Expression of the two nested overlapping reading frames of turnip yellow mosaic virus RNA is enhanced by a 5′ cap and by 5′ and 3′ viral sequences
Expression of the two nested overlapping reading frames of turnip yellow mosaic virus RNA is enhanced by a 5′ cap and by 5′ and 3′ viral sequences
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DOI：
10.1128/jvi.78.17.9325-9335.2004
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发表时间：
2004-09-01
影响因子：
5.4
通讯作者：
Dreher, TW
中科院分区：
文献类型：
--
作者：
Matsuda, D;Bauer, L;Dreher, TW
mRNA分子的翻译效率通常由其5 '-和/或3'-非翻译区（UTR）决定。先前，我们已经发现芜菁黄花叶病毒（TYMV）RNA的3 '-UTR与5'帽协同增强翻译。在此，我们在豇豆原生质体中使用荧光素酶报告系统来显示来自TYMV基因组RNA的5' 217个核苷酸相对于载体衍生的17个核苷酸的5'-UTR增强表达。从具有帽和5'和3' TYMV序列的RNA观察到最大表达。在成对的报道构建体中，携带UTR的5 ′ 217个核苷酸和两个重叠的TYMV开放阅读框（ORF）ORF-69和ORF-206的前43或41个密码子分别与荧光素酶基因框内融合。这允许从每个ORF的起始密码子（AUG（69）和AUG（206））的表达被单独监测，但从正常序列环境监测。来自两个AUG密码子的表达严重依赖于5'帽，在基因组3'-UTR存在下，来自AUG（69）的表达比来自AUG（206）的表达高三倍。中断cap/3 '-UTR协同作用的变化（即，去除帽或TYMV 3 '-UTR）导致从AUG起始的比例更高（206）。突变3 '-UTR以防止氨酰化，以及缺失75%的5'-UTR，同样导致AUG（69）相对于AUG（206）的表达比率较低。每个AUG起始密码子的突变增加了另一个的起始。总的来说，这些结果并不完全符合标准泄漏核糖体扫描的预期，并留下了核糖体定型到AUG 69和AUG的精确机制（206）。然而，我们的观察结果不支持最近基于体外研究的提议，其中提议3 '-UTR特异性地在AUG（206）而不是在AUG（69）指导帽非依赖性起始。
The translation efficiency of an mRNA molecule is typically determined by its 5'- and/or 3'-untranslated regions (UTRs). Previously, we have found that the 3'-UTR of Turnip yellow mosaic virus (TYMV) RNA enhances translation synergistically with a 5' cap. Here, we use a luciferase reporter system in cowpea protoplasts to show that the 5' 217 nucleotides from TYMV genomic RNA enhance expression relative to a vector-derived 17-nucleotide 5'-UTR. Maximum expression was observed from RNAs with a cap and both 5' and 3' TYMV sequences. In paired reporter constructs, the 5' 217 nucleotides harboring the UTR and the first 43 or 41 codons of the two overlapping TYMV open reading frames (ORFs), ORF-69 and ORF-206, respectively, were fused in frame with the luciferase gene. This allowed expression from the initiation codon of each ORF (AUG(69) and AUG(206)) to be monitored separately but from the normal sequence environment. Expression from both AUG codons was heavily dependent on a 5' cap, with a threefold-higher expression occurring from AUG(69) than from AUG(206) in the presence of the genomic 3'-UTR. Changes that interrupted the cap/3'-UTR synergy (i.e., removal of the cap or TYMV 3'-UTR) resulted in a higher proportion of initiation from AUG(206). Mutation of the 3'-UTR to prevent aminoacylation, as well as deletion of 75% of the 5'-UTR, likewise resulted in a lower ratio of expression from AUG(69) relative to AUG(206). Mutation of each AUG initiation codon increased initiation from the other. Taken together, these results do not fully conform to the expectations of standard leaky ribosomal scanning and leave open the precise mechanism of ribosome commitment to AUG 69 and AUG(206). However, our observations do not support a recent proposal based on in vitro studies in which the 3'-UTR is proposed to direct cap-independent initiation specifically at AUG(206) and not at AUG(69).