Negulescu D, Leong L E, Chandy K G, Semler B L, Gutman G A
Department of Physiology and Biophysics, College of Medicine, University of California, Irvine, California 92697, USA.
J Biol Chem. 1998 Aug 7;273(32):20109-13. doi: 10.1074/jbc.273.32.20109.
The mammalian Kv1.4 voltage-gated potassium channel mRNA contains an unusually long (1.2 kilobases) 5'-untranslated region (UTR) and includes 18 AUG codons upstream of the authentic site of translation initiation. Computer-predicted secondary structures of this region reveal complex stem-loop structures that would serve as barriers to 5' --> 3' ribosomal scanning. These features suggested that translation initiation in Kv1.4 might occur by the mechanism of internal ribosome entry, a mode of initiation employed by a variety of RNA viruses but only a limited number of vertebrate genes. To test this possibility we introduced the 5'-UTR of mouse Kv1.4 mRNA into the intercistronic region of a bicistronic vector containing two tandem reporter genes, chloramphenicol acetyltransferase and luciferase. The control construct translated only the upstream chloramphenicol cistron in transiently transfected mammalian cells. In contrast, the construct containing the mKv1.4 UTR efficiently translated the luciferase cistron as well, demonstrating the presence of an internal ribosome entry segment. Progressive 5' --> 3' deletions localized the activity to a 3'-proximal 200-nucleotide fragment. Suppression of cap-dependent translation by extracts from poliovirus-infected HeLa cells in an in vitro translation assay eliminated translation of the upstream cistron while allowing translation of the downstream cistron. Our results indicate that the 5'-untranslated region of mKv1.4 contains a functional internal ribosome entry segment that may contribute to unusual and physiologically important modes of translation regulation for this and other potassium channel genes.
哺乳动物的Kv1.4电压门控钾通道mRNA含有一个异常长（1.2千碱基）的5'非翻译区（UTR），并且在真正的翻译起始位点上游包含18个AUG密码子。该区域的计算机预测二级结构揭示了复杂的茎环结构，这些结构将作为5'→3'核糖体扫描的障碍。这些特征表明，Kv1.4中的翻译起始可能通过内部核糖体进入机制发生，这是多种RNA病毒采用的一种起始模式，但只有有限数量的脊椎动物基因使用。为了测试这种可能性，我们将小鼠Kv1.4 mRNA的5'UTR引入到一个双顺反子载体的顺反子间区域，该载体包含两个串联的报告基因，氯霉素乙酰转移酶和荧光素酶。对照构建体在瞬时转染的哺乳动物细胞中仅翻译上游的氯霉素顺反子。相比之下，包含mKv1.4 UTR的构建体也有效地翻译了荧光素酶顺反子，证明存在内部核糖体进入片段。5'→3'的逐步缺失将活性定位到一个3'近端的200核苷酸片段。在体外翻译试验中，脊髓灰质炎病毒感染的HeLa细胞提取物对帽依赖性翻译的抑制消除了上游顺反子的翻译，同时允许下游顺反子的翻译。我们的结果表明，mKv1.4的5'非翻译区包含一个功能性的内部核糖体进入片段，这可能有助于该钾通道基因以及其他钾通道基因的异常和生理上重要的翻译调控模式。
