RNA Transport Within the Nucleus

描述（由申请人提供）：本研究项目涉及一个团队， 两名细胞生物学家和一名生物药理学家， 最近和现在正在联合起来进行RNA定位的研究 和细胞核中的交通。这项研究几乎是独一无二的， 核内RNA的运动是在活细胞中追踪的，而不是通过需要化学固定的定位方法。这一点的重要性 这种方法不能被夸大，因为它打开了体内 研究核内RNA转运的动力学途径。两大 实验策略采取，无论是在P.I？的实验室， 是内源性核RNA的荧光标记和显微注射 荧光RNA进入哺乳动物细胞核。一个基于扩散的 核RNA运输已经出现了从这个初步的工作，这将是 进一步调查。拟议的研究将详细描述 核糖体RNA以及β-珠蛋白的核内运输模式， c-fos mRNA。将研究突变的β-珠蛋白RNA， 核内运输和定位缺陷。研究还将 进行剪接和多聚腺苷酸化，但核限制的RNA，Xist， 这是一种由失活的X染色体产生的不寻常的转录物， 预测显示出非常不同的核内运输性质， mRNA。该项目的另一个方面将解决核内交通的问题， 这些RNA与已知参与核输入的蛋白质有关， RNA输出，即Ran-GTTR和TAP。

DESCRIPTION (provided by applicant): This research project involves a team of two cell biologists and a biophysicist who have productively collaborated recently and now are joining together to undertake studies of RNA localization and traffic in the cell nucleus. The proposed research is virtually unique in that intranuclear RNA movements are tracked in living cells, rather than by localization methods that require chemical fixation. The importance of this approach cannot be overstated, since it opens the door to the in vivo investigation of dynamic pathways of intranuclear RNA transport. The two major experimental strategies to be taken, both developed in the P.I?s laboratory, are the fluorescent tagging of endogenous nuclear RNAs and the microinjection of fluorescent RNAs into mammalian cell nuclei. A diffusion-based model for nuclear RNA transport has emerged from this initial work and this will be further investigated. The proposed studies will characterize in detail the intranuclear trafficking patterns of ribosomal RNA as well as beta-globin and c-fos mRNAs. Mutant beta-globin RNAs will be investigated to relate processing defects to intranuclear transport and localization. Studies will also be undertaken of the spliced and polyadenylated but nucleus-restricted RNA, Xist, which is an unusual transcript produced by the inactive X chromosome and is predicted to display very different intranuclear transport properties than mRNAs. Another aspect of the project will address the intranuclear traffic of these RNAs in relation to proteins known to be involved in nuclear import or RNA export, i.e. the Ran-GTPase and TAP.