Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5′ untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated
Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5′ untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated
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DOI：
10.1128/mcb.02138-06
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发表时间：
2007-07-01
影响因子：
5.3
通讯作者：
Shatsky, Ivan N.
中科院分区：
文献类型：
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作者：
Dmitriev, Sergey E.;Andreev, Dmitri E.;Shatsky, Ivan N.
反转录转座子L1是LINE家族的一个移动的遗传元件，在哺乳动物基因组中极为广泛。它编码双顺反子mRNA，这在真核细胞mRNA中是非常罕见的。极长且富含GC的L1 5'非翻译区（5' UTR）指导每个mRNA合成大量RNA结合蛋白ORF 1 p拷贝。可能提示L1 mRNA的5 'UTR含有强大的内部核糖体进入位点（IRES）元件。使用加帽或未加帽的聚腺苷酸化单顺反子（L1 5 'UTR-Fluc）或双顺反子（Rluc-L1 5' UTR-Fluc）RNA构建体转染培养的细胞，已经确定L1的5 'UTR不含IRES。去帽使L1 5 'UTR的起始活性降低至背景的起始活性。此外，翻译被5 'UTR中的上游AUG密码子抑制。然而，L1 5 'UTR的这种帽依赖性起始活性出乎意料地高，并且类似于β-肌动蛋白5' UTR（84个核苷酸长）的帽依赖性起始活性。令人惊讶的是，L1 5 'UTR的核苷酸序列的多达80%的缺失及其大部分茎环不会显著改变其翻译起始效率。这些数据可以修改目前关于40 S核糖体亚基科普复杂5 'UTR的机制的想法，并质疑每个高效率工作的长的富含GC的5' UTR必须包含IRES的概念。我们的数据还表明，ORF 2的翻译起始不是由内部启动，无论是。这是非常低效的，大概是基于重新启动事件。
Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.