CSDE1 as a Post Transcriptional Regulator of the LDLR

CSDE1 作为 LDLR 的转录后调节因子

• 批准号: 10447206
• 负责人: John S Chorba
• 金额: $ 62.7万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10447206
• 关键词: 3' Untranslated Regions; Address; Affect; Atherosclerosis; Berberine; Binding; Biological; Blood Circulation; CRISPR interference; Cardiovascular system; Cause of Death; Cell Culture Techniques; Cell membrane; Cell model; Cell surface; Cells; Cellular biology; Cholesterol; Cholesterol Homeostasis; Clinic; Coronary heart disease; Data; Development; Disease; Drug Targeting; Elements; Endotoxins; Environment; Event; Future; Gene Expression Profiling; Genes; Genetic Transcription; Goals; Health; Heart Diseases; Hepatic; Human; Immunoprecipitation; Inflammatory; Lipids; Liver; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Maintenance; Mass Spectrum Analysis; Mediating; Messenger RNA; Mission; Modeling; Molecular Chaperones; Pathway interactions; Pharmaceutical Preparations; Plasma; Play; Post-Transcriptional Regulation; Proteins; RNA; RNA Binding; Regulation; Research Design; Risk Factors; Role; Specificity; Surface; System; Therapeutic; Tissues; Transcript; Transcriptional Regulation; United States; United States National Institutes of Health; Virus; Whole Organism; Work; blood lipoprotein; crosslink; genome-wide; improved; in vivo; inhibitor; innovation; insight; knock-down; mRNA Decay; mRNA Stability; modifiable risk; mouse model; novel; overexpression; public health relevance; receptor expression; recruit; therapeutic target; tissue culture; transcriptome; transcriptome sequencing; uptake

Low-density lipoprotein (LDL) is a key risk factor for atherosclerotic heart disease, the leading cause of death in the US. Therapies that upregulate the hepatic LDLR lower LDL and protect against cardiovascular events. Moreover, the LDLR also plays a fundamental role in maintenance of intracellular cholesterol homeostasis. Therefore, the mechanisms that regulate the LDLR are important to understand. Via an innovative genome- wide CRISPR interference screen, we identified CSDE1 as a novel post-transcriptional modulator of LDLR mRNA stability. In tissue culture, knockdown of CSDE1 is as powerful as knockdown of the targets of statins or PCSK9 inhibitors, the most effective LDL lowering therapies in the clinic. Moreover, CSDE1 also acts independently of these targets. However, both the mechanism of and specificity for CSDE1's effect on the hepatic LDLR mRNA remain unknown. Therefore, the overall goal of this proposal is to establish the role of CSDE1 as a specific regulator of the hepatic LDLR and circulating LDL. In Aim 1, we will establish the mechanism by which hepatic CSDE1 regulates LDLR mRNA stability. Because CSDE1 binds the 3' UTR of LDLR, but its effects are tissue-dependent, we hypothesize that CSDE1 recruits other factors required for LDLR mRNA decay. We will therefore establish the dynamic range of CSDE1's effect through overexpression studies, use mass spectrometry approaches to identify the additional protein components required for CSDE1- mediated LDLR mRNA decay, and place CSDE1 in the context of a natural compound, berberine, which affects LDLR mRNA stability through specific elements in the LDLR 3' UTR. In Aim 2, we will identify the role of hepatic CSDE1 in post-transcriptional regulation of the LDLR. We will use global translational profiling (RNA- seq) to evaluate the effects of CSDE1 knockdown and overexpression on the hepatic transcriptome. We will combine this with cross-linking and immunoprecipitation (CLIP) studies to identify the direct RNA interactors, and their binding motifs, of hepatic CSDE1. In Aim 3, we will establish whether the effect of hepatic CSDE1 persists in vivo. We will use a mouse model to evaluate the effect of Csde1 knockdown and overexpression on plasma lipids and in vivo hepatic Ldlr regulation. Together, this project will identify the mechanism and fidelity of a promising new regulator of the LDLR, an extremely important player in the development of atherosclerosis. The results will establish CSDE1 as a promising therapeutic target. Moreover, they will provide insight into the mechanism of specificity of a general mechanism of mRNA regulation, with implications for fundamental cell biology and other disease states.

低密度脂蛋白（LDL）是动脉粥样硬化性心脏病的关键危险因素，而动脉粥样硬化性心脏病是美国的主要死亡原因。上调肝脏 LDLR 的疗法可降低 LDL 并预防心血管事件。此外，LDLR 在维持细胞内胆固醇稳态方面也发挥着重要作用。因此，了解调节 LDLR 的机制非常重要。通过创新的全基因组 CRISPR 干扰筛选，我们确定 CSDE1 是 LDLR mRNA 稳定性的新型转录后调节剂。在组织培养中，CSDE1 的敲低与他汀类药物或 PCSK9 抑制剂靶标的敲低一样强大，后者是临床上最有效的 LDL 降低疗法。此外，CSDE1 的作用也独立于这些目标。然而，CSDE1 对肝脏 LDLR mRNA 影响的机制和特异性仍然未知。因此，该提案的总体目标是确立 CSDE1 作为肝脏 LDLR 和循环 LDL 特异性调节剂的作用。在目标 1 中，我们将建立肝脏 CSDE1 调节 LDLR mRNA 稳定性的机制。由于 CSDE1 结合 LDLR 的 3' UTR，但其作用具有组织依赖性，因此我们假设 CSDE1 招募了 LDLR mRNA 衰减所需的其他因子。因此，我们将通过过表达研究确定 CSDE1 作用的动态范围，使用质谱方法来识别 CSDE1 介导的 LDLR mRNA 衰减所需的其他蛋白质成分，并将 CSDE1 置于影响 LDLR mRNA 的天然化合物小檗碱的背景下通过 LDLR 3' UTR 中的特定元件实现稳定性。在目标 2 中，我们将确定肝脏 CSDE1 在 LDLR 转录后调节中的作用。我们将使用全局翻译分析 (RNA-seq) 来评估 CSDE1 敲低和过表达对肝转录组的影响。我们将其与交联和免疫沉淀 (CLIP) 研究相结合，以确定肝 CSDE1 的直接 RNA 相互作用因子及其结合基序。在目标 3 中，我们将确定肝脏 CSDE1 的作用在体内是否持续存在。我们将使用小鼠模型来评估 Csde1 敲低和过表达对血浆脂质和体内肝脏 Ldlr 调节的影响。该项目将共同确定 LDLR 的一种有前途的新型调节剂的机制和保真度，LDLR 在动脉粥样硬化的发展中起着极其重要的作用。结果将确立 CSDE1 作为一个有前途的治疗靶点。此外，它们还将深入了解 mRNA 调节一般机制的特异性机制，对基础细胞生物学和其他疾病状态具有影响。