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 研究开创的逆转录病毒调控系统分析揭示了核 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重要的见解。逆转录病毒模型系统，特别是 HIV-1 的调控，在 mRNA 代谢领域取得了重大发现。 HIV-1 Rev是第一个被识别的病毒mRNA输出因子，它的发现有助于发现介导基因表达转录后控制的分子机制，以及我们开发增加病毒蛋白表达的方法，即开发RNA优化（也称为密码子优化），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 向量。 Rev的研究还促使我们得出这样一个概念：所有逆转录病毒/逆转录元件都使用对其复制至关重要的转录后控制机制。这些对照需要病毒 RNA 元件和细胞/病毒因子的组合，能够有效地将病毒 mRNA 与核输出途径连接起来并促进翻译。逆转录病毒/逆转录元件输出的研究为理解细胞基因表达中的基本和复杂步骤提供了重要工具。这一策略导致我们过去对 NXF1 进行了鉴定和表征，NXF1 是细胞 mRNA 输出的关键核受体，其辅因子是 RNA 结合基序 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，因为它与 DBP5 和 NXF1 结合，因此，它充当 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 D 型逆转录病毒转录物的 mRNA 输出要求，该要求由 SRV/D 相关逆转录病毒和一些鼠 LTR 逆转录元件中存在的顺式作用 RNA 输出元件 (CTE) 及其结合伴侣细胞蛋白 NXF1 介导，并且对于这些逆转录病毒的表达和迁移性至关重要。我们还发现，鼠LTR逆转录元件的不同亚类的表达和移动性取决于不同的顺式作用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 输出途径某些关键组件的高亲和力 RNA 配体趋同进化。因此，尽管有复杂的进化历史，逆转录元件/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，从而促进特定 KSHV mRNA 靶标（包括 ORF59 mRNA）的积累。我们发现 RBM15 和 OTT3 参与 ORF57 增强的 KSHV ORF59 表达，并提供与 NXF 输出途径的联系，NXF 输出途径是不同疱疹家族成员之间保守的相互作用。我们最近证明，ORF57 虽然非常能够促进许多 KSHV 基因的表达，但不能提供 RNA 输出功能。尽管不是真正的输出因子，ORF57 与其他疱疹病毒的相关蛋白一样，在许多病毒基因的转录后调控中发挥着关键作用，并且对于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提供了独特的工具来解决控制病毒和细胞 mRNA 表达的分子步骤的复杂网络，这导致了关于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表达的调节，这些基因在癌症免疫治疗和作为 DNA 疫苗方案的分子佐剂方面具有实际应用。研究发现，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反应的数量并改变免疫反应的质量。为了最佳地利用这些细胞因子，我们正在研究它们的调节，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节步骤转录后和翻译后调节策略。为了充分利用 IL-12 DNA 的潜力，我们研究了这种糖基化 70 kDa 异二聚细胞因子的生物学，以最大限度地提高细胞因子的产量。尽管每个亚基的产生是独立调节的，但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至关重要。在我们对 IL-15 和 IL15Ra 交叉稳定的关键细胞内调节步骤的发现的推动下，我们研究了 p35 和 p40 亚基的转录后调节和相互作用，从而导致最佳的 IL-12p70 产生。通过研究控制 IL-12p70 生物合成的分子步骤，我们发现 RNA 优化的基因序列的组合，以及重要的是，细胞内两个亚基相对表达水平的微调，导致 IL-12p70 异二聚体的产量增加了 1 个对数。重要的是，我们发现 p40 亚基在增强 p35 稳定性和促进其通过跨高尔基体网络的细胞内运输方面发挥着关键作用，从而形成稳定、有效分泌的 IL-12p70 复合物。基于这些观察，设计了 IL-12p70 的双表达质粒以获得两个亚基的有利相对水平和最佳的 IL-12 表达。与目前临床试验中使用的表达天然序列的 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，并具有广阔的未来转化应用前景。新型 HIV DNA 疫苗质粒的开发。理想的 HIV 疫苗应针对所有 HIV-1 变种提供保护。 HIV序列多样性和潜在免疫显性“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，我们正在探索最大化免疫强度和广度的方法，重点关注艾滋病毒的高度保守区域，以诱导对几乎不变的蛋白质组片段的免疫反应，这对病毒的功能至关重要，同时排除对可变的和潜在的免疫显性“诱饵”表位的反应。受益于我们在 RNA 和蛋白质表达和运输方面的经验，我们开发了一种针对 p24gag 内区域的原型疫苗。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行了进一步测试。在小鼠和猕猴的概念验证研究中，我们证明用这种 DNA 进行免疫可引发针对 CE 的强大细胞和体液免疫反应，这是 p55gag DNA 疫苗接种无法实现的。这一新概念的转化目前正在 HVTN/DAIDS 支持的临床试验中进行。