Posttranscriptional control of gene expression

我们的研究集中在基因表达的调控，特别是 控制细胞和病毒mRNA表达的机制。mRNA的关键步骤 代谢是mRNA从细胞核到细胞质的转运。分析 由HIV-1研究开创的逆转录病毒系统，已经揭示了一些重要的 核mRNA输出的方面，并提供了重要的见解， 细胞mRNA和蛋白质转运。我们正在利用逆转录病毒系统来识别和研究 利用生物化学、功能基因组学和 蛋白质组学对转录后调控机制的剖析， 大分子的核质运输与理解 在细胞基因表达以及病毒表达中。我们鉴定了mRNA的输出 由顺式作用RNA介导的猿猴D型逆转录病毒转录物的需要 输出元件（CTE）及其结合伴侣，细胞蛋白NXF 1。我们进一步发现， 细胞NXF 1蛋白作为细胞mRNA的关键核受体， 功能在后生动物中是保守的。我们发现老鼠的活动性 LTR-逆转录元件（脑池内A颗粒逆转录元件）取决于 顺式作用RNA转运元件RTE。逆转录因子转录本的输出和表达 取决于RTE。因此，RTE的作用类似于CTE，是细胞RNA输出的有效信号 因子这一发现表明，尽管有着复杂的进化历史， 逆转录病毒都依赖于转录后调节。我们鉴定了RNA 结合基序15（RBM 15）蛋白作为结合和输出含有RTE的 mRNA。RBM 15是一种新的mRNA输出因子，属于SPEN蛋白家族， 在后生动物中是保守的重要的是，我们发现RBM 15作为分子连接和系链 将RTE-mRNA转移到NXF 1输出途径。因此，这些实验已经确定了另一个 mRNA输出途径的重要因素。由于HIV gag和env mRNA在大肠杆菌中表达不足， 由于缺乏有效转录后Rev-RRE调控，这些mRNA作为优异的 报道者研究在转录后水平上mRNA表达的改善。我们 证明了RNA输出元件CTE和RTE的组合协同地改善了RNA输出元件CTE和RTE之间的相互作用。 gag和env表达。因此，这一发现为我们提供了一种简单的新技术， 用于DNA介导基因转移应用的改进的基因表达。我们还研究了 调节的细胞mRNA表达，即细胞因子基因的表达的实例， 特别是白细胞介素15（IL-15）。IL-15是一种多功能细胞因子，在许多细胞中表达。 组织中其作为分子佐剂在疫苗和癌症免疫治疗中的应用是有希望的。在 除了在转录水平上的调节，IL-15的表达还在几个转录水平上受到控制。 转录后和翻译后步骤如mRNA稳定性，翻译， 细胞内运输和分泌。作为我们研究逆转录病毒的直接延伸 mRNA，我们研究了IL-15的表达。天然mRNA的表达很差， RNA优化我们发现对于鼠、恒河猴和人IL-15有很大的改善。因此，在本发明中， 用于优化HIV基因表达方法也可以提高IL-15的表达 从简单的DNA载体转录后和翻译后的结合 修饰导致产生高效的表达质粒，产生数百个 生物活性IL-15的水平高一倍。这种表达质粒是我们的鸡尾酒的一部分， DNA质粒用于SIV的预防性和免疫接种方案。这些 优化的表达载体在疫苗和免疫治疗中具有潜在的应用价值 艾滋病和癌症的治疗方法。

Summary Our research focuses on the regulation of gene expression, in particular the mechanisms controlling cellular and viral mRNA expression. A critical step in the mRNA metabolism is the transport of the mRNA from the nucleus to the cytoplasm. Analysis of retroviral systems, pioneered by research on HIV-1, have shed light into some important aspects of nuclear mRNA export and have provided critical insights into mechanisms governing cellular mRNA and protein transport. We are utilizing retroviral systems to identify and study mechanisms of mRNA metabolism using a combination of biochemistry, functional genomics, and proteomics. The dissection of the mechanisms of posttranscriptional control and nucleocytoplasmic trafficking of macromolecules are relevant to understand processes involved in cellular gene expression as well as virus expression. We identified the mRNA export requirement of the simian type D retroviral transcript which is mediated by the cis acting RNA export element (CTE) and its binding partner, the cellular protein NXF1. We further found that the cellular NXF1 protein acts as the key nuclear receptor for cellular mRNAs, and that this function is conserved in metazoa. We identified that the mobility of the murine LTR-retroelements (Intracisternal A particle retroelements) depends on the presence of the cis-acting RNA transport element RTE. The export and expression of the retroelement transcript depends on RTE. Thus, RTE acts like the CTE and is a potent signal for a cellular RNA export factor. This finding reveals that, despite a complex evolutionary history, retroelements and retroviruses share the dependency on posttranscriptional regulation. We identified the RNA binding motif 15 (RBM15) protein as the cellular factor that binds and exports RTE-containing mRNAs. RBM15, a novel mRNA export factor, belongs to the SPEN family of proteins and is conserved among metazoa. Importantly, we found that RBM15 acts as molecular link and tethers the RTE-mRNAs to the NXF1 export pathway. Thus, these experiments have identified another important factor of the mRNA export route. Since HIV gag and env mRNAs are poorly expressed in the absence of potent posttranscriptional Rev-RRE regulation, these mRNAs serve as excellent reporters to study improvement of mRNA expression at the posttranscriptional level. We demonstrated that the combination of RNA export elements CTE and RTE synergistically improves gag and env expression. Thus, this discovery provided us with a simple novel technology to improve gene expression for DNA mediated gene transfer applications. We also studied an example of regulated cellular mRNA expression, namely the expression of cytokine genes, in particular interleukin 15 (IL-15). IL-15 is a multifunctional cytokine expressed in many tissues. Its use as molecular adjuvant in vaccine and in cancer immunotherapy is promising. In addition to regulation at the transcriptional level, IL-15 expression is controlled at several posttranscriptional and posttranslational steps such as mRNA stability, translation, intracellular trafficking and secretion. As a direct extension of our research of retroviral mRNAs, we studied expression of IL-15. Expression from the native mRNA is poor and upon RNA-optimization we found great improvement for murine, rhesus macaque and human IL-15. Thus, the methodology utilized to optimize HIV gene expression also let to improved IL-15 expression from simple DNA vectors. The combination of posttranscriptional and posttranslational modification led to the generation of efficient expression plasmids producing several hundred fold higher levels of bioactive IL-15. Such expression plasmids are part of our cocktail of DNA plasmids used in preventive and immunotherapeutic vaccination protocols in SIV. These optimized expression vectors have potential applications in vaccine and immunotherapy approaches against AIDS and cancer.