Movement and localization of RNA in the cell nucleus.
Movement and localization of RNA in the cell nucleus.
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RNA 在细胞核中的运动和定位。
DOI：
10.1096/fasebj.13.9002.s238
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Pederson,T
中科院分区：
文献类型：
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作者：
Pederson,T
各种RNA从它们的染色体合成位点移动到它们在细胞中的功能位置是真核基因读出的重要步骤，尽管与RNA的转录、RNA加工和各种功能相比，这一步骤还不太清楚。从物理化学的观点来看，许多种类的RNA被分离到细胞中合适的位置是一个值得注意的现象。本文总结了我和我的同事在过去7年中进行的研究，以描述活细胞中RNA的细胞内运输和定位。我们已经开发的一种方法是玻璃针显微注射100.01 μ l的荧光RNA溶液到培养的哺乳动物细胞的细胞核或细胞质中。这种“荧光RNA细胞化学”方法已经解决了前信使RNA（前mRNA）具有高亲和力的核内位点（“斑点”），并揭示了某些其他RNA从其核质注射位点到核仁的非常快速的运动。这些快速运输的核仁RNA之一是信号识别颗粒（SRP）RNA，进一步的结果表明，核仁是一个网站的SRP RNA加工或核糖核蛋白组装之前输出到细胞质。在这些荧光RNA显微注射研究中，我们还使用了突变的RNA分子来鉴定特定的核苷酸序列，这些序列作为RNA在其各自的核内位点定位的靶向元件。在第二种方法中，我们使用了荧光相关光谱（FCS），一种经典的生物物理方法，用于测量体外分子运动，再加上共聚焦荧光显微镜来测量细胞核中的多聚（A）RNA的运动，有趣的发现是，这些RNA似乎在细胞核内移动的速度与水溶液中的扩散速度相当。使用光漂白后荧光恢复法（FRAP）的平行实验显示，核内多聚（A）RNA的扩散系数接近FCS测量的扩散系数。这些结果与核质基质的结构有关-这是细胞生物学中一个极具争议和未解决的问题（29）。我们开发的方法和这些初步结果代表了全面了解细胞核中RNA运输的第一个重要步骤。Pederson，T. RNA在细胞核中的移动和定位.FASEB J.13（增刊），S238-S242（1999）
The movement of various RNAs from their sites of chromosomal synthesis to their functional locations in the cell is an important step in eukaryotic gene readout, though one less well understood than the transcription, RNA processing, and various functions of RNA. The segregation of the many classes of RNA out into to their appropriate sites in the cell is, from a physical chemical point of view, a remarkable phenomenon. This paper summarizes investigations my colleagues and I have undertaken over the past 7 years to describe the intracellular traffic and localization of RNA in living cells. One approach we have developed is to glass‐needle microinject ∼0.01 pl of fluorescent RNA solutions into the nucleus or cytoplasm of cultured mammalian cells. This ‘fluorescent RNA cytochemistry’ approach has resolved intranuclear sites (‘speckles’) for which premessenger RNAs (pre‐mRNA) have high affinity and has revealed very rapid movements of certain other RNAs from their nucleoplasmic injection sites to the nucleoli. One of these rapidly trafficking nucleolar RNAs is the signal recognition particle (SRP) RNA, and further results indicate that the nucleolus is a site of SRP RNA processing or ribonucleoprotein assembly prior to export to the cytoplasm. In these fluorescent RNA microinjection studies, we have also used mutant RNA molecules to identify specific nucleotide sequences that function as targeting elements for the localization of RNAs at their respective intranuclear sites. In a second approach, we have used fluorescent correlation spectroscopy (FCS), a classical biophysical method for measuring molecular motionin vitro, coupled with confocal fluorescence microscopy to measure the movement of poly(A) RNA in the nucleus, with the interesting finding that these RNAs appear to move about inside the nucleus at rates comparable to diffusion in aqueous solution. Parallel experiments using the method of fluorescence recovery after photobleaching (FRAP) revealed a diffusion coefficient for intranuclear poly(A) RNA close to that measured by FCS. These results bear on the structure of the nucleoplasmic ground substance‐an extremely controversial and unsolved problem in cell biology (29). The methods we have developed and these initial results represent the first major step toward a comprehensive understanding of RNA traffic in the cell nucleus.—Pederson, T. Movement and localization of RNA in the cell nucleus.FASEB J.13 (Suppl.), S238‐S242 (1999)