IDENTIFICATION OF XIST RNA INTERACTING PROTEINS

XIST RNA 相互作用蛋白的鉴定

• 批准号: 2773473
• 负责人: DAVID WARSHAWSKY
• 金额: $ 2.14万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-08 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2773473
• 关键词: RNA; affinity chromatography; antisense nucleic acid; genetic techniques; heterochromatin; intermolecular interaction; ribonucleoproteins; sex chromosomes

X-inactivation is a mammalian mechanism to compensate for dosage differences between females with two X-chromosomes and males with one X chromosome. X-inactivation requires the Xist gene, a gene encoding a large structural RNA that coats the inactive X-chromosome and interacts with the nuclear matrix. This research plan is based on the hypothesis that Xist RNA plays a structural and functional role in forming the heterochromatic X-chromosome and occurs within a ribonucleoprotein (RNP) complex. To understand how Xist functions and how heterochromatin is constructed in X-inactivation, I will identify factors which interact with Xist RNA, focusing specifically on protein components of the RNP. To maximize the chances of isolating Xist RNA interacting proteins, two complementary strategies will be taken in parallel: 1) The yeast three hybrid system will be used to screen for interacting proteins using Xist RNA fragments as "bait". This novel genetic approach takes advantage of the existing expertise and reagents in Dr. Lee's laboratory. 2) A biochemical approach based on affinity chromatography. Xist RNA-containing complexes will be purified by utilizing the ability of Xist RNA to bind antisense Xist cDNA immobilized on magnetic beads. Since Xist RNA is large, it is likely to bind many factors in vivo. Using both a genetic and a biochemical approach is important because their differing methodologies may yield the identification of different interacting partners for Xist RNA.

X灭活是一种补偿剂量的哺乳动物机制 两个X染色体和男性的女性之间的差异 X染色体。 X灭活需要Xist基因，一种编码的基因 一个大的结构RNA，可覆盖不活动的X染色体和 与核基质相互作用。 该研究计划基于 Xist RNA在 形成异光体X染色体，发生在 核糖核蛋白（RNP）复合物。 了解XIST的功能和 如何在X灭活中构建异染色质，我将确定 与Xist RNA相互作用的因素，专门针对蛋白质 RNP的组件。 为了最大化隔离Xist RNA的机会 相互作用的蛋白质，将采取两种互补策略 并行：1）酵母三个混合动力系统将用于筛选 使用Xist RNA片段作为“诱饵”进行相互作用的蛋白质。 这本小说 遗传方法利用现有的专业知识和试剂 在李博士的实验室中。 2）基于亲和力的生化方法 色谱法。 Xist含RNA的复合物将通过 利用Xist RNA结合反义Xist cDNA的能力 固定在磁珠上。 由于Xist RNA很大，因此很可能 在体内结合许多因素。 同时使用遗传和生化 方法很重要，因为它们的不同方法可能会产生 识别Xist RNA的不同相互作用伙伴。