IDENTIFICATION OF XIST RNA INTERACTING PROTEINS

X失活是哺乳动物的一种补偿剂量的机制 具有两条X染色体的女性与只有一条X染色体的男性的差异 X染色体。X失活需要Xist基因，一种编码 一种覆盖在不活跃的X染色体上的大型结构RNA 与核基质相互作用。本研究计划是基于 Xist RNA在结构和功能上起作用的假说 形成异染色质X染色体，并发生在 核糖核蛋白(RNP)复合体。要了解Xist的工作原理和 异染色质是如何在X失活中构建的，我将确定 与Xist RNA相互作用的因子，特别是蛋白质 RNP的组件。为了最大限度地分离Xist RNA 相互作用的蛋白质，将采取两种互补的策略 平行：1)酵母三杂交系统将用于筛选 以Xist RNA片段为“诱饵”的相互作用蛋白质。这部小说 遗传方法利用了现有的专业知识和试剂 在李博士的实验室里。2)基于亲和力的生化方法 层析法。含Xist RNA的络合物将通过以下方式纯化 利用Xist RNA结合反义Xist基因的能力 固定在磁珠上。由于Xist RNA很大，很可能 在体内结合许多因子。同时使用基因和生物化学 方法很重要，因为它们不同的方法可能会产生 Xist RNA不同相互作用伙伴的鉴定。

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.