Mutational analysis of fibrillarin and its mobility in living human cells.
Mutational analysis of fibrillarin and its mobility in living human cells.
复制标题
DOI：
10.1083/jcb.151.3.653
复制
发表时间：
2000-10-30
影响因子：
7.8
通讯作者：
Dirks, R W
中科院分区：
文献类型：
--
作者：
Snaar, S;Wiesmeijer, K;Jochemsen, A G;Tanke, H J;Dirks, R W
Cajal小体(CBS)是一种亚核细胞器，含有RNA转录和RNA加工中许多不同途径的成分。CBS已经与核仁等其他亚核细胞器联系在一起，但CBS中存在核仁蛋白的原因尚不清楚。在这里，我们使用全长纤维蛋白和截短纤维蛋白突变体与绿色荧光蛋白(GFP)融合来证明纤维蛋白的特定结构域是纤维蛋白正确定位到核仁和CBS所必需的。第二个间隔区和羧基末端的α-螺旋结构域似乎分别针对核仁转录中心和CBS。RNP结构域的存在似乎是正确靶向纤维蛋白的先决条件。对稳定表达纤维蛋白-绿色荧光蛋白的人类细胞的延时共聚焦显微镜显示，CBS融合和分裂，尽管频率很低。光漂白后纤维蛋白-绿色荧光蛋白在核仁和胞核中的荧光恢复表明，它在两个细胞器中都有很高的迁移率(估计的扩散常数∼为0.02μm2 S−1)，并且在胞质中的迁移率明显大于在核仁中的迁移率。
Cajal bodies (CBs) are subnuclear organelles that contain components of a number of distinct pathways in RNA transcription and RNA processing. CBs have been linked to other subnuclear organelles such as nucleoli, but the reason for the presence of nucleolar proteins such as fibrillarin in CBs remains uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that specific structural domains of fibrillarin are required for correct intranuclear localization of fibrillarin to nucleoli and CBs. The second spacer domain and carboxy terminal alpha-helix domain in particular appear to target fibrillarin, respectively, to the nucleolar transcription centers and CBs. The presence of the RNP domain seems to be a prerequisite for correct targeting of fibrillarin. Time-lapse confocal microscopy of human cells that stably express fibrillarin-GFP shows that CBs fuse and split, albeit at low frequencies. Recovered fluorescence of fibrillarin-GFP in nucleoli and CBs after photobleaching indicates that it is highly mobile in both organelles (estimated diffusion constant ∼0.02 μm2 s−1), and has a significantly larger mobile fraction in CBs than in nucleoli.