Guo L, Allen E, Miller W A
Interdepartmental Genetics, Plant Pathology Department, Iowa State University, Ames 50011, USA.
RNA. 2000 Dec;6(12):1808-20. doi: 10.1017/s1355838200001539.
Barley yellow dwarf virus RNA lacks both a 5' cap and a poly(A) tail, yet it is translated efficiently. It contains a cap-independent translation element (TE), located in the 3' UTR, that confers efficient translation initiation at the AUG closest to the 5' end of the mRNA. We propose that the TE must both recruit ribosomes and facilitate 3'-5' communication. To dissect its function, we determined the secondary structure of the TE and roles of domains within it. Nuclease probing and structure-directed mutagenesis revealed that the 105-nt TE (TE105) forms a cruciform secondary structure containing four helices connected by single-stranded regions. TE105 can function in either UTR in wheat germ translation extracts. A longer viral sequence (at most 869 nt) is required for full cap-independent translation in plant cells. However, substantial translation of uncapped mRNAs can be obtained in plant cells with TE105 combined with a poly(A) tail. All secondary structural elements and most primary sequences that were mutated are required for cap-independent translation in the 3' and 5' UTR contexts. A seven-base loop sequence was needed only in the 3' UTR context. Thus, this loop sequence may be involved only in communication between the UTRs and not directly in recruiting translational machinery. This structural and functional analysis provides a framework for understanding an emerging class of cap-independent translation elements distinguished by their location in the 3' UTR.
大麦黄矮病毒RNA既没有5'帽结构也没有聚腺苷酸尾巴，但它仍能高效翻译。它含有一个位于3'非翻译区（UTR）的不依赖帽结构的翻译元件（TE），该元件能在最靠近mRNA 5'端的AUG处实现高效的翻译起始。我们提出，该TE必须既能招募核糖体又能促进3'-5'通讯。为了剖析其功能，我们确定了TE的二级结构及其内部结构域的作用。核酸酶探测和结构导向诱变表明，105个核苷酸的TE（TE105）形成了一种十字形二级结构，包含由单链区域连接的四个螺旋。TE105在小麦胚芽翻译提取物的任一UTR中都能发挥作用。在植物细胞中进行完全不依赖帽结构的翻译需要更长的病毒序列（最多869个核苷酸）。然而，将TE105与聚腺苷酸尾巴结合，在植物细胞中可实现无帽mRNA的大量翻译。所有发生突变的二级结构元件和大多数一级序列在3'和5'UTR背景下进行不依赖帽结构的翻译时都是必需的。一个七碱基环序列仅在3'UTR背景下是必需的。因此，该环序列可能仅参与UTR之间的通讯，而不直接参与招募翻译机器。这种结构和功能分析为理解一类新兴的、以其在3'UTR中的位置为特征的不依赖帽结构的翻译元件提供了一个框架。
