CSDE1 as a Post Transcriptional Regulator of the LDLR

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，动脉粥样硬化性心脏病是美国的主要死因。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。通过创新的全基因组CRISPR干扰筛选，我们将CSDE 1鉴定为LDLR mRNA稳定性的新型转录后调节剂。在组织培养中，CSDE 1的敲除与他汀类药物或PCSK 9抑制剂的靶点敲除一样强大，这是临床上最有效的LDL降低疗法。此外，CSDE 1也独立于这些靶标发挥作用。然而，CSDE 1对肝脏LDLR mRNA影响的机制和特异性仍然未知。因此，本提案的总体目标是确定CSDE 1作为肝脏LDLR和循环LDL的特异性调节剂的作用。在目的1中，我们将建立肝CSDE 1调节LDLR mRNA稳定性的机制。因为CSDE 1结合LDLR的3' UTR，但其作用是组织依赖性的，我们假设CSDE 1募集LDLR mRNA衰变所需的其他因子。因此，我们将通过过表达研究建立CSDE 1作用的动态范围，使用质谱法鉴定CSDE 1介导的LDLR mRNA衰减所需的其他蛋白质组分，并将CSDE 1置于天然化合物小檗碱的背景下，小檗碱通过LDLR 3' UTR中的特定元件影响LDLR mRNA的稳定性。在目标2中，我们将确定肝脏CSDE 1在LDLR转录后调节中的作用。我们将使用全局翻译谱（RNA-seq）来评估CSDE 1敲低和过表达对肝转录组的影响。我们将结合联合收割机与交联和免疫沉淀（CLIP）的研究，以确定直接的RNA相互作用，和他们的结合基序，肝CSDE 1。在目标3中，我们将确定肝CSDE 1的作用是否在体内持续。我们将使用小鼠模型来评估Csde 1敲低和过表达对血脂和体内肝脏Ldlr调节的影响。总之，该项目将确定一种有前途的新的LDLR调节剂的机制和保真度，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CSDE 1作为一个有前途的治疗靶点。此外，他们将提供深入了解mRNA调控的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的影响。

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.