Al-Maghrebi May, Brulé Hervé, Padkina Marina, Allen Carrie, Holmes W Michael, Zehner Zendra E
Department of Biochemistry and Molecular Biophysics and the Massey Cancer Center, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, VA 23298-0614, USA.
Nucleic Acids Res. 2002 Dec 1;30(23):5017-28. doi: 10.1093/nar/gkf656.
Previously, we have shown that the vimentin 3' untranslated region (3'UTR) contains a highly conserved region, which is sufficient for the perinuclear localization of a reporter mRNA. This region was shown to specifically bind protein(s) by band shift analyses. UV-cross-linking studies suggest these proteins are 46- and 35-kDa in mass. Here, we have used this sequence as 'bait' to isolate RNA binding proteins using the yeast three-hybrid method. This technique relies on a functional assay detecting bona fide RNA-protein interaction in vivo. Three cDNA isolates, HAX-1, eEF-1gamma and hRIP, code for proteins of a size consistent with in vitro cross- linking studies. In all cases, recombinant proteins were capable of binding RNA in vitro. Although hRIP is thought to be a general mRNA binding protein, this represents an unreported activity for eEF-1gamma and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin's 3'UTR. Both in vivo synthesized eEF-1gamma and HAX-1 proteins were 'pulled out' of HeLa whole cell extracts by binding to a RNA affinity column comprised of vimentin's 3'UTR. Moreover, size-fractionation of extracts results in the separation of large complexes containing either eEF-1gamma or HAX-1. Thus, in addition to their known functions, both eEF-1gamma and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.
此前，我们已经表明波形蛋白3'非翻译区（3'UTR）包含一个高度保守的区域，该区域足以使报告基因mRNA定位于细胞核周。通过凝胶迁移分析表明该区域能特异性结合蛋白质。紫外线交联研究表明这些蛋白质的分子量分别为46 kDa和35 kDa。在此，我们使用该序列作为“诱饵”，通过酵母三杂交方法分离RNA结合蛋白。该技术依赖于一种在体内检测真实RNA - 蛋白质相互作用的功能分析方法。三个cDNA分离物，即HAX - 1、eEF - 1γ和hRIP，编码的蛋白质大小与体外交联研究结果一致。在所有情况下，重组蛋白在体外都能够结合RNA。虽然hRIP被认为是一种普通的mRNA结合蛋白，但这代表了eEF - 1γ和HAX - 1一种未被报道的活性。此外，HAX - 1的结合似乎对波形蛋白的3'UTR具有特异性。通过与由波形蛋白3'UTR组成的RNA亲和柱结合，体内合成的eEF - 1γ和HAX - 1蛋白都从HeLa全细胞提取物中“被拉出”。此外，提取物的大小分级分离导致含有eEF - 1γ或HAX - 1的大复合物分离。因此，除了它们已知的功能外，eEF - 1γ和HAX - 1都是RNA结合蛋白，这表明它们在mRNA翻译和/或细胞核周定位中具有新的作用。
