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.

项目概要/赠款摘要 低密度脂蛋白（LDL）是动脉粥样硬化性心脏病的一个关键危险因素，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的治疗可以降低LDL并防止心血管事件。 此外，LDLR还在维持细胞内胆固醇稳态方面发挥重要作用。 因此，了解调节LDLR的机制很重要。通过创新的基因组- 通过广泛的CRISPR干扰筛选，我们将CSDE1鉴定为LDLR的新型转录后调节剂 mRNA稳定性在组织培养中，CSDE1的敲低与他汀类药物或抗肿瘤药物的靶点的敲低一样强大。 PCSK9抑制剂是临床上最有效的LDL降低疗法。此外，CSDE1还起作用， 独立于这些目标。然而，CSDE1的作用机制和特异性均不清楚。 肝LDLR mRNA仍未知。因此，本提案的总体目标是确立 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CSDE1的3 'UTR， LDLR，但其影响是组织依赖性的，我们假设CSDE1招募所需的其他因素， LDLR mRNA衰减。因此，我们将建立CSDE1通过过表达的作用的动态范围。 研究，使用质谱法来确定CSDE1所需的其他蛋白质组分。 介导的LDLR mRNA衰减，并将CSDE1置于天然化合物小檗碱的背景下， 通过LDLR 3 'UTR中的特异性元件影响LDLR mRNA稳定性。在目标2中，我们将确定 肝CSDE1在LDLR转录后调节中的作用。我们将使用全局翻译谱（RNA- seq）以评估CSDE1敲低和过表达对肝转录组的影响。我们将 联合收割机将其与交联和免疫沉淀（CLIP）研究相结合，以鉴定直接RNA相互作用物， 和它们的结合基序。在目标3中，我们将确定肝CSDE1的作用是否 在体内持续存在。我们将使用小鼠模型来评估Csde 1敲低和过表达对人乳腺癌细胞系的影响。 血浆脂质和体内肝脏Ldlr调节。总之，这个项目将确定机制和保真度 LDLR是动脉粥样硬化发展中极其重要的参与者。 这些结果将确立CSDE1作为一个有前途的治疗靶点。此外，他们将提供洞察力， mRNA调控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。