The Hrp65 self-interaction is mediated by an evolutionarily conserved domain and is required for nuclear import of Hrp65 isoforms that lack a nuclear localization signal
The Hrp65 self-interaction is mediated by an evolutionarily conserved domain and is required for nuclear import of Hrp65 isoforms that lack a nuclear localization signal
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DOI：
10.1242/jcs.00690
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发表时间：
2003-10-01
影响因子：
4
通讯作者：
Visa, N
中科院分区：
文献类型：
--
作者：
Kiesler, E;Miralles, F;Visa, N
Hrp65是摇蚊摇蚊的一种进化保守的RNA结合蛋白，具有一个保守的DBHS（Drosophila behavior，human splicing）结构域，该结构域也存在于几种哺乳动物蛋白中。在酵母双杂交筛选中，我们发现Hrp65可以与自身相互作用。在这里，我们证实了Hrp65的自我相互作用，在体外下拉实验和地图的序列负责的相互作用的区域，我们称之为位于DBHS结构域内的蛋白质结合结构域。我们还表明，果蝇NonA和人类PSF的蛋白质结合结构域，其他两种蛋白质与保守的DBHS结构域，结合到酵母双杂交系统中的Hrp65。这些观察结果表明，蛋白质结合结构域可以介导Hrp65的同源二聚化以及不同的含DBHS的蛋白质之间的异源二聚化。此外，通过凝胶过滤色谱法对重组Hrp65的分析表明，Hrp65不仅可以二聚化，而且可以寡聚化成至少3至6个分子的复合物。此外，我们分析了Hrp65自身相互作用在共转染试验中的功能意义，我们的结果表明，不同的Hrp65亚型之间的相互作用是至关重要的细胞内定位。
Hrp65, an evolutionary conserved RNA-binding protein from the midge Chironomus tentans, has a conserved DBHS (Drosophila behavior, human splicing) domain that is also present in several mammalian proteins. In a yeast two-hybrid screening we found that Hrp65 can interact with itself. Here we confirm the Hrp65 self-interaction by in vitro pull-down experiments and map the sequences responsible for the interaction to a region that we refer to as the protein-binding domain located within the DBHS domain. We also show that the protein-binding domains of Drosophila NonA and human PSF, two other proteins with conserved DBHS domains, bind to Hrp65 in the yeast two-hybrid system. These observations indicate that the protein-binding domain can mediate homodimerization of Hrp65 as well as heterodimerization between different DBHS-containing proteins. Moreover, analyses of recombinant Hrp65 by gel-filtration chromatography show that Hrp65 can not only dimerize but also oligomerize into complexes of at least three to six molecules. Furthermore, we have analyzed the functional significance of the Hrp65 self-interaction in cotransfection assays, and our results suggest that the interaction between different Hrp65 isoforms is crucial for their intracellular localization.