Shen Zhifa, Paquin Nicolas, Forget Amélie, Chartrand Pascal
Département de Biochimie, Université de Montréal, Canada.
Mol Biol Cell. 2009 Apr;20(8):2265-75. doi: 10.1091/mbc.e08-11-1151. Epub 2009 Feb 25.
The transport and localization of mRNAs results in the asymmetric synthesis of specific proteins. In yeast, the nucleocytoplasmic shuttling protein She2 binds the ASH1 mRNA and targets it for localization at the bud tip by recruiting the She3p-Myo4p complex. Although the cytoplasmic role of She2p in mRNA localization is well characterized, its nuclear function is still unclear. Here, we show that She2p contains a nonclassical nuclear localization signal (NLS) that is essential for its nuclear import via the importin alpha Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS leads to defective ASH1 mRNA localization and Ash1p sorting. Interestingly, these phenotypes mimic knockouts of LOC1 and PUF6, which encode for nuclear RNA-binding proteins that bind the ASH1 mRNA and control its translation. We find that She2p interacts with both Loc1p and Puf6p and that excluding She2p from the nucleus decreases this interaction. Absence of nuclear She2p disrupts the binding of Loc1p and Puf6p to the ASH1 mRNA, suggesting that nuclear import of She2p is necessary to recruit both factors to the ASH1 transcript. This study reveals that a direct coupling between localization and translation regulation factors in the nucleus is required for proper cytoplasmic localization of mRNAs.
mRNA的运输和定位导致特定蛋白质的不对称合成。在酵母中，核质穿梭蛋白She2与ASH1 mRNA结合，并通过招募She3p-Myo4p复合体将其靶向定位到芽尖。尽管She2p在mRNA定位中的细胞质作用已得到充分表征，但其核功能仍不清楚。在这里，我们表明She2p含有一个非经典核定位信号（NLS），该信号对于其通过输入蛋白α Srp1p进行核输入至关重要。通过对其NLS进行诱变将She2p排除在细胞核外会导致ASH1 mRNA定位缺陷和Ash1p分选异常。有趣的是，这些表型类似于LOC1和PUF6的敲除，它们编码与ASH1 mRNA结合并控制其翻译的核RNA结合蛋白。我们发现She2p与Loc1p和Puf6p都相互作用，并且将She2p排除在细胞核外会减少这种相互作用。细胞核中缺乏She2p会破坏Loc1p和Puf6p与ASH1 mRNA的结合，这表明She2p的核输入对于将这两种因子募集到ASH1转录本上是必要的。这项研究表明，细胞核中定位和翻译调控因子之间的直接偶联对于mRNA在细胞质中的正确定位是必需的。
