REGULATION OF CHOLESTEROL HOMEOSTASIS BY NONCODING RNAS

描述(由申请人提供)：细胞胆固醇稳态在多个胆固醇转移步骤中被调节，并通过一个负反馈回路对内质网(ER)中膜胆固醇的升高做出反应。这些类固醇感知和转运途径的改变有助于人类先天的新陈代谢错误和后天疾病状态。为了阐明控制这些关键的胆固醇稳态途径的机制，我们进行了一项功能遗传筛选，导致分离出具有细胞内胆固醇运输缺陷的细胞系，这些缺陷富含突变，并破坏了长非编码RNA(NcRNA)基因。这些基因表现出进化上保守的外显子/内含子组织和核心启动子区域，缺乏显著的开放阅读框架或外显子内的核苷酸同源性，并包含含有小核仁RNA(SnoRNA)物种的高度保守的内含子区域。这类长的ncRNAs似乎主要作为宿主基因来促进孤儿snoRNAs的表达和处理。我们推测，经过处理的snoRNA可能通过控制剪接事件或翻译来调节参与胆固醇稳态的基因的表达，从而代表了一种先前未知的细胞胆固醇稳态调节模式。该建议的具体目的是(1)鉴定通过我们的遗传筛选发现的对于维持胆固醇稳态至关重要的ncRNA基因，(2)确定snoRNA宿主基因在调节细胞内胆固醇转移和细胞胆固醇稳态中的作用，(3)确定孤儿snoRNA元件控制胆固醇调节途径的分子机制(S)，以及(4)通过将我们的基于细胞的研究扩展到体内动物模型来研究通过遗传筛选发现的孤儿snoRNA和基因通路的生理作用。提出的研究是创新的，因为他们探索了一种新的小RNA依赖的途径，以前没有涉及到细胞胆固醇稳态的调节。这些研究具有非常重要的意义，因为阐明这一RNA调节途径有可能为操纵细胞处理胆固醇提供新的分子靶点。这一建议与动脉粥样硬化高度相关，动脉粥样硬化是一种以胆固醇稳态失调为特征的常见疾病，也与罕见的、通常是致命的类固醇代谢先天错误有关。 公共卫生相关性：调节细胞胆固醇平衡的途径的改变有助于人类遗传性疾病和获得性疾病状态，如动脉粥样硬化。我们实验室的初步研究已经发现了一组以前没有涉及到这些胆固醇调节途径的基因。这项提议的目标是研究这些基因如何调节细胞中胆固醇的分布和水平。拟议的研究可能会为降低疾病状态下的胆固醇的药物开发确定新的靶点。

DESCRIPTION (provided by applicant): Cellular cholesterol homeostasis is regulated at multiple cholesterol transfer steps and through a negative feedback loop that responds to elevations of membrane cholesterol in the endoplasmic reticulum (ER). Alterations in these sterol sensing and trafficking pathways in contribute to human inborn errors of metabolism and to acquired disease states. To elucidate mechanisms governing these critical cholesterol homeostatic pathways, we performed a functional genetic screen that led to isolation of cell lines with intracellular cholesterol trafficking defects that were enriched in mutants with disruption of long non-coding RNA (ncRNA) genes. These genes exhibit evolutionarily conserved exon/intron organization and core promoter regions, lack significant open reading-frames or nucleotide homology within exons, and contain short, highly conserved intronic regions that harbor small nucleolar RNA (snoRNA) species. This class of long ncRNAs appears principally to serve as host genes to facilitate expressing and processing of the orphan snoRNAs. We hypothesize that the processed snoRNAs modulate expression of genes involved in cholesterol homeostasis, possibly through control of splicing events or translation, and thus represent a previously unrecognized mode of regulation for cellular cholesterol homeostasis. The Specific Aims of this proposal are (1) To characterize ncRNA genes identified by our genetic screen that are critical for maintenance of cholesterol homeostasis, (2) To determine the role of the snoRNA host genes in regulation of intracellular cholesterol transfer and cellular cholesterol homeostasis, (3) To determine the molecular mechanism(s) through which the orphan snoRNA elements exert control over cholesterol regulatory pathways, and (4) To examine the physiological role of the orphan snoRNAs and gene pathways identified through the genetic screen by extending our cell-based studies to in vivo animal models. The proposed studies are innovative in that they explore a novel small RNA-dependent pathway not previously implicated in regulation of cellular cholesterol homeostasis. These studies are highly significant because elucidation of this RNA regulatory pathway has the potential to provide new molecular targets for manipulation of the cellular handling of cholesterol. This proposal is highly relevant to atherosclerosis, a common disease characterized by dysregulation of cholesterol homeostasis, as well as to rare, often fatal inborn errors of sterol metabolism. PUBLIC HEALTH RELEVANCE: Alterations in pathways that regulate cellular cholesterol balance contribute to human genetic diseases and to acquired disease states, such as atherosclerosis. Preliminary studies in our laboratory have identified a set of genes not previously implicated in these cholesterol regulatory pathways. The goal of this proposal is to study how these genes modulate the distribution and level of cholesterol in cells. The proposed studies may identify new targets for drug development for lowering cholesterol in disease states.