A Eukaryotic (Insect) Tricistronic mRNA Encodes Three Proteins Selected by Context-dependent Scanning
A Eukaryotic (Insect) Tricistronic mRNA Encodes Three Proteins Selected by Context-dependent Scanning
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DOI：
10.1074/jbc.m110.180398
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发表时间：
2010-11-19
影响因子：
4.8
通讯作者：
Kamimura, Manabu
中科院分区：
文献类型：
--
作者：
Kanamori, Yasushi;Hayakawa, Yoichi;Kamimura, Manabu
真核生物的mRNAs通常被认为是单顺反子，只编码一种蛋白质。虽然在真菌、植物和动物中发现了编码两种蛋白质的双顺反子mRNAs，但到目前为止，在任何真核生物中编码两种以上蛋白质的多顺反子mRNAs仍然是难以捉摸的。在这里，我们证明了来自家蚕的单个mRNA编码昆虫细胞因子麻痹肽(PP)的前体和两个新的细胞因子前体蛋白uENF1和uENF2。RT-PCR分析表明，该基因在成虫体内广泛保守。Western印迹分析和用萤火虫荧光素酶ORF替换三个ORF产生的修饰的mRNAs的报告分析表明，在细胞系、幼虫组织和无细胞系统中，所有三种蛋白质都是从这个mRNAs翻译而来的。使用Renilla荧光素酶ORF或茎环的插入实验排除了内部核糖体进入位点参与三种蛋白质翻译的可能性。另一方面，对5‘-近端uENF1 ORF翻译起始序列的系统突变分析表明，下游uENF2和PP ORF的翻译涉及上下文相关的泄漏扫描机制。在体外，与uENF1成熟相对应的合成肽和合成的PP一样刺激血细胞的扩散，而uENF2的合成肽拮抗PP和uENF1的刺激活性，提示这三种蛋白相互作用控制细胞免疫。因此，真核生物有一种细胞三顺反子mRNA，编码三种功能相关的蛋白质，就像操纵子一样。
Eukaryotic mRNAs are generally considered monocistronic and encode only one protein. Although dicistronic mRNAs encoding two proteins were found in fungi, plants, and animals, polycistronic mRNAs encoding more than two proteins have remained elusive so far in any eukaryote. Here we demonstrate that a single mRNA from silkworm encodes the precursor of an insect cytokine paralytic peptide (PP) and two new cytokine precursor-like proteins, uENF1 and uENF2. RT-PCR analysis showed that this mRNA is widely conserved in moths. Western blot analyses and reporter assays using its modified mRNAs, created by replacing each one of the three ORFs with the firefly luciferase ORF, showed that all three proteins were translated from this mRNA in cell lines, larval tissues, and cell-free systems. Insertion experiments using the Renilla luciferase ORF or a stem loop ruled out the possible involvement of internal ribosome entry site in the three protein translation. On the other hand, systematic mutation analysis of the translation initiation sequence of the 5'-proximal uENF1 ORF suggested that the context-dependent leaky-scanning mechanism is involved in translation of the downstream uENF2 and PP ORFs. In vitro, a synthetic peptide corresponding to the putative mature form of uENF1 stimulated spreading of hemocytes as did the synthetic PP, whereas that of uENF2 antagonized the stimulating activities of PP and the uENF1 peptide, suggesting that the three proteins control cellular immunity interactively. Thus, eukaryotes have a cellular tricistronic mRNA that encodes three functionally related proteins as in an operon.