The role of SRSF1 in liver function and NASH

SRSF1在肝功能和NASH中的作用

• 批准号: 9900785
• 负责人: Waqar Arif
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-16 至 2021-05-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900785
• 关键词: 5' Splice Site; Ablation; Affect; Alcohol consumption; Alternative Splicing; American; Arginine; Bioinformatics; Biological Assay; Biological Models; Cell Death; Cell physiology; Cells; Computer Analysis; Cytoplasm; Data; Defect; Dependovirus; Development; Dipeptides; Disease; Disease Progression; Etiology; Event; Family; Fatty Liver; Fatty acid glycerol esters; Fibrinogen; Fibrosis; Gene Expression; General Population; Genes; Global Change; Goals; Hepatic; Hepatocyte; Histology; Homeostasis; Human; In Vitro; Individual; Infiltration; Inflammation; Injections; Knock-out; Knockout Mice; Knowledge; Lead; Length; Leukocytes; Lipid Peroxidation; Lipids; Liver; Liver Cirrhosis; Liver Failure; Liver diseases; MALAT1 gene; Mediating; Messenger RNA; Metabolic Diseases; Methods; Modeling; Molecular; Monitor; Mus; Non-Insulin-Dependent Diabetes Mellitus; Nonsense-Mediated Decay; Nuclear; Obesity; Oxidative Stress; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Physiology; Play; Population; Prevalence; Protein Family; Proteins; RNA; RNA Processing; RNA Recognition Motif; RNA Splicing; RNA-Protein Interaction; Reactive Oxygen Species; Recombinants; Regulation; Regulator Genes; Research; Resolution; Role; Serine; Spliced Genes; Structure; Tail; Tertiary Protein Structure; Therapeutic; Tissues; Transcript; Translations; Treatment Efficacy; Untranslated RNA; Up-Regulation; Veins; Viral; Wild Type Mouse; cell injury; cytokine; disease phenotype; early onset; effective therapy; endoplasmic reticulum stress; experimental study; genetic regulatory protein; in vivo; insight; liver function; liver injury; mRNA Precursor; member; mouse model; mutant; nonalcoholic steatohepatitis; nucleocytoplasmic transport; oxidative damage; public health relevance; response; therapeutic development; therapeutic target; transcriptome; transcriptome sequencing

 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.

 描述（由申请人提供）：非酒精性脂肪性肝炎（NASH）正在成为美国人群中最常见的肝脏疾病之一。它是一种代谢紊乱，其中肝脏内的脂肪积聚（脂肪变性）与炎症、肝损伤 和肝硬化而没有大量饮酒。尽管影响2-5%的美国人口，但目前没有有效的NASH治疗方法。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）是无症状的，难以检测。此外，由于缺乏可行且稳健的模型系统，针对NASH病理学的有效治疗剂的开发一直缓慢。我们已经发现，SRSF 1（SRSF 1 HKO）的肝细胞特异性消融，剪接调节蛋白，触发严重的NASH表型和早期发作。SRSF 1在组成性剪接和选择性剪接中都起着直接作用，并且最近已被证明还调节特定转录物的翻译和无义介导的衰变。尽管SRSF 1在剪接中的结构和功能作用被广泛表征，但其在组织生理学中的作用还不清楚。本提案的总体目标是确定SRSF 1缺失导致NASH的致病机制。目标1.我们将首先确定在我们的小鼠模型中促进肝损伤的潜在分子不规则性。使用体内病毒介导的SRSF 1 HKO模型，我将确定哪些激活的机制是主要与次要的SRSF 1活性损失的反应。我们也有初步的数据表明，MALAT 1，一个长的非编码RNA，已经失去了与SRSF 1 HKO肝细胞核斑点的关联。斑点是富含前mRNA剪接因子、RNA加工因子和RNA分子（包括MALAT 1）的高度动态核结构域。我们将进一步研究SRSF 1 HKO肝细胞中的斑点组成，并确定MALAT 1定位于斑点的丢失是否直接影响NASH病理学的发展。目标2.其次，我们将通过对从野生型和SRSF 1 HKO小鼠分离的肝细胞进行高分辨率RNA-Seq来构建SRSF 1调控的基因网络。我们还将使用iCLIP-Seq确定SRSF 1的直接mRNA靶点，iCLIP-Seq是一种用于鉴定活细胞中蛋白质-RNA相互作用的方法。这两种方法的数据将允许构建一个强大的基因调控网络。该网络将提供对导致目标1中确定的细胞反应激活的分子机制的见解。目标3。最后，我们将确定SRSF 1的功能，剪接或翻译调节，是维持正常肝细胞功能的关键。这将使用具有改变的剪接和/或翻译调节活性的突变体构建体来实现。我们将在体内将这些构建体引入SRSF 1 HKO肝脏，并进行目标1中描述的类似测定。这些实验的结果将进行比较，以确定SRSF 1的剪接和翻译调节功能在维持肝脏稳态的贡献。