Alternative Splicing in Regulation of Cholesterol Synthesis and Uptake

胆固醇合成和摄取调节中的选择性剪接

基本信息

批准号：
8114155
负责人：
Marisa Wong Medina
金额：
$ 40.13万
依托单位：
CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8114155
关键词：
25-hydroxycholesterol 3&apos Untranslated Regions 3-hydroxy-3-methylglutaryl-coenzyme A Affect Alternative Splicing Binding Cardiovascular Diseases Cell Line Cell surface Cells Cholesterol Cholesterol Homeostasis Complementary DNA Computer Simulation DNA Development Dose Enzymes Exons Gene Expression Genes Genetic Genetic Determinism Genetic Polymorphism Genetic Transcription Genome Goals Hepatocyte Human In Vitro Incubated Individual Individual Differences Introns LDL Cholesterol Lipoproteins Lead Link Lipids Literature Low Density Lipoprotein Receptor Low-Density Lipoproteins Lymphocyte Measurement Measures Mediating Mevalonate kinase Molecular Oxidoreductase Pathway interactions Phenotype Physiological Plasma Polypyrimidine Tract-Binding Protein Process Proteins RNA Splicing Regulation Research Risk Role SNP genotyping Simvastatin Single Nucleotide Polymorphism Site-Directed Mutagenesis Small Interfering RNA Spliced Genes Sterols Testing Trans-Activators Transfection Variant base cardiovascular disorder risk cholesterol biosynthesis genome-wide improved in vivo inhibitor/antagonist knock-down mRNA Precursor non-genetic novel overexpression public health relevance response uptake

项目摘要

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.

描述（由申请人提供）：LDL胆固醇水平对于确定心血管疾病的风险至关重要。最近，对细胞内胆固醇的两个最关键调节剂的替代剪接，3-羟基-3-甲基戊二酰 - 凝集酶辅酶A还原酶（HMGCR）（HMGCR）（HMGCR），胆固醇生物合成的速率限制酶，以及LDL受体（ldllr）的ldlmmam andmmam andmme andme a inmmam andme a inmmam以及辛伐他汀减少LDL的大小。最近的证据表明，参与胆固醇代谢的四个基因的替代剪接（HMGCR，LDLR，HMG-COA合酶和甲龙酸酯激酶）由固醇协同调节，从而通过固醇载荷增加了替代性剪接，而固醇减少了stterol Deppletion抑制替代性剪接。此外，辛伐他汀的全基因组转录分析孵育的人淋巴细胞细胞系表明，〜300个已知成分的95个已知成分对他汀类药物有反应（FDR <0.0001）。其中，几个剪接因子与其他证据的介导替代剪接的调节有关，包括：（1）基因表达变异与细胞表面LDLR和血浆LDL浓度的变异相关；（2）与血浆LDL水平相关的DNA多态性；（3）siRNA敲低导致前mRNA剪接变化；（4）在已知结合基序的计算机预测中。这些发现导致了以下假设：细胞内胆固醇水平调节剪接因子，从而在胆固醇合成和摄取的多个基因的替代剪接中产生协调的变化，并且该过程的变化是细胞和等离子胆固醇代谢的决定因素。因此，该提案的总体目标是：（1）证明替代剪接是调节细胞胆固醇合成和摄取以及血浆LDL水平的一种新型机制；（2）确定此过程的非遗传和遗传修饰符。确定固醇调节的替代剪接是否发生在胆固醇生物合成途径中的大量基因中；替代剪接的变化将在HEPG2细胞，原发性人肝细胞和永生化的人类淋巴细胞细胞系中进行定量，这些细胞用该途径的特定抑制剂和产物处理（AIM 1）。使用siRNA，过表达构建体和迷你基因构建体（AIM 2），将确定和验证负责策划这些协调变化的剪接因子。最后，这些观察结果的生理相关性将通过测试遗传调节的替代剪接与体内血浆LDL水平和体外胆固醇相关的表型的关联进行评估。 SNP功能将通过位点的定向小型基因构建体确认（AIM 3）。证明替代剪接在调节胆固醇代谢的调节中的作用以及该过程的遗传决定因素的鉴定将有助于描述有助于血浆LDL的个体间变化的分子途径，从而提高我们对心血管疾病发育和风险的理解。公共卫生相关性：该提案的目的是测试替代剪接是否是调节胆固醇稳态的新型机制。具体而言，我们寻求确定固醇调节的替代剪接的程度，涉及胆固醇生物合成和摄取的基因，以及确定与体内和细胞胆固醇代谢相关的该反应的遗传和非遗传调节剂。因此，这项研究将有助于我们理解血浆LDL胆固醇变异的分子决定因素，从而进一步增加了我们对心血管疾病风险和发育的了解。