Beobachtung einzelner mRNA Transkripte im Zellkern und während des Kernporenkomplex vermittelten Exports in lebenden Zellen

观察细胞核中以及活细胞中核孔复合物介导的输出过程中的单个 mRNA 转录本

• 批准号: 52507302
• 负责人: Dr. David Grünwald
• 金额: --
• 依托单位: Kavli Institute of NanoScience
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/52507302?language=en
• 关键词: Beobachtung; einzelner; mRNA; Transkripte; im

The genetic information of DNA is transcribed into mRNA (messenger RNA), which gets exported into the cytoplasm. There mRNA is translated into proteins by a special machinery, called Ribosome. The control of mRNA availability is a central regulatory mechanism for cellular metabolism. Within this project single mRNA molecules shall be followed from their birth to their appearance in the cytoplasm. While transcribed, RNA gets modified and packed. RNA does exist as mRNP (messenger Ribo-Nucleo-Particle) in the nucleus. How and than these mRNPs gets modified to be recognized by the export machinery is currently not known. The time of one export event, the orientation of the mRNA during export, the question if the mRNA gets unfolded and how transport is terminated are important to elucidate our understanding of cellular function. Before it was impossible to label mRNA for microscopic applications inside living cells. Using a new viral system (MS2), it is possible to modify a gene so its mRNA contains a sequence which is highly specifically bound by the MS2 binding protein (MBP). MBP is not endogenous for mammalian cells and can be conjugated to a fluorescent protein to be used as marker for the mRNA. To track such MS2-tagged mRNAs in living cells a new multiplexing high speed microscope will be built, allowing the observation of at least three different fluorescence channels with millisecond time resolution.

DNA的遗传信息被转录成信使RNA(Messenger RNA)，输出到细胞质中。在那里，信使核糖体通过一种特殊的机制被翻译成蛋白质。控制信使核糖核酸的可获得性是细胞代谢的中心调节机制。在这个项目中，单个信使核糖核酸分子从它们出生到它们出现在细胞质中都将被跟踪。在转录的过程中，RNA被修饰和包装。核糖核酸确实以mRNP(信使核仁)的形式存在于细胞核中。目前尚不清楚这些mRNPs是如何被修改以被出口机构识别的。一次输出事件的时间、输出过程中信使核糖核酸的取向、信使核糖核酸是否展开以及运输如何终止的问题，对于阐明我们对细胞功能的理解是很重要的。以前，不可能在活细胞内标记用于显微应用的信使核糖核酸。使用一种新的病毒系统(MS2)，可以修改一个基因，使其mRNA包含一个高度特异地与MS2结合蛋白(MBP)结合的序列。MBP对哺乳动物细胞来说不是内源性的，可以与一种荧光蛋白结合，作为该mRNA的标记。为了在活细胞中追踪这种标记了MS2的mRNAs，将建造一台新的多路高速显微镜，允许以毫秒的时间分辨率观察至少三个不同的荧光通道。