Alternative Splicing in Regulation of Cholesterol Synthesis and Uptake

描述（由申请人提供）：LDL胆固醇水平在确定心血管疾病风险方面具有根本重要性。最近，细胞内胆固醇的两个最关键的调节剂3-羟基-3-甲基戊二酰辅酶A还原酶（HMGCR）（胆固醇生物合成的限速酶）和负责LDL摄取的LDL受体（LDLR）的选择性剪接与血浆LDL的变化以及辛伐他汀降低LDL的幅度相关。最近的证据表明，参与胆固醇代谢的四个基因（HMGCR，LDLR，HMG-CoA合成酶和甲羟戊酸激酶）的选择性剪接是由甾醇协同调节的，使得甾醇负载增加选择性剪接，而甾醇消耗抑制选择性剪接。此外，辛伐他汀孵育的人淋巴细胞系的全基因组转录分析表明，约300种已知的超剪接体组分中有95种对他汀有反应（FDR<0.0001）。其中，几种剪接因子涉及介导选择性剪接的固醇调节，基于另外的证据，包括：（1）基因表达的变化与细胞表面LDLR和血浆LDL浓度的相关性;（2）与血浆LDL水平相关的DNA多态性;（3）siRNA敲低导致前mRNA剪接的变化;和（4）已知结合基序的计算机预测。这些发现导致了这样的假设，即细胞内胆固醇水平调节剪接因子，以产生参与胆固醇合成和摄取的多个基因的选择性剪接的协调变化，并且该过程中的变化是细胞和血浆胆固醇代谢的决定因素。因此，本提案的总体目标是：（1）证明选择性剪接是一种参与调节细胞胆固醇合成和摄取以及血浆LDL水平的新机制;（2）鉴定该过程的非遗传和遗传修饰剂。为了确定胆固醇生物合成途径中大量基因中是否存在甾醇调节的可变剪接;将在用该途径的特异性抑制剂和产物处理的HepG 2细胞、原代人肝细胞和永生化人淋巴细胞系中定量可变剪接的变化（目的1）。负责协调这些协调变化的剪接因子将使用siRNA、过表达构建体和微基因构建体进行鉴定和验证（Aim 2）。最后，将通过检测基因调控的可变剪接与体内血浆LDL水平和体外胆固醇相关表型的相关性，评估这些观察结果的生理相关性。SNP功能性将通过微基因构建体的定点诱变来确认（目的3）。选择性剪接在调节胆固醇代谢中的作用的证明和这一过程的遗传决定因素的鉴定将有助于描绘有助于血浆LDL个体间变异的分子途径，从而提高我们对心血管疾病发展和风险的理解。 公共卫生相关性：这项提案的目的是测试选择性剪接是否是一种调节胆固醇稳态的新机制。具体而言，我们试图确定参与胆固醇生物合成和摄取的基因的固醇调节的选择性剪接的程度，以及确定与体内和细胞胆固醇代谢相关的这种反应的遗传和非遗传调节因子。因此，这项研究将有助于我们了解血浆LDL胆固醇变化的分子决定因素，进一步增加我们对心血管疾病风险和发展的认识。

DESCRIPTION (provided by applicant): LDL cholesterol levels are of fundamental importance in determining risk for cardiovascular disease. Recently, alternative splicing of the two most critical regulators of intracellular cholesterol, 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGCR), the rate-limiting enzyme of cholesterol biosynthesis, and the LDL receptor (LDLR), responsible for uptake of LDL, have been associated with variation in plasma LDL as well as with the magnitude of LDL reduction by simvastatin. Recent evidence has indicated that alternative splicing of four genes involved in cholesterol metabolism (HMGCR, LDLR, HMG-CoA synthase and mevalonate kinase) is coordinately regulated by sterols such that sterol loading increases alternative splicing while sterol depletion suppresses alternative splicing. In addition, genome-wide transcription analysis of simvastatin incubated human lymphocyte cell lines demonstrated that 95 of ~300 known components of supraspliceosomes were responsive to statin (FDR<0.0001). Among these, several splicing factors were implicated in mediating sterol regulation of alternative splicing on the basis of additional lines of evidence including: (1) correlations of variation in gene expression with both cell surface LDLR and plasma LDL concentrations; (2) DNA polymorphisms associated with plasma LDL levels; (3) siRNA knock-down resulting in changes in pre-mRNA splicing; and (4) in silico prediction of known binding motifs. These findings lead to the hypotheses that intracellular cholesterol levels regulate splicing factor(s) to generate coordinated changes in alternative splicing of multiple genes involved in cholesterol synthesis and uptake, and that variation in this process is a determinant of cellular and plasma cholesterol metabolism. Thus, the overall objectives of this proposal are: (1) to demonstrate that alternative splicing is a novel mechanism involved in regulating cellular cholesterol synthesis and uptake as well as plasma LDL levels; and (2) to identify non-genetic and genetic modifiers of this process. To determine if sterol regulated alternative splicing occurs in a larger number of genes in the cholesterol biosynthesis pathway; changes in alternative splicing will be quantified in HepG2 cells, primary human hepatocytes, and immortalized human lymphocyte cell lines treated with specific inhibitors and products of this pathway (Aim 1). The splicing factors responsible for orchestrating these coordinated changes will be identified and validated using siRNA, overexpression constructs and mini-gene constructs (Aim 2). Lastly, the physiological relevance of these observations will be assessed by testing for associations of genetically regulated alternative splicing with both in vivo plasma LDL levels and in vitro cholesterol-related phenotypes. SNP functionality will be confirmed by site directed mutagenesis of mini-gene constructs (Aim 3). Demonstration of the role of alternative splicing in the regulation of cholesterol metabolism and identification of genetic determinants of this process will aid in delineating molecular pathways contributing to inter-individual variation in plasma LDL and thus improve our understanding of cardiovascular disease development and risk. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to test if alternative splicing is a novel mechanism of regulating cholesterol homeostasis. Specifically, we seek to determine the extent of sterol regulated alternative splicing of genes involved in cholesterol biosynthesis and uptake, as well as to identify both genetic and non-genetic regulators of this response that are associated with both in vivo and cellular cholesterol metabolism. Thus, this research will contribute to our understanding of molecular determinants of variation in plasma LDL cholesterol, further adding to our knowledge of cardiovascular disease risk and development.