Eukaryotic translational coupling in UAAUG stop-start codons for the bicistronic RNA translation of the non-long terminal repeat retrotransposon SART1
Eukaryotic translational coupling in UAAUG stop-start codons for the bicistronic RNA translation of the non-long terminal repeat retrotransposon SART1
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DOI：
10.1128/mcb.25.17.7675-7686.2005
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发表时间：
2005-09-01
影响因子：
5.3
通讯作者：
Fujiwara, H
中科院分区：
文献类型：
--
作者：
Kojima, KK;Matsumoto, T;Fujiwara, H
大多数真核细胞mRNA是单顺反子的;然而，许多逆转录病毒和长末端重复序列（LTR）逆转录转座子使用核糖体移码在单个RNA转录物上编码多个蛋白质。非长末端重复序列（non-LTR）反转录转座子被认为是LTR反转录转座子和逆转录病毒的祖先，但其双顺反子RNA的翻译机制尚不清楚。我们利用杆状病毒表达系统大量表达了家蚕非LTR反转录转座子SART 1的双顺反子RNA，并通过Western blotting检测到了第二个开放阅读框架蛋白（ORF 2）。ORF 2蛋白被翻译为独立的蛋白，而不是ORF 1-ORF 2融合蛋白。我们通过诱变发现，UAAUG重叠的终止-起始密码子和下游RNA二级结构是有效的ORF 2翻译所必需的。增加ORF 1终止密码子和ORF 2起始密码子之间的距离会降低翻译效率。这些结果不同于酵母GCN 4基因所代表的真核翻译再起始机制，在该机制中，再起始的概率随着两个ORF之间的距离增加而增加。SART 1 ORF 2的翻译机制类似于在原核生物和病毒中观察到的翻译偶联。我们的研究结果表明，翻译偶联是双顺反子RNA翻译的一般机制。
Most eukaryotic cellular mRNAs are monocistronic; however, many retroviruses and long terminal repeat (LTR) retrotransposons encode multiple proteins on a single RNA transcript using ribosomal frameshifting. Non-long terminal repeat (non-LTR) retrotransposons are considered the ancestor of LTR retrotransposons and retroviruses, but their translational mechanism of bicistronic RNA remains unknown. We used a baculovirus expression system to produce a large amount of the bicistronic RNA of SART1, a non-LTR retrotransposon of the silkworm, and were able to detect the second open reading frame protein (ORF2) by Western blotting. The ORF2 protein was translated as an independent protein, not as an ORF1-ORF2 fusion protein. We revealed by mutagenesis that the UAAUG overlapping stop-start codon and the downstream RNA secondary structure are necessary for efficient ORF2 translation. Increasing the distance between the ORF1 stop codon and the ORF2 start codon decreased translation efficiency. These results are different from the eukaryotic translation reinitiation mechanism represented by the yeast GCN4 gene, in which the probability of reinitiation increases as the distance between the two ORFs increases. The translational mechanism of SART1 ORF2 is analogous to translational coupling observed in prokaryotes and viruses. Our results indicate that translational coupling is a general mechanism for bicistronic RNA translation.