Zaidi S H, Denman R, Malter J S
Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison 53792-2472.
J Biol Chem. 1994 Sep 30;269(39):24000-6.
Growing evidence suggests that Alzheimer's disease results from dysregulated production and deposition of beta-amyloid in the central nervous system. beta-Amyloid is derived from proteolytic processing of one of multiple amyloid precursor protein (APP) isoforms. The production of APP in many somatic tissues and tumor cell lines provides a more accessible model to study the regulation of APP gene expression. Recent data suggest that APP mRNAs accumulate in activated lymphocytes and neuronal tumor lines. We are interested in defining the contribution of alterations in stability to changes in steady-state APP mRNA levels in these model systems. Herein we demonstrate by mobility shift assay that the 3'-untranslated region of APP RNAs which contain a contiguous 29-base region interacts in vitro with multiple mRNA-binding proteins found in cytosolic lysates prepared from normal and transformed human cells. UV cross-linking of radiolabeled APP RNAs to cytosolic protein extracts followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified six distinct RNA-protein complexes of 42, 47, 65, 73, 84, and 104 kDa. Competition assays with APP, AU-rich, or irrelevant RNAs demonstrated that binding was specific and in some cases preferential for AU- or U-rich sequences by which we tentatively place the binding site of the proteins along the 29-base region. APP mRNA-binding proteins were constitutively active in all tumor lines examined as well as at diminished levels in whole human brain cytosolic lysates. The core element is AU-rich and highly conserved between human and some murine APP mRNAs. In the accompanying paper (Zaidi, S. H. E. and Malter, J. S. (1994) J. Biol. Chem. 269, 24007-24013) we show that this 29-base element in the 3'-untranslated region regulates the stability of APP mRNA. Cumulatively these data suggest that steady-state APP mRNA levels are modulated by cytosolic protein-RNA interactions.
越来越多的证据表明，阿尔茨海默病是由中枢神经系统中β-淀粉样蛋白的产生失调和沉积所致。β-淀粉样蛋白源自多种淀粉样前体蛋白（APP）异构体之一的蛋白水解加工。在许多体细胞组织和肿瘤细胞系中产生的APP为研究APP基因表达的调控提供了一个更容易获取的模型。最近的数据表明，APP mRNA在活化的淋巴细胞和神经元肿瘤细胞系中积累。我们感兴趣的是确定在这些模型系统中，稳定性改变对稳态APP mRNA水平变化的作用。在此，我们通过迁移率变动分析证明，APP RNA的3'-非翻译区包含一个连续的29个碱基的区域，在体外与从正常和转化的人类细胞制备的胞质裂解物中发现的多种mRNA结合蛋白相互作用。将放射性标记的APP RNA与胞质蛋白提取物进行紫外线交联，随后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，鉴定出六种不同的RNA-蛋白质复合物，分子量分别为42、47、65、73、84和104 kDa。用APP、富含AU的RNA或无关RNA进行的竞争分析表明，结合是特异性的，在某些情况下，对富含AU或U的序列具有优先性，据此我们初步确定了蛋白质沿着29个碱基区域的结合位点。APP mRNA结合蛋白在所有检测的肿瘤细胞系中均呈组成性活性，在全人类脑胞质裂解物中的水平也有所降低。核心元件富含AU，在人类和一些小鼠APP mRNA之间高度保守。在随附的论文中（Zaidi, S. H. E.和Malter, J. S.（1994年）《生物化学杂志》269, 24007 - 24013），我们表明3'-非翻译区的这个29个碱基的元件调节APP mRNA的稳定性。这些数据累积起来表明，稳态APP mRNA水平受胞质蛋白-RNA相互作用的调节。
