Ozawa Takeaki, Umezawa Yoshio
Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan.
Methods Mol Biol. 2011;714:175-88. doi: 10.1007/978-1-61779-005-8_11.
Localization of mRNAs plays pivotal roles in different cell types, including neurons and the cells in the developing stages. To visualize the dynamic movements of mRNAs in living cells, many methods have been emerged in the past decade. However, it has not been realized to visualize endogenous mRNAs with genetically encoded fluorescent probes. We recently developed fluorescent protein-based RNA probes for characterizing the localization and dynamics of mRNAs in single living cells. The probes consist of two RNA-binding domains of human PUMILIO1, each connected with split fragments of a fluorescent protein capable of reconstitution upon binding to a target mRNA. The probes are modified to specifically recognize a 16-base sequence of an mRNA of interest and to target into organelles by means of a short signal peptide. We have shown that ND6 mRNA is concentrated particularly on mitochondrial DNA (mtDNA) and movement of the mRNA is restricted in mitochondria. The probes provide a general means to study spatial and temporal mRNA localization and dynamics in intracellular compartments in living cells.
mRNA的定位在包括神经元和发育阶段细胞在内的不同细胞类型中起着关键作用。为了可视化活细胞中mRNA的动态运动，在过去十年中出现了许多方法。然而，利用基因编码的荧光探针来可视化内源性mRNA尚未实现。我们最近开发了基于荧光蛋白的RNA探针，用于表征单个活细胞中mRNA的定位和动态。这些探针由人PUMILIO1的两个RNA结合结构域组成，每个结构域与一个荧光蛋白的分裂片段相连，该荧光蛋白在与靶mRNA结合时能够重新组装。这些探针经过修饰，能够特异性识别感兴趣mRNA的16个碱基序列，并通过短信号肽靶向细胞器。我们已经表明，ND6 mRNA特别集中在线粒体DNA（mtDNA）上，并且mRNA在线粒体中的移动受到限制。这些探针为研究活细胞内细胞区室中mRNA的时空定位和动态提供了一种通用方法。
