Posttranscriptional control of gene expression

基因表达的转录后控制

• 批准号: 8552670
• 负责人: BARBARA K FELBER
• 金额: $ 46.57万
• 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/8552670
• 关键词: ATP phosphohydrolase; Address; Adjuvant; Affinity; Animal Model; Antigens; Binding; Biochemical; Biochemistry; Biological; Biological Models; Boxing; Cell Nucleus; Chemical Vaccines; Codon Nucleotides; Comparative Study; Complex; Cytokine Gene; Cytomegalovirus; Cytoplasm; DNA; Dependency; Development; Dissection; Elements; Embryo; Family; Family member; Future; Gene Expression; Gene Expression Regulation; Genetic; Genomics; Goals; HIV; HIV vaccine; HIV-1; Herpesviridae; Herpesvirus Type 3; Homologous Gene; Human Herpesvirus 4; Human Herpesvirus 8; Immune response; Interleukin-12; Interleukin-15; Ligands; Light; Link; Mason-Pfizer monkey virus; Mediating; Messenger RNA; Metabolism; Methodology; Methods; Modeling; Molecular; Mus; Nuclear; Nuclear Export; Nuclear Pore Complex; Nuclear Receptors; Pathway interactions; Plasmids; Post-Translational Regulation; Process; Production; Protein Family; Proteins; Proteomics; RNA; RNA Binding; RNA Transport; Recording of previous events; Regulation; Regulatory Element; Reporting; Research; Retroelements; Retroviridae; Role; SIV; SRV-1; SRV-2; Simplexvirus; Specificity; Structure; System; Technology; Testing; Tissues; Transcript; Translations; Vaccine Adjuvant; Vaccines; Viral; Viral Proteins; Virus; Work; cancer immunotherapy; cofactor; cytokine; expression vector; functional genomics; gene therapy; improved; in vivo; insight; knock-down; mRNA Export; mRNA Expression; macromolecule; method development; novel; protein transport; rev Protein; tool; trafficking; viral RNA

Our research focuses on the regulation of gene expression, in particular the mechanisms controlling cellular and viral mRNA expression. 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. The dissection of the mechanisms of posttranscriptional control and nucleocytoplasmic trafficking of macromolecules is relevant to understanding the processes involved in cellular gene expression as well as virus expression. Understanding the basic mechanisms of mRNA expression led to the development of RNA/codon optimization as a key strategy to improve gene expression, which also led to the development of efficient expression vectors for both HIV/SIV antigens and cytokines.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. The study of Rev also prompted us to derive the concept that all retroviruses and 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 and retroelement export could provide important tools to understand essential and complex steps in cellular gene expression. In fact, this strategy resulted in our past identification and characterization of NXF1, which is the key nuclear receptor for the export of cellular mRNAs. 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. CTE is a highly conserved RNA element located next to the 3LTR in SRV-1, SRV-2, MPMV/SRV-3, and the recently discovered SRV-4 as well as in some murine LTR-retroelements. CTE is essential for the expression and mobility of these retroviruses. We discovered that cellular NXF1 protein acts as the key nuclear receptor for cellular mRNAs and that this function is conserved in metazoa and is essential. We also identified that the expression and mobility of a different subclass of murine LTR-retroelements depends on the presence of a distinct cis-acting RNA transport element, RTE, which acts like CTE but does not share its sequence or structural features. Thus, despite a complex evolutionary history, retroelements and retroviruses share a dependency on posttranscriptional regulation, but the detailed molecular mechanisms are distinct. We identified the mechanism that promotes the export of the RTE RNA export and reported that the RNA binding motif 15 (RBM15) protein acts as the cellular factor that binds and exports RTE-containing mRNAs via the NXF1 export pathway. RBM15, a novel mRNA export factor, belongs to the SPEN family of proteins and is conserved among metazoa. We reported that another SPEN protein, RBM15b/OTT3, acts as an RNA export co-factor like RBM15. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knock-down of RBM15 in mouse is embryonic lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, likely have distinct biological roles. Since RBM15 andOTT3 are cofactors of NXF1, we speculate that they may be part of a developmental or/and tissue-specific switch that controls mRNA export rates or/and specificity. How does NXF1 guide the transcripts through the nuclear pore complex (NPC)? The DEAD-box ATPase DBP5, located at the NPC, is thought to mediate the directional passage of mRNA through the NPC via an unknown mechanism, since it does not bind to NXF1. We reported that RBM15 provides the missing link, since it binds to both DBP5 and NXF1 and thus, it acts as molecular link to the NXF1 export pathway.We also identified a RNA export element present in another class of retroelements (mus D retroelements). This export element (termed MTE) is distinct in sequence and structure compared to the previously identified export elements. We identified that structure of MTE and identified two classes of tertiary interactions, namely kissing loops and a pseudoknot. We showed that the complex tertiary structure allows for distinct long-range molecular interactions that are essential for function. This work constitutes the first example of an RNA transport element requiring such structural motifs to mediate nuclear export. 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.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. Interestingly, we found that the RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59 and provide a link to the NXF export pathway. We further noted that RBM15 and OTT3 also interact with the ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69, demonstrating conservation of the interaction of RBM15 and OTT3 with the posttranscriptional regulators of different herpes family members.Thus, despite a complex evolutionary history, 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 are highly regulated at several posttranscriptional as well as posttranslational steps. 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 posttranscriptional and posttranslational regulation strategies. As a result, the formation and secretion of the subunits and heterodimers are highly regulated steps. Using this information, we have generated optimized expression vectors, which allow their efficient use in animal models. These optimized cytokine DNAs provide us with important molecular tools to be tested as molecular adjuvants in vaccine and in cancer immunotherapy, with promising future translational applications.

我们的研究重点是基因表达的调节，特别是控制细胞和病毒mRNA表达的机制。对HIV-1的研究率高的逆转录病毒调节系统的分析已阐明了核mRNA导出的重要方面，并为控制细胞mRNA和蛋白质转运的机制提供了关键的见解。分解后控制后控制和大分子核质流量的机制与了解细胞基因表达和病毒表达涉及的过程有关。理解mRNA表达的基本机制导致RNA/密码子优化作为改善基因表达的关键策略，这也导致了HIV/SIV抗原和细胞因子的有效表达载体的发展。逆转录病毒模型系统，特别是HIV-1的调节，导致了MRNA代理领域的主要发现。 HIV-1 Rev是第一个鉴定出的病毒mRNA输出因子，其发现对介导基因表达的转录后控制的分子机制以及我们的发展方法来提高病毒蛋白的表达的发展，即在RNA优化的开发（也称为密码子优化）中，该方法适用于许多Gene the，许多Gene cye cye cye cye cye cye cye cye cye cye cye canse consention covension tene consection。对REV的研究还促使我们得出了这样的概念，即所有逆转录病毒和重新元素都使用转录后控制机制，对它们的复制必不可少。这些对照需要将病毒RNA元件和能够有效将病毒mRNA连接到核出口途径并促进翻译的细胞/病毒因子组合。对逆转录病毒和追溯出口的研究可以提供重要的工具，以了解细胞基因表达的基本和复杂步骤。实际上，这一策略导致了我们过去的鉴定和表征NXF1，这是导出细胞mRNA的关键核受体。我们鉴定了D型D型逆转录病毒转录物的mRNA输出需求，该需求是由顺式作用RNA输出元件（CTE）及其结合伴侣介导的细胞蛋白NXF1。 CTE是一个高度保守的RNA元件，位于SRV-1，SRV-2，MPMV/SRV-3中的3LTR旁边，最近发现的SRV-4以及一些鼠LTR重元中。 CTE对于这些逆转录病毒的表达和活动性至关重要。我们发现细胞NXF1蛋白充当细胞mRNA的关键核受体，并且该功能在后生动物中是保守的，并且是必不可少的。 我们还确定，不同鼠LTR重新元素不同子类的表达和迁移率取决于存在独特的顺式作用RNA传输元件RTE的存在，该元件的作用类似于CTE，但不具有其序列或结构特征。因此，尽管有复杂的进化史，但逆转录病毒和逆转录病毒对转录后调节有着依赖性，但是详细的分子机制是不同的。我们确定了促进RTE RNA导出的导出的机制，并报告说RNA结合基序15（RBM15）蛋白是通过NXF1导出途径结合和导出含RTE的mRNA的细胞因子。 RBM15是一种新型的mRNA输出因子，属于蛋白质的Spen家族，并在后唑萨中保守。 我们报道说，另一种SPEN蛋白RBM15B/OTT3充当RNA输出的co因子，例如RBM15。生化和亚细胞定位研究表明，OTT3和RBM15在体内也相互相互作用，进一步支持共享功能。小鼠中RBM15的遗传敲低是胚胎致死的，表明OTT3无法补偿RBM15损失，这支持了这样一种观念，即除了共享相似的活性外，这些蛋白质可能具有不同的生物学作用。 由于RBM15和3是NXF1的辅助因子，因此我们推测它们可能是控制mRNA输出速率或/和特异性的发育或/和组织特异性开关的一部分。 NXF1如何通过核孔复合物（NPC）指导转录本？位于NPC的Dead-Box ATPase DBP5被认为可以通过未知机制介导mRNA通过NPC的方向传递，因为它不与NXF1结合。我们报告说，RBM15提供了缺失的链接，因为它与DBP5和NXF1均结合，因此它是与NXF1导出途径的分子链接。我们还确定了另一类的重新元素中存在的RNA导出元件（mus d retroements）。与先前确定的导出元件相比，该导出元件（称为MTE）在序列和结构上是不同的。我们确定了MTE的结构，并确定了两类的三级相互作用，即接吻循环和伪诺。我们表明，复杂的三级结构允许对功能必不可少的独特的远程分子相互作用。这项工作构成了RNA传输元件的第一个例子，需要这种结构基序以介导核出口。我们的发现表明，现代恢复元件中的转录后调节元素随着NXF1 mRNA出口途径的某些关键组成部分的高亲和力RNA配体的趋于趋于发展。POSTSTRANSCTRANSTIONSCONLUTIONAL CONLUTIONS也是控制病毒的产生，例如Kaposi的Sarcoma-sarmaMAseed iS.sseed-exed iS.Ksshvirus（Ksshvirus）（kaposi sherpesvirus）（Ksshvirus）（Ksshvirus）（Ksshvirus）（kaposi sherpecectres）（kaposi）。包括ORF59 mRNA在内的特定KSHV mRNA靶标的积累。有趣的是，我们发现RBM15和OTT3参与KSHV ORF59的ORF57增强表达，并提供了与NXF出口途径的链接。我们进一步指出，RBM15和OTT3还与ORF57同源物Epstein-Barr病毒（EBV）EB2相互作用，单纯疱疹病毒（HSV）ICP27，Varicella-Zoster病毒（VZV）IE4/ORF4和compomegalovirus（CMV）UL69 UL69 UL69 UL69，INBM 1不同疱疹家族成员的转录后调节剂。尽管如此，尽管有复杂的进化史，恢复元素，逆转录病毒和病毒（例如疱疹病毒家族），但具有同样的基本概念，对后期后的调节具有相同的基本概念。总之，对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表达的复杂分子步骤网络，这导致了有关调节细胞核出口机制的因素的重大发现。我们还研究了某些细胞因子基因表达的调节，这些基因表达在几种后记录后以及转运后的几个。发现使用细胞因子DNA（IL-12和IL-15）作为分子疫苗佐剂可改善数量并改变免疫反应的质量。 为了最佳使用这些细胞因子，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转录后和翻译后调节策略。结果，亚基和异二聚体的形成和分泌是高度调节的步骤。使用这些信息，我们生成了优化的表达向量，从而使它们在动物模型中有效使用。这些优化的细胞因子DNA为我们提供了重要的分子工具，可以在疫苗和癌症免疫疗法中作为分子佐剂进行测试，并具有有希望的未来翻译应用。