Posttranscriptional control of gene expression

我们的研究集中于基因表达的调控，特别是控制细胞和病毒mRNA表达的机制。信使核糖核酸代谢的一个关键步骤是信使核糖核酸从细胞核到细胞质的运输。通过对HIV-1的研究，对逆转录病毒系统的分析已经揭示了核mRNA输出的一些重要方面，并为控制细胞mRNA和蛋白质运输的机制提供了关键的见解。我们正在利用逆转录病毒系统，结合生物化学、功能基因组学和蛋白质组学来识别和研究信使核糖核酸代谢的机制。对大分子转录后调控和核质运输机制的剖析对于理解细胞基因表达和病毒表达的过程是相关的。我们鉴定了猴D型逆转录病毒转录本的mRNA输出需求，它是由顺式作用的RNA输出元件(CTE)及其结合伙伴细胞蛋白Nxf1介导的。我们进一步发现，细胞Nxf1蛋白是细胞mRNAs的关键核受体，这一功能在后生动物中是保守的。我们发现，小鼠LTR逆转录元件(脑池内A粒子逆转录元件)的迁移率取决于顺式作用的RNA转运元件RTE的存在。反转录元件转录本的输出和表达依赖于RTE。因此，RTE的作用类似于CTE，是细胞RNA输出因子的有效信号。这一发现表明，尽管有一个复杂的进化史，逆转录元件和逆转录病毒都对转录后调控有着共同的依赖。我们确定RNA结合基序15(RBM15)蛋白是结合和输出含有RTE的mRNAs的细胞因子。RBM15是一种新的信使核糖核酸输出因子，属于SPEN蛋白家族，在后生动物中保守。重要的是，我们发现RBM15作为分子链，将RTE-mRNAs连接到Nxf1输出途径。因此，这些实验确定了信使核糖核酸输出途径的另一个重要因素。由于HIV Gag和env的mRNAs在缺乏有效的转录后REV-RRE调节的情况下表达很低，这些mRNAs是在转录后水平上研究mRNA表达改善的极好的报告者。我们证明了RNA输出元件CTE和RTE的结合协同提高了Gag和env的表达。因此，这一发现为我们提供了一种简单的新技术来提高DNA介导的基因转移应用中的基因表达。我们还研究了一个受调控的细胞mRNA表达的例子，即细胞因子基因的表达，特别是白介素15(IL-15)。IL-15是一种在多种组织中表达的多功能细胞因子。它作为分子佐剂在疫苗和癌症免疫治疗中的应用前景广阔。除了在转录水平上的调控外，IL-15的表达还在转录后和翻译后的几个步骤中受到控制，如mRNA的稳定、翻译、细胞内转运和分泌。作为我们对逆转录病毒mRNAs研究的直接延伸，我们研究了IL-15的表达。在RNA优化后，我们发现小鼠、恒河猴和人IL-15的表达水平有了很大的提高。因此，用来优化HIV基因表达的方法也可以从简单的DNA载体中提高IL-15的表达。转录后修饰和翻译后修饰的结合导致了高效表达质粒的产生，产生的生物活性IL-15水平要高出数百倍。这些表达质粒是我们用于SIV预防和免疫治疗疫苗接种方案的DNA质粒鸡尾酒的一部分。这些优化的表达载体在艾滋病和癌症的疫苗和免疫治疗方法中具有潜在的应用前景。

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.