Characterization of cyclin L1 as an immobile component of the splicing factor compartment
Characterization of cyclin L1 as an immobile component of the splicing factor compartment
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DOI：
10.1096/fj.07-8377com
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发表时间：
2007-10-01
期刊：
影响因子：
4.8
通讯作者：
Becker, Walter
中科院分区：
文献类型：
--
作者：
Herrmann, Andreas;Fleischer, Katrin;Becker, Walter
细胞周期蛋白 L1 和细胞周期蛋白 L2 是细胞周期蛋白家族的两个密切相关的成员，包含 C 端富含精氨酸和丝氨酸 (RS) 的结构域，并且位于剪接因子区室（核斑点）中。在这里，我们应用光漂白技术来表明，与细胞周期蛋白 L2 相比，细胞周期蛋白 L1 的绿色荧光蛋白 (GFP) 融合蛋白在活 COS7 细胞的细胞核内不可移动。本研究的目的是 1) 表征不同细胞状态下细胞周期蛋白 L1 的核内定位和移动特性，2) 剖析固定细胞周期蛋白 L1 所需的结构元件。放线菌素 D 的转录抑制导致 GFP-细胞周期蛋白 L2 在圆形且增大的核斑点中积累，但不影响 GFP-细胞周期蛋白 L1 的亚核分布模式。尽管 GFP-cyclin L1 在细胞周期的大多数阶段不流动，但在中期细胞的细胞质中广泛分布且高度流动。通过对一系列嵌合体、缺失构建体和点突变体的分析，确定了细胞周期蛋白 L1 的 RS 结构域内的一个片段对于核斑点中蛋白质的固定性是必需的。这项研究首次表征了核散斑的固定成分。
Cyclin L1 and cyclin L2 are two closely related members of the cyclin family that contain C-terminal arginine-and serine-rich (RS) domains and are localized in the splicing factor compartment (nuclear speckles). Here we applied photobleaching techniques to show that a green fluorescent protein (GFP) fusion protein of cyclin L1, in contrast to cyclin L2, was not mobile within the nucleus of living COS7 cells. The objectives of this study were to 1) characterize the intranuclear localization and mobility properties of cyclin L1 in different cellular states, and 2) dissect the structural elements required for immobilization of cyclin L1. Transcriptional arrest by actinomycin D caused accumulation of GFP-cyclin L2 in rounded and enlarged nuclear speckles but did not affect the sub-nuclear pattern of distribution of GFP-cyclin L1. Although immobile in most phases of the cell cycle, GFP-cyclin L1 was diffusely distributed and highly mobile in the cytoplasm of metaphase cells. By analysis of a series of chimeras, deletion constructs, and a point mutant, a segment within the RS domain of cyclin L1 was identified to be necessary for the immobility of the protein in nuclear speckles. This study provides the first characterization of an immobile component of nuclear speckles.