Correct cellular localization is essential for the function of many eukaryotic proteins and hence cell physiology. Here, we present a synthetic genetic device that allows to control nuclear and cytosolic localization based on controlled alternative splicing in human cells. The device is based on the fact that an alternative 3' splice site is located within a TetR aptamer that in turn is positioned between the branch point and the canonical splice site. The novel splice site is only recognized when the TetR repressor is bound. Addition of doxycycline prevents TetR aptamer binding and leads to recognition of the canonical 3' splice site. It is thus possible to produce two independent splice isoforms. Since the terminal loop of the aptamer may be replaced with any sequence of choice, one of the two isoforms may be extended by the respective sequence of choice depending on the presence of doxycycline. In a proof-of-concept study, we fused a nuclear localization sequence to a cytosolic target protein, thus directing the protein into the nucleus. However, the system is not limited to the control of nuclear localization. In principle, any target sequence can be integrated into the aptamer, allowing not only the production of a variety of different isoforms on demand, but also to study the function of mislocalized proteins. Moreover, it also provides a valuable tool for investigating the mechanism of alternative splicing in human cells.

中文翻译：

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通过 TetR 适体控制的 3' 剪接位点选择诱导核输入

正确的细胞定位对于许多真核蛋白质的功能以及细胞生理学至关重要。在这里，我们提出了一种合成遗传装置，可以基于人类细胞中受控的选择性剪接来控制核和胞质定位。该装置基于以下事实：替代的 3' 剪接位点位于 TetR 适体内，而 TetR 适体又位于分支点和规范剪接位点之间。只有当 TetR 阻遏物结合时，新的剪接位点才会被识别。添加多西环素可防止 TetR 适体结合并导致经典 3' 剪接位点的识别。因此可以产生两种独立的剪接同工型。由于适体的末端环可以用任何选择的序列替换，因此两种同工型之一可以根据多西环素的存在通过相应的选择序列延伸。在一项概念验证研究中，我们将核定位序列与胞质靶蛋白融合，从而引导蛋白质进入细胞核。然而，该系统并不局限于核定位的控制。原则上，任何靶序列都可以整合到适配体中，不仅可以按需生产多种不同的亚型，还可以研究错误定位蛋白质的功能。此外，它还为研究人类细胞中选择性剪接的机制提供了一个有价值的工具。