CSDE1 as a Post Transcriptional Regulator of the LDLR - Diversity Supplement

CSDE1 作为 LDLR 的转录后调节因子 - 多样性补充剂

• 批准号: 10635281
• 负责人: John S Chorba
• 金额: $ 9.71万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-19 至 2022-08-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10635281
• 关键词: 3' Untranslated Regions; Address; Affect; Atherosclerosis; Berberine; Binding; Blood Circulation; CRISPR interference; Cardiovascular system; Cause of Death; Cell Culture Techniques; Cellular biology; Cholesterol; Cholesterol Homeostasis; Clinic; Coronary heart disease; Development; Disease; Drug Targeting; Elements; Event; Future; Genes; Genetic Transcription; Goals; Guidelines; Health; Hepatic; Human; Immunoprecipitation; Lipids; Low-Density Lipoproteins; Maintenance; Mass Spectrum Analysis; Mediating; Messenger RNA; Mission; National Heart, Lung, and Blood Institute; Pharmaceutical Preparations; Plasma; Play; Post-Transcriptional Regulation; Proteins; RNA; Regulation; Research; Research Design; Risk Factors; Role; Specificity; Tissues; United States National Institutes of Health; Whole Organism; Work; crosslink; genome-wide; improved; in vivo; inhibitor; innovation; insight; knock-down; mRNA Decay; mRNA Stability; mouse model; novel; overexpression; parent grant; public health relevance; recruit; therapeutic target; tissue culture; transcriptome; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT OF PARENT GRANT 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.

项目摘要/家长补助金摘要 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导致死亡的主要原因 在美国，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。 通过广泛的 CRISPR 干扰筛选，我们发现 CSDE1 是一种新型的 LDLR 转录后调节剂 在组织培养中，CSDE1 的敲低与他汀类药物或他汀类药物靶标的敲低一样强大。 PCSK9 抑制剂是临床上最有效的 LDL 降低疗法，此外，CSDE1 也起作用。 然而，CSDE1 对这些目标的影响的机制和特异性。 肝脏 LDLR mRNA 仍然未知，因此，该提案的总体目标是确定 LDLR 的作用。 CSDE1 作为肝脏 LDLR 和循环 LDL 的特异性调节剂 在目标 1 中，我们将建立 肝脏 CSDE1 调节 LDLR mRNA 稳定性的机制，因为 CSDE1 结合 3' UTR。 LDLR，但其作用是组织依赖性的，我们追求CSDE1招募所需的其他因子 因此，我们将通过过度表达来确定 CSDE1 效应的动态范围。 研究中，使用质谱方法来鉴定 CSDE1- 所需的其他蛋白质成分 介导 LDLR mRNA 衰减，并将 CSDE1 置于天然化合物小檗碱的背景下，该化合物 通过 LDLR 3' UTR 中的特定元件影响 LDLR mRNA 稳定性 在目标 2 中，我们将确定 LDLR 的作用。 肝脏 CSDE1 在 LDLR 转录后调控中的作用 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。 将其与交联和免疫沉淀 (CLIP) 研究相结合，以确定直接的 RNA 相互作用因子， 及其结合基序，肝脏 CSDE1。 在目标 3 中，我们将确定肝脏 CSDE1 是否有影响。 我们将使用小鼠模型来评估 Csde1 敲低和过表达对体内的影响。 该项目将共同确定血浆脂质和体内肝脏 Ldlr 的调节机制和保真度。 LDLR 是一种有前途的新型调节剂，LDLR 在动脉粥样硬化的发展中起着极其重要的作用。 这些结果将确立 CSDE1 作为一个有前途的治疗靶点，此外，它们还将提供对 CSDE1 的深入了解。 mRNA 调节一般机制的特异性机制，对基础细胞的影响 生物学和其他疾病状态。