shows that MALAT1, a long non-coding RNA, has lost association to nuclear speckles in the SRSF1 HKO hepatocytes. Speckles are highly dynamic nuclear domains enriched with pre-mRNA splicing factors, RNA processing factors and RNA molecules including MALAT1. We will further investigate speckle composition in SRSF1 HKO hepatocytes and determine if loss of MALAT1 localization to speckles has direct implications in the development of NASH pathology.
Aim 2. Secondly, we will construct the gene network regulated by SRSF1 by performing high-resolution RNA-Seq on hepatocytes isolated from wildtype and SRSF1 HKO mice. We will also determine direct mRNA targets of SRSF1 using iCLIP-Seq, a method used to identify protein-RNA interactions in living cells. Data from both of these approaches will allow for the construction ofa robust gene regulatory network. This network will provide insights into the molecular mechanisms resulting in the activation of cellular responses identified in Aim 1.
Aim 3. Finally, we will determine which of SRSF1's functions, splicing or translation regulation, is crucial for maintaining normal hepatocyte function. This will be achieved using mutant constructs, which have either altered splicing and/or translation regulation activities. We will introduce these constructs to SRSF1 HKO livers in vivo and perform similar assays described in Aim 1. Results of these experiments will be compared to determine the contributions of SRSF1's splicing and translation regulatory functions in maintaining liver homeostasis.
Non-alcoholic steatohepatitis (NASH) is an increasingly prevalent fatty liver disease which currently has no effective treatments. We have identified a mouse model which mimics the pathological phenotype of this disease. This research seeks to provide detailed understanding of the mechanism causing this disease progression in the mouse model and may offer insight into effective targets for development of therapeutics against NASH
|Bangru, Sushant; Arif, Waqar; Seimetz, Joseph et al. (2018) Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote liver regeneration. Nat Struct Mol Biol 25:928-939|
|Arif, Waqar; Datar, Gandhar; Kalsotra, Auinash (2017) Intersections of post-transcriptional gene regulatory mechanisms with intermediary metabolism. Biochim Biophys Acta Gene Regul Mech 1860:349-362|