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，而动脉粥样硬化性心脏病是美国的主要死亡原因。上调肝脏低密度脂蛋白、降低低密度脂蛋白和预防心血管事件的治疗方法。此外，LDLR在维持细胞内胆固醇稳态方面也起着重要作用。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，我们发现CSDE1是LDLR mRNA稳定性的一种新型转录后调节剂。在组织培养中，敲低CSDE1的作用与敲低他汀类药物或PCSK9抑制剂的作用一样强大，这是临床上最有效的降低LDL的治疗方法。此外，CSDE1也独立于这些目标而起作用。然而，CSDE1对肝脏LDLR mRNA影响的机制和特异性尚不清楚。因此，本提案的总体目标是确定CSDE1作为肝脏LDLR和循环LDL的特异性调节剂的作用。在Aim 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，但其作用是组织依赖性的，我们假设CSDE1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CSDE1作用的动态范围，使用质谱方法确定CSDE1介导的LDLR mRNA衰变所需的额外蛋白质成分，并将CSDE1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Aim 2中，我们将确定肝脏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敲低和过表达对肝脏转录组的影响。我们将结合交联和免疫沉淀（CLIP）研究来鉴定肝脏CSDE1的直接RNA相互作用物及其结合基序。在Aim 3中，我们将确定肝脏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Csde1敲低和过表达对血浆脂质和体内肝脏Ldlr调节的影响。总之，该项目将确定一种有前途的LDLR新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作为一个有前景的治疗靶点。此外，它们将为mRNA调控的一般机制的特异性机制提供见解，对基础细胞生物学和其他疾病状态具有指导意义。

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.