Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export.
Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export.
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DOI：
10.1083/jcb.200108007
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发表时间：
2001-10-29
期刊：
影响因子：
--
通讯作者：
Forbes DJ
中科院分区：
文献类型：
--
作者：
Vasu S;Shah S;Orjalo A;Park M;Fischer WH;Forbes DJ
进行核输出的RNA首先遇到核孔的篮。两个篮子蛋白，Nup 98和Nup 153，是mRNA输出所必需的，但它们在孔内的分子伴侣在很大程度上是未知的。因为只要脊椎动物重要的孔蛋白仍未被发现，RNA输出的机制就将受到质疑，我们着手寻找它们的伴侣。Nup 98和Nup 153的片段用于爪蟾卵提取物的下拉实验，其含有丰富的解体的核孔。引人注目的是，Nup 98和Nup 153各自结合相同的四种大蛋白。纯化和序列分析表明，两个是已知的脊椎动物核孔蛋白，Nup 96和Nup 107，而两个映射到未知功能的ORF。克隆了编码新蛋白的基因，并制备了抗体。免疫荧光显示它们是新的核孔蛋白，命名为Nup 160和Nup 133，它们在孔的篮侧上可接近。核孔蛋白Nup 160、Nup 133、Nup 107和Nup 96以复合物形式存在于爪蟾卵提取物和组装孔中，现在称为Nup 160复合物。Sec 13在Nup 98和Nup 153下拉中表现突出，我们发现它是Nup 160复合体的一员。我们已经绘制了结合Nup 160亚复合物所需的位点，并发现在Nup 98中，结合位点用于将Nup 98拴在细胞核上;在Nup 153中，结合位点将Nup 153靶向核孔。通过转染和体内转运试验，我们发现特定的Nup 160和Nup 133片段阻断poly[A]+ RNA的输出，但不阻断蛋白质的输入或输出。这些结果表明，两个新的脊椎动物核孔蛋白，Nup 160和Nup 133，不仅与Nup 98和Nup 153相互作用，但本身发挥作用的mRNA输出。
RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.
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