The role of SRSF1 in liver function and NASH

 描述(申请人提供)：非酒精性脂肪性肝炎(NASH)是美国人群中最常见的肝病之一。这是一种代谢紊乱，肝脏中的脂肪堆积(脂肪变性)与炎症、肝脏损伤有关。 和肝硬变，但不会大量饮酒。尽管影响了2%-5%的美国人口，但目前还没有有效的治疗NASH的方法。目前对这种疾病的认识是有限的，因为早期阶段(单纯性脂肪变性)没有症状，很难发现。此外，由于缺乏可行和稳健的模型系统，针对NASH病理的有效治疗方法的开发一直很缓慢。我们发现，肝细胞特异性的SRSF1(SRSF1 HKO)是一种剪接调节蛋白，它会触发严重的和早期的NASH表型。SRSF1在结构性剪接和选择性剪接中发挥直接作用，最近被证明还调节特定转录本的翻译和无义介导的衰退。虽然SRSF1在剪接中的结构和功能已经被广泛地描述，但它在组织生理学中的作用还不是很清楚。这项建议的总体目标是确定SRSF1缺失导致NASH的致病机制(S)。目的1.我们将首先在我们的小鼠模型中确定潜在的促进肝损伤的分子异常。使用体内病毒介导的SRSF1 HKO模型，我将确定哪些激活机制是对SRSF1活性丧失的主要和次要反应。我们也有初步的数据表明，MALAT1，一个长的非编码RNA，已经失去了与SRSF1HKO肝细胞中的核斑点的联系。斑点是一种高度动态的核结构域，富含前mRNA剪接因子、RNA加工因子和包括MALAT1在内的RNA分子。我们将进一步研究SRSF1HKO肝细胞中的斑点成分，并确定MALAT1对斑点的定位缺失是否在NASH病理的发展中有直接意义。目的2.其次，我们将通过对野生型和SRSF1 HKO小鼠的肝细胞进行高分辨RNA-Seq来构建SRSF1调控的基因网络。我们还将使用iCLIP-Seq来确定SRSF1的直接mRNA靶点，这是一种用于识别活细胞中蛋白质-RNA相互作用的方法。来自这两种方法的数据将使构建一个强大的基因调控网络成为可能。这个网络将提供对导致激活目标1中确定的细胞反应的分子机制的洞察。目标3。最后，我们将确定SRSF1的S功能中的哪一个功能，剪接调控或翻译调控，对维持正常的肝细胞功能至关重要。这将通过突变结构实现，突变结构改变了剪接和/或翻译调节活动。我们将把这些构建物引入体内的SRSF1HKO肝脏，并进行目标1中描述的类似分析。这些实验的结果将进行比较，以确定SRSF1的S剪接和翻译调节功能在维持肝脏动态平衡方面的贡献。

 DESCRIPTION (provided by applicant): 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.