Dimaano C, Ball J R, Prunuske A J, Ullman K S
Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112, USA.
J Biol Chem. 2001 Nov 30;276(48):45349-57. doi: 10.1074/jbc.M102592200. Epub 2001 Sep 20.
Traffic between the nucleus and cytoplasm takes place through a macromolecular structure termed the nuclear pore complex. To understand how the vital process of nucleocytoplasmic transport occurs, the contribution of individual pore proteins must be elucidated. One such protein, the nucleoporin Nup153, is localized to the nuclear basket of the pore complex and has been shown to be a central component of the nuclear transport machinery. Perturbation of Nup153 function was demonstrated previously to block the export of several classes of RNA cargo. Moreover, these studies also showed that Nup153 can stably associate with RNA in vitro. In this study, we have mapped a domain within Nup153, encompassing amino acids 250-400 in human Nup153, that is responsible for RNA association. After cloning this region of Xenopus Nup153, we performed a cross-species analysis. Despite variation in sequence conservation between Drosophila, Xenopus, and human, this domain of Nup153 displayed robust RNA binding activity in each case, indicating that this property is a hallmark feature of Nup153 and pointing toward a subset of amino acid residues that are key to conferring this ability. We have further determined that a recombinant fragment of Nup153 can bind directly to RNA and that this fragment can interact with endogenous RNA targets. Our findings identify a functionally conserved domain in Nup153 and suggest a role for RNA binding in Nup153 function at the nuclear pore.
细胞核与细胞质之间的物质运输通过一种称为核孔复合体的大分子结构进行。为了理解核质运输这一重要过程是如何发生的,必须阐明单个孔蛋白的作用。其中一种蛋白,核孔蛋白Nup153,定位于孔复合体的核篮,并且已被证明是核运输机制的核心成分。先前已证明Nup153功能的扰动会阻断几类RNA货物的输出。此外,这些研究还表明Nup153在体外能与RNA稳定结合。在本研究中,我们在Nup153中定位了一个结构域,该结构域在人类Nup153中包含氨基酸250 - 400,它负责与RNA结合。克隆非洲爪蟾Nup153的这一区域后,我们进行了跨物种分析。尽管果蝇、非洲爪蟾和人类之间的序列保守性存在差异,但Nup153的这一结构域在每种情况下都表现出强大的RNA结合活性,表明这一特性是Nup153的标志性特征,并指向赋予这种能力的关键氨基酸残基子集。我们进一步确定Nup153的一个重组片段可以直接与RNA结合,并且该片段可以与内源性RNA靶点相互作用。我们的研究结果确定了Nup153中一个功能保守的结构域,并提示RNA结合在核孔处Nup153功能中的作用。