CONTROL OF C-MYC MESSENGER-RNA HALF-LIFE INVITRO BY A PROTEIN CAPABLE OF BINDING TO A CODING REGION STABILITY DETERMINANT
CONTROL OF C-MYC MESSENGER-RNA HALF-LIFE INVITRO BY A PROTEIN CAPABLE OF BINDING TO A CODING REGION STABILITY DETERMINANT
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DOI：
10.1101/gad.6.4.642
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发表时间：
1992-04-01
影响因子：
10.5
通讯作者：
ROSS, J
中科院分区：
文献类型：
--
作者：
BERNSTEIN, PL;HERRICK, DJ;ROSS, J
多核糖体相关的c-myc mRNA在细胞中和在含有来自培养的哺乳动物细胞的提取物的体外mRNA衰减系统中相对快速地降解。使用该系统，设计了一种竞争/筛选试验来搜索与多聚体相关的c-myc mRNA的特定区域结合并从而改变其半衰期的因子。首先在含有对应于c-myc mRNA本身部分的外源竞争RNA的反应中测定mRNA稳定性。从c-myc编码区的羧基末端部分添加182个核苷酸的正义链片段使c-myc mRNA不稳定至少8倍。该RNA片段对所测试的其他mRNA的稳定性没有影响。此外，c-myc mRNA在含有不相关的竞争者RNA或来自c-myc 5'区的有义链RNA的反应中不稳定。含有c-myc编码区片段的多核糖体相关珠蛋白mRNA在体外也被182个核苷酸的RNA破坏稳定。如通过UV-交联实验所确定的，182个核苷酸的RNA片段被识别并结合到约75-kD的多聚体相关蛋白。根据这些数据加上c-myc mRNA衰变产物的北方印迹分析，我们认为，约75 kD的蛋白质通常结合到一个c-myc编码区的决定簇，并保护该区域的mRNA从核酸内切酶攻击。保护蛋白，翻译，核糖体暂停，和c-myc mRNA营业额之间可能存在的联系进行了讨论。
Polysome-associated c-myc mRNA is degraded relatively rapidly in cells and in an in vitro mRNA decay system containing extracts from cultured mammalian cells. Using this system, a competition/screening assay was devised to search for factors that bind to specific regions of polysome-associated c-myc mRNA and thereby alter its half-life. mRNA stability was first assayed in reactions containing exogenous competitor RNAs corresponding to portions of c-myc mRNA itself. The addition of a 182-nucleotide sense strand fragment from the carboxy-terminal portion of the c-myc-coding region destabilized c-myc mRNA by at least eightfold. This RNA fragment had no effect on the stability of other mRNAs tested. Moreover, c-myc mRNA was not destabilized in reactions containing unrelated competitor RNAs or sense strand RNA from the c-myc 5' region. Polysome-associated globin mRNA containing the c-myc-coding region segment in-frame was also destabilized in vitro by the 182-nucleotide RNA. As determined by UV-cross-linking experiments, the 182-nucleotide RNA fragment was recognized by and bound to an approximately 75-kD polysome-associated protein. On the basis of these data plus Northern blotting analyses of c-myc mRNA decay products, we suggest that the approximately 75-kD protein is normally bound to a c-myc-coding region determinant and protects that region of the mRNA from endonuclease attack. Possible links between the protective protein, translation, ribosome pausing, and c-myc mRNA turnover are discussed.