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mRNA（信使RNA），然后输出到细胞质中。在那里，mRNA通过一种称为核糖体的特殊机制翻译成蛋白质。mRNA可用性的控制是细胞代谢的中心调节机制。在这个项目中，单个mRNA分子将从它们的出生到它们在细胞质中的出现进行跟踪。在转录过程中，RNA被修饰和包装。RNA在细胞核中以mRNP（信使核糖核颗粒）的形式存在。目前还不知道这些mRNP如何被修改以被出口机构认可。一个输出事件的时间，输出过程中mRNA的方向，mRNA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非常重要。在此之前，不可能在活细胞内标记mRNA以用于微观应用。使用一种新的病毒系统（MS2），有可能修饰一个基因，使其mRNA含有一个被MS2结合蛋白（MBP）高度特异性结合的序列。MBP对于哺乳动物细胞不是内源性的，并且可以与荧光蛋白缀合以用作mRNA的标记物。为了在活细胞中追踪这种MS2标记的mRNA，将建立一种新的多路复用高速显微镜，允许以毫秒时间分辨率观察至少三种不同的荧光通道。