You Y, Chen C Y, Shyu A B
Department of Biochemistry and Molecular Biology, University of Texas, Health Science Center, Houston Medical School 77030.
Mol Cell Biol. 1992 Jul;12(7):2931-40. doi: 10.1128/mcb.12.7.2931-2940.1992.
Rapid decay of the c-fos transcript plays a critical role in controlling transforming potential of the c-fos proto-oncogene. One of the mRNA instability determinants is a 75-nucleotide AU-rich element (ARE) present in the 3' untranslated region of the c-fos transcript. It appears to control two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require the AUUUA motifs, and subsequent degradation of deadenylated mRNA, which appears to be dependent on the AUUUA motifs. In this study, we report the identification of four U-rich sequence binding proteins (URBPs) that specifically interact with a 20-nucleotide U-rich sequence within the c-fos ARE. Gel mobility shift assay and competition experiments showed that these protein factors form three specific band-shifted complexes with the c-fos ARE. Binding activity of one of the protein factors, a 37-kDa protein, is significantly affected by serum induction and by pretreatment of cells with drugs known to stabilize many of the immediate-early gene mRNAs. Combining UV cross-linking with a new approach, designated sequential RNase digestion, we were able to better determine the molecular masses of these cellular proteins. The binding sites for the four proteins were all mapped to a 20-nucleotide U-rich sequence located at the 3' half of the c-fos ARE, which contains no AUUUA pentanucleotides but stretches of uridylate residues. Single U-to-A point mutations in each of the three AUUUA motifs within the c-fos ARE have little effect on formation of the mobility-shifted complexes. Our data indicate c-fos ARE-protein interaction involves recognition of U stretches rather than recognition of the AUUUA motifs. We propose that UTBP binding may be involved in the first step, removal of the Poly(A) tail, in the c-fos ARE-mediated decay pathway.
c-fos转录本的快速降解在控制c-fos原癌基因的转化潜能中起着关键作用。mRNA不稳定的决定因素之一是存在于c-fos转录本3'非翻译区的一个75个核苷酸的富含AU元件（ARE）。它似乎控制着c-fos mRNA降解过程中的两个步骤：去除多聚（A）尾，这不需要AUUUA基序；以及随后对去腺苷酸化mRNA的降解，这似乎依赖于AUUUA基序。在本研究中，我们报告鉴定了四种富含U序列结合蛋白（URBP），它们与c-fos ARE内一个20个核苷酸的富含U序列特异性相互作用。凝胶迁移率变动分析和竞争实验表明，这些蛋白质因子与c-fos ARE形成三种特异性的带迁移复合物。其中一种蛋白质因子，一种37 kDa的蛋白质，其结合活性受到血清诱导以及用已知能稳定许多早期即刻基因mRNA的药物对细胞进行预处理的显著影响。将紫外线交联与一种新方法（称为顺序核糖核酸酶消化）相结合，我们能够更好地确定这些细胞蛋白质的分子量。这四种蛋白质的结合位点都定位到位于c-fos ARE 3'半部分的一个20个核苷酸的富含U序列，该序列不包含AUUUA五核苷酸，但有一段尿苷酸残基。c-fos ARE内三个AUUUA基序中的每一个的单个U到A点突变对迁移率变动复合物的形成影响很小。我们的数据表明c-fos ARE与蛋白质的相互作用涉及对U序列的识别而不是对AUUUA基序的识别。我们提出UTBP结合可能参与c-fos ARE介导的降解途径中的第一步，即去除多聚（A）尾。
