Posttranscriptional control of gene expression

基因表达的转录后控制

• 批准号: 8937714
• 负责人: BARBARA K FELBER
• 金额: $ 51.33万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937714
• 关键词: AIDS Vaccines; Address; Adjuvant; Affinity; Anabolism; Antigens; Binding; Biochemistry; Biological Models; Biology; Cell Nucleus; Cells; Chemical Vaccines; Clinical Trials; Codon Nucleotides; Comparative Study; Complex; Cytokine Gene; Cytoplasm; DNA; DNA Vaccines; Dependency; Development; Dissection; Elements; Epitopes; Family; Family member; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Goals; HIV; HIV Vaccine Trials Network; HIV vaccine; HIV-1; Herpesviridae; Human; Human Herpesvirus 8; Immune response; Immunization; Immunotherapy; Interleukin-12; Interleukin-15; Ligands; Light; Link; Macaca; Mediating; Messenger RNA; Metabolism; Methodology; Methods; Modeling; Molecular; Mus; Nuclear; Nuclear Export; Nuclear Receptors; Nuclear Translocation; Pathway interactions; Plasmids; Play; Post-Translational Regulation; Process; Production; Proteins; Proteome; Proteomics; RNA; RNA Binding; RNA Transport; Recording of previous events; Regimen; Regulation; Regulatory Element; Relative (related person); Research; Retroelements; Retroviridae; Role; SIV; System; Technology; Testing; Transcript; Translations; Vaccination; Vaccine Adjuvant; Vaccines; Variant; Viral; Viral Genes; Viral Proteins; Virus; base; cancer immunotherapy; cofactor; cytokine; design; experience; expression vector; functional genomics; gene therapy; improved; insight; mRNA Export; mRNA Expression; macromolecule; member; method development; novel; practical application; protein expression; protein transport; prototype; response; rev Protein; tool; trafficking; trans-Golgi Network; vector; vector vaccine; viral RNA

Our research focuses on the regulation of gene expression, in particular the mechanisms controlling cellular and viral mRNA expression, and on the development of DNA vectors for vaccine and immunotherapy. Analyses of retroviral regulatory systems, pioneered by research on HIV-1, have shed light into important aspects of nuclear mRNA export and provided critical insights into mechanisms governing cellular mRNA and protein transport. Retroviral model systems, and in particular the regulation of HIV-1, have led to major discoveries in the field of mRNA metabolism. HIV-1 Rev was the first identified viral mRNA export factor, and its discovery was instrumental in the discovery of molecular mechanisms mediating posttranscriptional control of gene expression as well as our development of methods to increase the expression of viral proteins, i.e. development of RNA optimization (also referred to as codon optimization), which is presently a key technology for many gene therapy applications, including HIV vaccines and cytokine vectors. The study of Rev also prompted us to derive the concept that all retroviruses/retroelements use posttranscriptional control mechanisms essential for their replication. These controls require a combination of viral RNA elements and cellular/viral factors able to efficiently link the viral mRNA to the nuclear export pathways and to promote translation. The study of retrovirus/retroelement export has provided important tools to understand essential and complex steps in cellular gene expression. This strategy resulted in our past identification and characterization of NXF1, which is the key nuclear receptor for the export of cellular mRNAs and its cofactor the RNA binding motif 15 (RBM15) protein and its related RBM15b/OTT3, both members of the SPEN family. RBM15 provides the missing link for nuclear translocation, since it binds to both DBP5 and NXF1 and thus, it acts as molecular link to the NXF1 export pathway. Both NXF1 and RBM15 are essential cellular proteins. We identified the mRNA export requirement of the simian type D retroviral transcript, which is mediated by the cis-acting RNA export element (CTE) present in SRV/D related retroviruses and in some murine LTR-retroelements, and its binding partner, the cellular protein NXF1, and is essential for the expression and mobility of these retroviruses. We also identified that the expression and mobility of a different subclasses of murine LTR-retroelements depends on the presence of a distinct cis-acting RNA transport element, RTE or MTE, which act like CTE but does not share its sequence or structural features. Our findings suggest that the posttranscriptional regulatory elements in modern retroelements evolved convergently as high-affinity RNA ligands of certain key components of the NXF1 mRNA export pathway. Thus, despite a complex evolutionary history, retroelements/retroviruses share a dependency on posttranscriptional regulation, but the detailed molecular mechanisms are distinct. Posttranscriptional regulation is also key to control the production of viruses such as the Kaposi's sarcoma-associated herpesvirus (KSHV) and is exerted via ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We found that the RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59 and provide a link to the NXF export pathway, a conserved interaction among different herpes family members. We recently demonstrated that ORF57, although very capable of promoting expression of many KSHV genes, is not able to provide RNA export function. Although not a bona fide export factor, ORF57, like the related proteins of other herpes viruses, plays a critical role in the posttranscriptional regulation of many viral genes and remains essential for the virus production. Thus, retroelements, retroviruses and virus like the Herpes virus family, share the same basic concepts of posttranscriptional regulation. Together, comparative studies of different viral models provide unique tools to address the complex network of molecular steps controlling viral and cellular mRNA expression and this has led to major discoveries on the factors regulating cellular nuclear export mechanisms. We also studied regulation of expression of some cytokine genes, which have practical applications for cancer immunotherapy and as molecular adjuvant for DNA vaccine regimens. The use of cytokine DNAs (IL-12 and IL-15) as molecular vaccine adjuvants was found to improve the quantity and alter the quality of the immune responses. To optimally use these cytokines, we are studying their regulation and have found that IL-15/IL-15Ra as well as the IL-12 cytokine family use similar highly regulated steps posttranscriptional and posttranslational regulation strategies. To use IL-12 DNA to its full potential, we studied the biology of this glycosylated 70 kDa heterodimeric cytokine to maximize cytokine production. Although the production of each subunit is independently regulated, coexpression of both molecules in the same cell is essential to form biologically active heterodimer. Prompted by our findings on the critical intracellular regulatory step of IL-15 and IL15Ra cross-stabilization, we investigated the posttranscriptional regulation and interaction of the p35 and p40 subunits leading to optimal IL-12p70 production. Investigating molecular steps controlling IL-12p70 biosynthesis, we found that the combination of RNA-optimized gene sequences, and importantly, fine-tuning of the relative expression levels of the two subunits within a cell resulted in a 1 log increased production of the IL-12p70 heterodimer. Importantly, we discovered that the p40 subunit plays a critical role in enhancing the p35 stability and promoting its intracellular trafficking, through the trans-Golgi network, resulting in formation of a stable, efficiently secreted IL-12p70 complex. Based on these observations, dual expression plasmids for IL-12p70 were designed to obtain favorable relative levels of the two subunits and optimal IL-12 expression. Our expression-optimized human IL-12 DNA showed higher cytokine production compared to DNAs expressing the native sequences that are currently employed in clinical trials. These optimized cytokine DNAs provide important molecular tools to be tested as molecular adjuvants in vaccine and in cancer immunotherapy, with promising future translational applications. Development of novel HIV DNA vaccine plasmids. An ideal HIV vaccine should provide protection against all HIV-1 variants. HIV sequence diversity and the presence of potential immunodominant "decoy" epitopes are hurdles in the development of an effective AIDS vaccine. To address these problems, we are exploring approaches to maximize immunological strength and breadth focusing on highly conserved regions of HIV to induce immune responses to nearly invariable proteome segments, essential for the function of the virus, while excluding responses to variable and potentially immunodominant "decoy" epitopes. We developed a prototype vaccine targeting regions within the p24gag, benefitting from our experience in RNA and protein expression and trafficking. Optimized plasmids were tested in mice and successful candidates were further tested in macaques. In proof-of-concept studies in mice and macaques, we demonstrated that immunization with this DNA elicits robust cellular and humoral immune responses against CE, which cannot be achieved by p55gag DNA vaccination. The translation of this novel concept is currently being pursued in an HVTN/DAIDS-supported clinical trial.

我们的研究重点是基因表达的调控，特别是控制细胞和病毒mRNA表达的机制，以及用于疫苗和免疫治疗的DNA载体的开发。由HIV-1研究开创的对逆转录病毒调控系统的分析，揭示了细胞核mRNA输出的重要方面，并为控制细胞mRNA和蛋白质运输的机制提供了关键的见解。逆转录病毒模型系统，特别是对HIV-1的调控，已经导致了mRNA代谢领域的重大发现。HIV-1 Rev是第一个被发现的病毒mRNA输出因子，它的发现有助于发现介导转录后基因表达控制的分子机制，以及我们开发增加病毒蛋白表达的方法，即RNA优化（也称为密码子优化）的开发，这是目前许多基因治疗应用的关键技术，包括HIV疫苗和细胞因子载体。Rev的研究还促使我们得出这样一个概念，即所有逆转录病毒/逆转录元件都使用转录后控制机制来进行复制。这些控制需要病毒RNA元件和细胞/病毒因子的结合，能够有效地将病毒mRNA连接到核输出途径并促进翻译。逆转录病毒/逆转录因子输出的研究为理解细胞基因表达的基本和复杂步骤提供了重要的工具。这种策略导致我们过去鉴定和表征NXF1，这是细胞mrna输出的关键核受体及其辅助因子RNA结合motif 15 （RBM15）蛋白及其相关的RBM15b/OTT3，两者都是SPEN家族的成员。RBM15提供了核易位缺失的环节，因为它结合DBP5和NXF1，因此，它作为NXF1输出途径的分子链接。NXF1和RBM15都是必需的细胞蛋白。我们确定了猿类D型逆转录病毒转录物的mRNA输出需求，这是由SRV/D相关逆转录病毒和一些小鼠ltr逆转录元件中存在的顺式作用RNA输出元件（CTE）及其结合伙伴细胞蛋白NXF1介导的，对这些逆转录病毒的表达和迁移至关重要。我们还发现，小鼠ltr -逆转录因子的不同亚类的表达和迁移取决于一种独特的顺式作用RNA转运元件RTE或MTE的存在，它们的作用类似于CTE，但不共享其序列或结构特征。我们的研究结果表明，现代逆转录元件中的转录后调控元件作为NXF1 mRNA输出通路某些关键组分的高亲和力RNA配体趋同进化。因此，尽管具有复杂的进化史，逆转录因子/逆转录病毒都依赖于转录后调控，但详细的分子机制是不同的。转录后调控也是控制卡波西肉瘤相关疱疹病毒（KSHV）等病毒产生的关键，并通过ORF57发挥作用，促进特定KSHV mRNA靶标的积累，包括ORF59 mRNA。我们发现RBM15和OTT3参与了KSHV ORF59的orf57增强表达，并提供了NXF输出通路的链接，这是不同疱疹家族成员之间的保守相互作用。我们最近证明ORF57虽然能够促进许多KSHV基因的表达，但不能提供RNA输出功能。虽然ORF57不是真正的输出因子，但与其他疱疹病毒的相关蛋白一样，ORF57在许多病毒基因的转录后调控中起着关键作用，对病毒的产生仍然至关重要。因此，逆转录因子、逆转录病毒和疱疹病毒家族等病毒具有相同的转录后调控基本概念。总之，不同病毒模型的比较研究为解决控制病毒和细胞mRNA表达的复杂分子步骤网络提供了独特的工具，这导致了对调节细胞核输出机制的因素的重大发现。我们还研究了一些细胞因子基因的表达调控，这些基因在癌症免疫治疗和DNA疫苗方案的分子佐剂中具有实际应用。使用细胞因子dna （IL-12和IL-15）作为分子疫苗佐剂可以提高免疫应答的数量和改变免疫应答的质量。为了更好地利用这些细胞因子，我们正在研究它们的调控，并发现IL-15/IL-15Ra以及IL-12细胞因子家族使用类似的高度调控步骤，转录后和翻译后调控策略。为了充分利用IL-12 DNA的潜力，我们研究了这种糖基化的70 kDa异二聚体细胞因子的生物学特性，以最大化细胞因子的产生。虽然每个亚基的产生都是独立调节的，但这两个分子在同一细胞中的共表达对于形成具有生物活性的异二聚体是必不可少的。基于我们对IL-15和IL15Ra交叉稳定的关键细胞内调控步骤的发现，我们研究了p35和p40亚基的转录后调控和相互作用，从而导致IL-12p70的最佳产生。研究控制IL-12p70生物合成的分子步骤，我们发现rna优化基因序列的组合，重要的是，两个亚基在细胞内的相对表达水平的微调导致IL-12p70异源二聚体的产量增加了1倍。重要的是，我们发现p40亚基通过反式高尔基网络在增强p35稳定性和促进其细胞内运输中起关键作用，从而形成稳定、有效分泌的IL-12p70复合物。基于这些观察结果，设计了IL-12p70的双表达质粒，以获得有利的两个亚基的相对水平和最佳的IL-12表达。与目前用于临床试验的表达天然序列的DNA相比，我们的表达优化的人IL-12 DNA显示出更高的细胞因子产量。这些优化后的细胞因子dna提供了重要的分子工具，可作为疫苗和癌症免疫治疗中的分子佐剂进行测试，具有很好的未来转化应用前景。新型HIV DNA疫苗质粒的研制。一种理想的艾滋病毒疫苗应该能够预防所有的艾滋病毒-1变异。HIV序列多样性和潜在的免疫优势“诱饵”表位的存在是开发有效艾滋病疫苗的障碍。为了解决这些问题，我们正在探索将免疫强度和广度最大化的方法，重点关注HIV的高度保守区域，以诱导对几乎不变的蛋白质组片段（对病毒的功能至关重要）的免疫反应，同时排除对可变和潜在的免疫优势“诱饵”表位的反应。我们开发了一种针对p24gag区域的原型疫苗，这得益于我们在RNA和蛋白质表达和运输方面的经验。优化的质粒在小鼠中进行了测试，成功的候选质粒在猕猴中进一步进行了测试。在小鼠和猕猴的概念验证研究中，我们证明了用这种DNA免疫可引起针对CE的强大的细胞和体液免疫反应，这是p55gag DNA疫苗无法实现的。目前，HVTN/ aids支持的一项临床试验正在对这一新颖概念进行翻译。