Imaging dynamics of endogenous mitochondrial RNA in single living cells
Imaging dynamics of endogenous mitochondrial RNA in single living cells
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DOI：
10.1038/nmeth1030
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发表时间：
2007-05-01
期刊：
影响因子：
48
通讯作者：
Umezawa, Yoshio
中科院分区：
文献类型：
--
作者：
Ozawa, Takeaki;Natori, Yutaka;Umezawa, Yoshio
我们开发了基因编码的RNA探针，用于表征单个活细胞中线粒体RNA（mtDNA）的定位和动态。该探针由两个RNA-binding domain的ILIO 1，每个连接分裂片段的荧光蛋白能够重组后结合到一个目标RNA。我们设计的探针可以特异性识别编码NADH脱氢酶亚基6（ND 6）的16个碱基的mtDNA序列，并靶向线粒体基质，从而可以对活细胞中ND 6 mtDNA的定位进行实时成像。我们发现ND 6 mtDNA定位于线粒体内，特别是集中在线粒体DNA（mtDNA）上。ND 6 mtDNA的运动受到限制，但氧化应激诱导mtDNA分散在线粒体中并逐渐分解。这些探针提供了一种手段来研究在活的哺乳动物细胞的细胞内区室的mRNA的空间和时间的动态。
We developed genetically encoded RNA probes for characterizing localization and dynamics of mitochondrial RNA (mtRNA) in single living cells. The probes consist of two RNA-binding domains of PUMILIO1, each connected with split fragments of a fluorescent protein capable of reconstituting upon binding to a target RNA. We designed the probes to specifically recognize a 16-base sequence of mtRNA encoding NADH dehydrogenase subunit 6 (ND6) and to be targeted into the mitochondrial matrix, which allowed real-time imaging of ND6 mtRNA localization in living cells. We showed that ND6 mtRNA is localized within mitochondria and concentrated particularly on mitochondrial DNA (mtDNA). Movement of the ND6 mtRNA is restricted but oxidative stress induces the mtRNA to disperse in the mitochondria and gradually decompose. These probes provide a means to study spatial and temporal mRNA dynamics in intracellular compartments in living mammalian cells.