Single-molecule imaging of processing and transport of mRNA within a living cell

活细胞内 mRNA 加工和运输的单分子成像

• 批准号: 11480196
• 负责人: FUNATSU Takashi
• 金额: $ 8.64万
• 依托单位: Waseda University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480196/
• 关键词: mRNA; single-molecule imaging; fluorescence microscopy; スプライシング; 蛍光エネルギー移動

Messenger RNA is transcribed and processed in the nucleus and transported to the cytoplasm. In order to clarify the molecular mechanism of these processes, we have developed video-rate confocal microscopy that enabled us to visualize single fluorescence molecules. Movement of individual mRNA molecules within living cell was visualized by this microscopy to distinguish whether mRNA is directed by active transport or moving around by diffusion. For this purpose, Cy3-labeled truncated human β-globin mRNA molecules were prepared in vitro and micro-injected into the nucleus of Xenopus A6 cell. Cy3-labeled mRNA molecules could be observed except nucleoli, indicating that mRNA could access most of the space, excluding nucleoli, in the nucleus. Some population of mRNA moved around in diffusion, and some remained stationary. Statistical analysis of individual mRNA trajectories revealed that about 50 % of mRNA is moving at the diffusion constant of 0.2[μm^2/s]. This value is about 1/40 of that in solution (9 [μm2/s]). Our results support the mechanism that mRNA reach nuclear pores by diffusion process.

信使RNA在细胞核中转录和加工，并转运到细胞质。为了阐明这些过程的分子机制，我们开发了视频共聚焦显微镜，使我们能够可视化单个荧光分子。通过这种显微镜观察活细胞内单个mRNA分子的运动，以区分mRNA是通过主动运输还是通过扩散四处移动。为此，我们体外制备了Cy 3标记的截短型人β-珠蛋白mRNA分子，并将其显微注射到非洲爪蟾A6细胞核中。Cy 3标记的mRNA分子可进入除核仁外的大部分核内空间。一些mRNA群体在扩散中四处移动，而另一些则保持静止。单个mRNA轨迹的统计分析显示，约50%的mRNA以0.2[μm^2/s]的扩散常数移动。该值约为溶液中的1/40（9 [μm2/s]）。我们的结果支持mRNA通过扩散过程到达核孔的机制。

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