TRANSLATIONAL CONTROL OF ONCOPROTEIN MDM2 SYNTHESIS

癌蛋白MDM 2是负反馈环的一个组成部分，调节肿瘤抑制因子p53的活性和细胞水平。 MDM 2的合成在静息成纤维细胞的翻译水平上受到调节，这些成纤维细胞被激活以重新进入细胞周期。 在一类软组织肿瘤中，MDM 2蛋白的合成增强了一到两个数量级，而mRNA水平没有相应的增加。 在所有检测的细胞类型中，存在两种形式的mdm 2 mRNA。 长型（L-mdm 2）含有287个核苷酸的5'前导序列，具有两个上游开放阅读框（uORF），衍生自外显子1，短型（S-mdm 2）具有64个核苷酸的前导序列，衍生自外显子2。L-mdm 2在正常成纤维细胞、HeLa细胞和绒毛膜癌细胞中不能有效地负载核糖体。 L-mdm 2前导序列在报告基因上赋予低效的核糖体加载，并且两个uORF协同合作以抑制翻译。 在绒毛膜癌细胞系中，其中MDM 2表达被抑制性地去抑制，L-mdm 2前导序列中的uORF之间的协同相互作用丧失。 S-mdm 2 mRNA在绒毛膜癌细胞中与核糖体负载良好，但在正常成纤维细胞和HeLa细胞中翻译较差。 含有S-mdm 2前导序列的融合构建体在所有细胞类型中均能很好地翻译，这表明正常细胞中的翻译抑制需要mRNA部分中除5'前导序列以外的另一元件。本申请提出了一系列实验，旨在确定mdm 2基因在正常和肿瘤细胞中的翻译控制机制。 L-mdm 2中两个uORF之间的协同相互作用特别令人感兴趣，首先是因为它在过表达MDM 2蛋白的肿瘤细胞中丢失，其次是因为没有哺乳动物模型可以容易地用于多个相互作用的uORF的调节机制的实验研究。 通过类比酵母GCN 4基因中多个uORF的调节，将测试eIF-α磷酸化在MDM 2翻译调节中的可能参与。 还将定义S-mdm 2 mRNA中负责其在正常细胞和肿瘤细胞之间翻译差异的序列。

Oncoprotein MDM2 is a component of a negative feedback loop that regulates the activity and cellular level of tumor suppressor p53. The synthesis of MDM2 is regulated at the translational level in resting fibroblasts that are activated to reenter the cell cycle. In one class of soft tissue tumors, synthesis of MDM2 protein is enhanced by one to two orders of magnitude without a corresponding increase in mRNA level. In all cell types examined, there are two forms of mdm2 mRNA. The long form (L-mdm2) contains a 287-nucleotide 5' leader, with two upstream open reading frames (uORFs), derived from exon 1 and the short form (S-mdm2) has a 64-nucleotide leader arising from exon 2. L-mdm2 is inefficiently loaded with ribosomes in normal fibroblasts, HeLa cells and choriocarcinoma cells. The L-mdm2 leader confers inefficient ribosome loading on a reporter gene and the two uORFs cooperate synergistically to suppress translation. In the choriocarcinoma cell line, in which MDM2 expression is translationally derepressed, the synergistic interaction between the uORFs in the L-mdm2 leader is lost. The S-mdm2 mRNA is well loaded with ribosomes in the choriocarcinoma cells, but is poorly translated in normal fibroblasts and HeLa cells. Fusion constructs containing the S-mdm2 leader are well translated in all cell types, suggesting that the translational suppression in normal cells requires another element in part of the mRNA other than the 5' leader. This application proposes a series of experiments designed to define the mechanisms of translational control of the mdm2 gene in normal and neoplastic cells. The synergistic interaction between the two uORFs in L-mdm2 is of particular interest, first because it is lost in tumor cells that overexpress MDM2 protein and, second, because there is no mammalian model that is readily accessible for experimental study of the mechanism of regulation by multiple, interacting uORFs. By analogy to the regulation by multiple uORFs in the yeast GCN4 gene, the possible involvement of phosphorylation of eIF-alpha in regulation of MDM2 translation will be tested. The sequences in the S-mdm2 mRNA that are responsible for the differences in its translation between normal and tumor cells will also be defined.