de Almeida Rogério Alves, Heuser Tanja, Blaschke Rüdiger, Bartram Claus R, Janssen Johannes W G
Institute of Human Genetics, University of Heidelberg, Germany.
J Biol Chem. 2006 Jan 13;281(2):695-704. doi: 10.1074/jbc.M511467200. Epub 2005 Nov 7.
The myeov gene has been isolated by the tumorigenicity assay and is localized at chromosome 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of various organs. The mechanisms of myeov regulation remain enigmatic. The 5'-untranslated region (5'-UTR) of the myeov gene is long, encompasses several upstream AUGs, and is predicted to fold in a strong secondary structure, suggesting that its translation might be regulated by an internal ribosomal entry site. Here we show that initial experiments using monocistronic and dicistronic reporter constructs supported this assumption. However, the application of in vitro transcription/translation assays, Northern blot analysis, and promoterless dicistronic constructs revealed promoter activity of the myeov 5'-UTR. DNA transfection of dicistronic DNA constructs, normal and mutated forms of myeov cDNA fragments cloned in a eukaryotic expression vector, and direct RNA transfection analysis revealed that upstream AUG triplets in the 5'-UTR of the myeov transcript abrogate translation. Alternative splicing mechanisms in specific cell types and/or developmental stage may evade this translation control. Control experiments suggest that the 5'-UTR from encephalomyocarditis virus, when inserted at the midpoint of a dicistronic vector, is also able to function as a cryptic promoter.
通过致瘤性分析分离出了myeov基因，它定位于11号染色体q13区，这是各种癌症和B细胞肿瘤中染色体重排的常见位点。此外，myeov与细胞周期蛋白D1共同扩增，并在各种器官的癌症中过表达。myeov的调控机制仍然不明。myeov基因的5'-非翻译区（5'-UTR）很长，包含几个上游AUG，预计会折叠成一个强二级结构，这表明其翻译可能受内部核糖体进入位点调控。在这里我们表明，最初使用单顺反子和双顺反子报告基因构建体的实验支持了这一假设。然而，体外转录/翻译分析、Northern印迹分析和无启动子双顺反子构建体的应用揭示了myeov 5'-UTR的启动子活性。双顺反子DNA构建体、克隆在真核表达载体中的正常和突变形式的myeov cDNA片段的DNA转染以及直接RNA转染分析表明，myeov转录本5'-UTR中的上游AUG三联体消除翻译。特定细胞类型和/或发育阶段的可变剪接机制可能逃避这种翻译控制。对照实验表明，脑心肌炎病毒的5'-UTR插入双顺反子载体中点时，也能够作为一个隐蔽启动子发挥作用。
