The role of SRSF1 in liver function and NASH

SRSF1在肝功能和NASH中的作用

基本信息

批准号：
9231266
负责人：
Waqar Arif
金额：
$ 4.9万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
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/9231266
关键词：
5&apos 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 Injection of therapeutic agent Injury to Liver Knock-out Knockout Mice Knowledge Lead Length Leukocytes Lipid Peroxidation Lipid Peroxides 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 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 Stress 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 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

项目摘要

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。它是一种代谢性疾病，肝脏内脂肪堆积（脂肪变性）与炎症、肝脏疾病有关。受伤尽管 NASH 影响了 2-5% 的美国人，但目前对这种疾病的了解有限，因为早期阶段（单纯脂肪变性）无症状且难以发现。此外，由于缺乏可行且稳健的模型系统，针对 NASH 病理学的有效疗法的开发一直很缓慢，我们发现 SRSF1（SRSF1 HKO）（一种剪接调节）的肝细胞特异性消融。 SRSF1 蛋白，触发 NASH 表型的严重和早期发作，在组成型剪接和选择性剪接中发挥直接作用，并且最近显示还调节特定转录本的翻译和无义介导的衰减。主要特征是，其在组织生理学中的作用尚不清楚。该提案的总体目标是确定 SRSF1 缺失导致 NASH 的致病机制。首先，使用体内病毒介导的 SRSF1 HKO 模型确定促进肝损伤的潜在不规则分子，我将确定哪些激活机制是对 SRSF1 活性丧失的主要反应和继发反应。显示 MALAT1（一种长非编码 RNA）已失去与 SRSF1 HKO 肝细胞中核斑点的关联。斑点是富含前 mRNA 剪接因子、RNA 加工因子和核斑点的高度动态核结构域。我们将进一步分析 SRSF1 HKO 肝细胞中的 RNA 分子组成，并确定 MALAT1 定位到斑点的缺失是否对 NASH 病理学的发展有直接影响。其次，我们将通过执行高水平构建 SRSF1 调控的基因网络。 -对从野生型和 SRSF1 HKO 小鼠分离的肝细胞进行分辨率 RNA-Seq 我们还将使用 iCLIP-Seq 确定 SRSF1 的直接 mRNA 靶标。一种用于识别活细胞中蛋白质-RNA 相互作用的方法，这两种方法的数据将允许构建强大的基因调控网络，该网络将提供对导致 Aim 中识别的细胞反应激活的分子机制的见解。 1 目标 3. 最后，我们将确定 SRSF1 的哪些功能（剪接或翻译调节）对于维持正常肝细胞功能至关重要，这将通过具有剪接和/或翻译调节活性的突变体构建体来实现。在体内构建 SRSF1 HKO 肝脏，并进行目标 1 中描述的类似测定。将比较这些实验的结果，以确定 SRSF1 的剪接和翻译调节功能在维持肝脏稳态中的贡献。