Institute of Molecular Genetics, Czech Academy of Science, Prague, Czech Republic.
Faculty of Science, Charles University, Prague 14220, Czech Republic.
J Cell Sci. 2022 Apr 15;135(8). doi: 10.1242/jcs.259587. Epub 2022 Apr 25.
Coilin is a conserved protein essential for integrity of nuclear membrane-less inclusions called Cajal bodies. Here, we report an amino acid substitution (p.K496E) found in a widely-used human EGFP-coilin construct that has a dominant-negative effect on Cajal body formation. We show that this coilin-K496E variant fails to rescue Cajal bodies in cells lacking endogenous coilin, whereas the wild-type construct restores Cajal bodies in mouse and human coilin-knockout cells. In cells containing endogenous coilin, both the wild-type and K496E variant proteins accumulate in Cajal bodies. However, high-level overexpression of coilin-K496E causes Cajal body disintegration. Thus, a mutation in the C-terminal region of human coilin can disrupt Cajal body assembly. Caution should be used when interpreting data from coilin plasmids that are derived from this variant (currently deposited at Addgene).
科尔林是一种保守的蛋白质，对于核膜无膜包含体（称为卡哈尔体）的完整性至关重要。在这里，我们报告了在广泛使用的人类 EGFP-科尔林构建体中发现的一个氨基酸取代（p.K496E），它对卡哈尔体形成具有显性负效应。我们表明，这种科尔林-K496E 变体不能挽救缺乏内源性科尔林的细胞中的卡哈尔体，而野生型构建体则可以在小鼠和人类科尔林敲除细胞中恢复卡哈尔体。在含有内源性科尔林的细胞中，野生型和 K496E 变体蛋白都在卡哈尔体中积累。然而，科尔林-K496E 的高表达会导致卡哈尔体解体。因此，人类科尔林 C 末端区域的突变会破坏卡哈尔体的组装。在解释源自该变体的科尔林质粒的数据时应谨慎（目前保存在 Addgene）。
