Translation of MMTV Gag requires nuclear events involving splicing motifs in addition to the viral Rem protein and RmRE
Translation of MMTV Gag requires nuclear events involving splicing motifs in addition to the viral Rem protein and RmRE
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DOI：
10.1186/1742-4690-9-8
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发表时间：
2012-01-25
期刊：
影响因子：
3.3
通讯作者：
West, John T.
中科院分区：
文献类型：
--
作者：
Boeras, Ioana;Sakalian, Michael;West, John T.
背景资料：逆转录病毒Gag蛋白在内含子中编码，并且由于这种定位，它们受到前mRNA剪接的默认途径的影响。逆转录病毒通过多种相互交织的机制调节剪接和翻译，包括5 '-转录后控制元件、3'-组成型转运元件和将未剪接和单剪接mRNA偶联至转运机制的病毒蛋白RNA相互作用。gag基因中被称为抑制或不稳定序列的序列似乎也会影响病毒mRNA的稳定性和翻译，这些序列的作用可以通过沉默突变或RNA相互作用蛋白（如HIV-1 Rev）的存在来抵消。在这里，我们使用体内和体外表达系统的组合探索了小鼠乳腺肿瘤病毒（MMTV）Gag表达的要求。我们发现，MMTV gag等位基因的翻译抑制，尽管拥有一个功能性的开放阅读框（ORF）。对表达的阻断是转录后的，靶向mRNA，但不是mRNA转运或稳定性的功能。使用双顺反子报告，我们表明，抑制gag表达赋予了一个块帽依赖和帽独立的翻译到mRNA上。在体外合成的gag mRNA的直接引入导致翻译，这意味着在抑制表达的核作用。表达的抑制被克服了完整的前病毒表达或侧翼加格与剪接位点结合的功能性Rem-Rem反应元件（RmRE）interaction.Conclusions：MMTV加格的表达需要涉及病毒Rem蛋白，其同源结合靶RmRE的核相互作用，令人惊讶的是，无论是剪接供体和受体序列，以实现适当的信号在细胞质中的mRNA的翻译。
Background: Retroviral Gag proteins are encoded in introns and, because of this localization, they are subject to the default pathways of pre-mRNA splicing. Retroviruses regulate splicing and translation through a variety of intertwined mechanisms, including 5'-post-transcriptional control elements, 3'-constitutive transport elements, and viral protein RNA interactions that couple unspliced and singly spliced mRNAs to transport machinery. Sequences within the gag gene termed inhibitory or instability sequences also appear to affect viral mRNA stability and translation, and the action of these sequences can be countered by silent mutation or the presence of RNA interaction proteins like HIV-1 Rev. Here, we explored the requirements for mouse mammary tumor virus (MMTV) Gag expression using a combination of in vivo and in vitro expression systems.Results: We show that MMTV gag alleles are inhibited for translation despite possessing a functional open reading frame (ORF). The block to expression was post-transcriptional and targeted the mRNA but was not a function of mRNA transport or stability. Using bicistronic reporters, we show that inhibition of gag expression imparted a block to both cap-dependent and cap-independent translation onto the mRNA. Direct introduction of in vitro synthesized gag mRNA resulted in translation, implying a nuclear role in inhibition of expression. The inhibition of expression was overcome by intact proviral expression or by flanking gag with splice sites combined with a functional Rem-Rem response element (RmRE) interaction.Conclusions: Expression of MMTV Gag requires nuclear interactions involving the viral Rem protein, its cognate binding target the RmRE, and surprisingly, both a splice donor and acceptor sequence to achieve appropriate signals for translation of the mRNA in the cytoplasm.