Posttranscriptional control of gene expression

基因表达的转录后控制

• 批准号: 7965261
• 负责人: BARBARA K FELBER
• 金额: $ 86.75万
• 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/7965261
• 关键词: Acquired Immunodeficiency Syndrome; Adjuvant; Alternative Splicing; Animal Model; Attenuated; Binding; Biochemical; Biochemistry; Biological; Biological Models; Cancer Model; Cell Nucleus; Cells; Chemical Vaccines; Complex; Cytokine Gene; Cytoplasm; DNA; DNA Vaccines; Data; Dependency; Development; Dissection; Elements; Embryo; Family; Family Study; Family member; Future; Gene Expression; Gene Expression Regulation; Generations; Genetic; Genomics; Goals; Gold; HIV; HIV-1; Half-Life; Hand; Immune response; Individual; Injection of therapeutic agent; Interleukin-12; Interleukin-15; Interleukin-2; Intracisternal A-Particle Elements; Knowledge; Life; Light; Link; Macaca mulatta; Mammals; Mediating; Messenger RNA; Metabolism; Molecular; Mus; Neoplasm Metastasis; Neuroblastoma; Nuclear; Nuclear Receptors; Pathogenesis; Pathway interactions; Peptide Signal Sequences; Plasma; Plasmids; Post-Translational Protein Processing; Post-Translational Regulation; Process; Protein Family; Protein Isoforms; Proteins; Proteomics; RNA; RNA Binding; RNA Splicing; RNA Transport; Recording of previous events; Regulation; Research; Retroelements; Retroviridae; Role; Route; SIV; Specificity; System; T-Lymphocyte; Tissues; Transcript; Translations; Vaccine Adjuvant; Vaccines; Variant; Vertebrates; Viral; Viremia; Virus; cancer immunotherapy; cytokine; expression vector; functional genomics; improved; in vivo; inhibitor/antagonist; insight; knock-down; mRNA Export; mRNA Expression; mRNA Stability; macromolecule; novel; polypeptide; protein transport; rev Protein; trafficking; vector

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 expression and mobility of the murine LTR-retroelements (Intracisternal A particle retroelements) depends on the presence of the cis-acting RNA transport element RTE, which acts like the CTE. 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 via the NXF1 export pathway. RBM15, a novel mRNA export factor, belongs to the SPEN family of proteins and is conserved among metazoa. We further identified that another SPEN protein, OTT3 acts as RNA export co-factor like RBM15. On the other hand, SHARP, another SPEN family member does not have export function. 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. Why vertebrates had evolved and maintain variants of the otherwise similar factors? Since RBM15 and OTT3 are factors of the NXF1 pathway, which controls the export of general mRNA, we speculated that they may be part of a developmental or/and tissue-specific switch that controls mRNA export rates or/and specificity. Importantly, we found that RBM15 and OTT3 act as molecular link to the NXF1 export pathway. Thus, our studies on retroviral RNA export led to identification important factors of the mRNA export route. We also studied regulation of expression of cytokine genes. 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 studied their regulation and 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 knowledge, we have generated optimized expression vectors, which allow their efficient use in animal models, and their use as molecular adjuvant in vaccine and in cancer immunotherapy is promising and could be important for future translational applications. IL-15 expression is controlled at several posttranscriptional and posttranslational steps such as mRNA stability, translation, intracellular trafficking and secretion. 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. There are two known isoforms of IL-15 containing either a long signal peptide (LSP) or a short signal peptide (SSP), and are produced by alternatively spliced transcripts. We have studied the function of these 2 variants of IL-15. We found that similar to LSP IL-15, the SSP IL-15 is stabilized and secreted efficiently upon coexpression of IL-15Ralpha. Coinjection of SSP IL-15- and IL-15Ralpha-expressing plasmids into mice resulted in increased plasma levels of bioactive heterodimeric IL-15 and mobilization and expansion of NK and T cells. Therefore, SSP IL-15 is secreted and bioactive when produced as a heterodimer with IL-15Ralpha in the same cell. The apparent half-life of this heterodimer is lower compared with LSP IL-15/IL-15Ralpha, due to different intracellular processing. Coexpression of both LSP IL-15 and SSP IL-15 in the presence of IL-15Ralpha results in lower levels of bioactive IL-15, indicating that LSP and SSP IL-15 compete for the binding to IL-15Ralpha when expressed in the same cell. Because the SSP IL-15 interaction to IL-15Ralpha leads to a complex with lower apparent stability, SSP IL-15 functions as competitive inhibitor of LSP IL-15. The data suggest that usage of alternative splicing is an additional level of control of IL-15 activity. Expression of both SSP and LSP forms of IL-15 appears to be conserved in many mammals, suggesting that SSP may be important for expressing a form of IL-15 with lower magnitude or duration of biological effects. We also study the family of IL-12 and related cytokines, which consist of 2 polypeptide chains. Our studies revealed an important aspect of the posttranslational control of these cytokines. Using the expression of individual subunits alone or in combinations, we found that one of the subunits was relatively unstable and limiting; expression of the other subunit in the same cell increased the stability and secretion of the unstable partner and the levels of the secreted heterodimer. This allowed use to generate optimized vectors to produce such cytokines for in vivo application. As an application in a cancer model, injection of optimized IL-27 DNA together with IL-2 successfully abolished neuroblastoma metastasis. Future studies will be directed to replacing IL-2 with other cytokines like IL-15/IL-15Ra in such cancer models.

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。mRNA代谢的一个关键步骤是mRNA从细胞核转运到细胞质。由HIV-1研究开创的对逆转录病毒系统的分析，揭示了细胞核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mRNA代谢机制。对转录后调控和大分子核胞质转运机制的剖析，对于理解细胞基因表达和病毒表达过程具有重要意义。我们确定了猿猴D型逆转录病毒转录物的mRNA输出需求，该转录物是由顺式作用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蛋白作为细胞mrna的关键核受体，并且这种功能在后生动物中是保守的。我们发现小鼠ltr -逆转录因子（内源性A颗粒逆转录因子）的表达和迁移取决于顺式作用RNA转运元件RTE的存在，RTE的作用类似于CTE。这一发现表明，尽管具有复杂的进化史，逆转录因子和逆转录病毒都依赖于转录后调控。我们发现RNA结合基序15 （RBM15）蛋白是通过NXF1输出途径结合和输出含有rte的mrna的细胞因子。RBM15是一种新的mRNA输出因子，属于SPEN蛋白家族，在后生动物中保守。我们进一步发现另一种SPEN蛋白OTT3像RBM15一样作为RNA输出辅助因子。另一方面，SPEN家族的另一个成员SHARP没有输出功能。生化和亚细胞定位研究表明，OTT3和RBM15在体内也相互作用，进一步支持共享功能。小鼠RBM15基因敲低是胚胎致死的，这表明OTT3不能弥补RBM15的缺失，这支持了这些蛋白除了具有相似的活性外，可能具有不同的生物学作用的观点。为什么脊椎动物进化并保持了其他相似因素的变体？由于RBM15和OTT3是控制一般mRNA输出的NXF1通路的因子，我们推测它们可能是发育或/和组织特异性开关的一部分，控制mRNA输出率或/和特异性。重要的是，我们发现RBM15和OTT3作为NXF1输出途径的分子链接。因此，我们对逆转录病毒RNA输出的研究导致了mRNA输出途径的重要因素的确定。我们还研究了细胞因子基因的表达调控。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。为了优化这些细胞因子的使用，我们研究了它们的调控，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控策略。因此，亚基和异源二聚体的形成和分泌是高度调控的步骤。利用这些知识，我们已经生成了优化的表达载体，使它们能够在动物模型中有效地使用，并且它们作为疫苗和癌症免疫治疗中的分子佐剂是有希望的，并且对于未来的翻译应用可能是重要的。IL-15的表达受到转录后和翻译后几个步骤的控制，如mRNA的稳定性、翻译、细胞内运输和分泌。转录后和翻译后修饰的结合导致高效表达质粒的产生，其生物活性IL-15的水平提高了数百倍。IL-15有两种已知的同工异构体，含有长信号肽（LSP）或短信号肽（SSP），它们是通过选择性剪接转录物产生的。我们研究了IL-15的这两种变体的功能。我们发现与LSP IL-15相似，SSP IL-15在IL-15Ralpha共表达后稳定并有效分泌。向小鼠共注射表达SSP IL-15和il - 15rα的质粒，可增加血浆中具有生物活性的异二聚体IL-15的水平，并可动员和扩增NK细胞和T细胞。因此，SSP IL-15与il - 15rα在同一细胞中以异源二聚体的形式产生时，可以分泌并具有生物活性。与LSP IL-15/ il - 15rα相比，该异源二聚体的表观半衰期较低，这是由于细胞内处理的不同。在IL-15Ralpha存在的情况下，LSP IL-15和SSP IL-15的共表达会导致较低的生物活性IL-15水平，这表明LSP和SSP IL-15在同一细胞中表达时，会竞争与IL-15Ralpha的结合。由于SSP IL-15与il - 15rα的相互作用导致复合物的表观稳定性较低，SSP IL-15作为LSP IL-15的竞争性抑制剂。这些数据表明，选择性剪接的使用是对IL-15活性的额外控制。IL-15的SSP和LSP形式的表达在许多哺乳动物中似乎都是保守的，这表明SSP可能对表达一种具有较低强度或持续时间的生物效应的IL-15形式很重要。我们还研究了IL-12家族及其相关细胞因子，它由2个多肽链组成。我们的研究揭示了这些细胞因子翻译后控制的一个重要方面。使用单个亚基单独或组合的表达，我们发现其中一个亚基相对不稳定和受限；另一个亚基在同一细胞中的表达增加了不稳定伴侣的稳定性和分泌以及分泌的异二聚体的水平。这允许使用生成优化的载体来产生用于体内应用的细胞因子。将优化后的IL-27 DNA与IL-2一起注射到肿瘤模型中，成功地消除了神经母细胞瘤的转移。未来的研究将着眼于用其他细胞因子如IL-15/IL-15Ra替代IL-2在这类癌症模型中。