In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains.
In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains.
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Y14和NXF1 mRNA输出复合物的体内BIFC分析：在SC35域内及其周围的优先定位。
DOI：
10.1083/jcb.200503061
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发表时间：
2006-01-30
影响因子：
7.8
通讯作者：
Lichter, Peter
中科院分区：
文献类型：
--
作者：
Schmidt, Ute;Richter, Karsten;Berger, Axel Bernhard;Lichter, Peter
应用双分子荧光互补（BiFC）分析法研究剪接因子Y14与核输出因子1（NXF1）之间的复合物形成，该复合物与核mRNA的功能相关。Y14连接到黄色荧光蛋白的COOH末端（YFP; YC-Y14），并且NXF1融合到在MCF7细胞中表达的YFP的NH 2末端（YN-NXF1），在特异性结合时产生BiFC。荧光积聚在核斑点内和周围，表明斑点参与mRNA加工和输出。因此，BiFC依赖于转录和全长NXF 1。YC-Y14与YN-NXF1、NXF1、Y14和RNA的免疫共沉淀表明YC-Y14和YN-NXF1与RNA功能相关。光漂白后的荧光恢复和光漂白中的荧光损失表明，大约一半的积累的BiFC复合物在体内是不动的。这种不动的部分很容易耗尽三磷酸腺苷（ATP）的透化细胞管理。这些结果表明，RNA的一部分，这仍然是在细胞核中的几个小时，尽管它与剪接和输出蛋白，积累斑点，因为ATP依赖的机制。
The bimolecular fluorescence complementation (BiFC) assay, which allows the investigation of interacting molecules in vivo, was applied to study complex formation between the splicing factor Y14 and nuclear export factor 1 (NXF1), which evidence indicates are functionally associated with nuclear mRNA. Y14 linked to the COOH terminus of yellow fluorescent protein (YFP; YC-Y14), and NXF1 fused to the NH2 terminus of YFP (YN-NXF1) expressed in MCF7 cells yielded BiFC upon specific binding. Fluorescence accumulated within and around nuclear speckles, suggesting the involvement of speckles in mRNA processing and export. Accordingly, BiFC depended on transcription and full-length NXF1. Coimmunoprecipitation of YC-Y14 with YN-NXF1, NXF1, Y14, and RNA indicated that YC-Y14 and YN-NXF1 functionally associate with RNA. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching revealed that roughly half of the accumulated BiFC complexes were immobile in vivo. This immobile fraction was readily depleted by adenosine triphosphate (ATP) administration in permeabilized cells. These results suggest that a fraction of RNA, which remains in the nucleus for several hours despite its association with splicing and export proteins, accumulates in speckles because of an ATP-dependent mechanism.