PNRC1核定位信号序列的鉴定

为鉴定富含脯氨酸核受体辅调节蛋白1(PNRC1)分子的核定位信号序列（nuclear localization signal sequence, NLS），在生物信息学方法预测的基础上，先构建野生型PNRC1及删除预测NLS的PNRC1突变体的绿色荧光蛋白（GFP）重组表达载体，转染细胞后通过激光共聚焦显微镜观察PNRC1分子在删除预测NLS后细胞内的定位变化.然后,将预测的NLS编码序列直接连到GFP表达载体上，以及将预测的NLS加到胞浆蛋白上构建其GFP重组表达载体，转染细胞，观察预测的NLS能否把构建的重组体都带到细胞核内.结果显示,删除PNRC1中预测的NLS后，其定位从细胞核中变为主要定位在细胞浆中，而预测的NLS能把GFP或胞浆中的蛋白带到细胞核中.研究表明,预测的NLS为PNRC1分子真正的NLS.

Identification of Nuclear Localization Signal Sequence of PNRC1

To identify nuclear localization signal sequence (NLS) of proline-rich nuclear receptor coregulator protein 1 (PNRC1), vectors expressing green fluorescence protein(GFP)-tagged PNRC1 and GFP-tagged PNRC1 mutant with the putative NLS(aa 94-101, PKKRRKKK) deletion were generated then transfected into mammalian cells. The subcellular localization of PNRC1 and putative NLS deleted PNRC1 were examined by confocal microscope. In addition, recombinants expressing GFP-tagged putative NLS and GFP-tagged cytoplasm protein fused to putative NLS were constructsed, the subcellular localization of these NLS fusion proteins were examined in the transfected cells accordingly. The results demonstrated the native PNRC1 localized to the nucleus as expected, whereas the PNRC1 mutant with the putative NLS deletion primarily localized in the cytoplasm. The putative NLS of PNRC1 was found to be able to drive GFP and other cytoplasm protein into nucleus when it was fused to these proteins. We conclude that the putative NLS within PNRC1 indeed has a potent activity to mediate protein nuclear transportation.