The Translational Regulation of Pro-apoptotic Genes

促凋亡基因的翻译调控

• 批准号: 10431915
• 负责人: Wayne Miles
• 金额: $ 34.97万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-14 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10431915
• 关键词: Affect; Alleles; Apoptosis; Apoptotic; Auxins; BRAT gene; Binding; Binding Proteins; Cancer Cell Growth; Cancer Model; Cell Cycle; Cell Death; Cell Proliferation; Cells; Complex; Coupling; Data; Development; E2F transcription factors; Gene Expression Regulation; Genes; Genetic Transcription; Genetically Engineered Mouse; Genomics; Homologous Protein; Human; Human Engineering; Human Genome; Individual; Kinetics; Link; Malignant Neoplasms; Mediating; Messenger RNA; Metabolic; Methods; MicroRNAs; Modeling; Mus; Necrosis; Neoplasm Metastasis; Neoplasms; Pathologic; Pathway interactions; Patients; Pattern; Play; Process; Production; Protein Biosynthesis; Protein Family; Proteins; RNA; RNA Stability; RNA analysis; RNA-Binding Proteins; Regulation; Retinoblastoma; Retinoblastoma Protein; Ribosomes; Role; Survival Rate; System; Testing; Transcript; Transcription Coactivator; Translation Initiation; Translational Regulation; Translations; Tumor Suppressor Proteins; Work; Zinc Fingers; cancer cell; cancer therapy; cell growth; cell growth regulation; cytotoxic; human disease; human model; lung cancer cell; lung small cell carcinoma; member; mutant; novel; prevent; protein complex; protein degradation; response; therapy design; tumor; tumorigenesis; uncontrolled cell growth

THE TRANSLATIONAL REGULATION OF PRO-APOPTOTIC GENES PROJECT SUMMARY Inactivation of the Retinoblastoma 1 protein (pRB) is one of the most common alterations in cancer. This is because pRB inhibits the activity of three E2F transcription factors, E2F1-3, that control the expression of numerous critical RB-E2F target genes that are required for cancer cell growth, including cell cycle and metabolic factors. In addition to tumor-promoting processes, E2F1-3 also regulates the transcription of tumor-inhibiting genes that play a central role in apoptosis and necrosis. This regulatory circuit links the expression of RB-E2F proliferation and cell death genes, ultimately preventing uncontrolled cell growth. The widespread loss of pRB in cancer underscores a significant puzzle in the field: why don’t pRB-deficient cells simply die? To understand the regulation of the RB-E2F target genes, we profiled RNA and protein changes following pRB- depletion and found that the pro-apoptotic genes were transcribed, but they were not translated into protein. A search for RNA-binding proteins (RBPs) that bound to and blocked the ribosome occupancy of these genes identified the Pumilio complex as direct translational regulators of RB-E2F pro-apoptotic mRNAs. Furthermore, co-deletion of the two core components of the Pumilio complex, PUM1 and PUM2, provoked the cell death of RB1-/- cancer cells via the translation of pro-apoptotic mRNAs. The human Pumilio complex is comprised of PUM and NANOS (NOS) proteins, however significant functional redundancy between the members of the PUM and NOS protein families has limited cellular and tumor studies of the Pumilio complex. To circumvent this issue, we have engineered human cells to express only a single PUM or NOS protein that we can specifically degrade utilizing a novel Auxin-inducible degron system. This technical advance has enabled us to conduct kinetic analysis of RNA stability, localization and translation initiation in the absence of PUM or NOS and to probe the role(s) of different Pumilio complexes in cellular regulation and tumorigenesis. In this new application we propose to: 1) Determine the mechanism(s) of Pumilio complex translational suppression of RB-E2F target pro-apoptotic genes, 2) Define the mechanism of Pumilio complex regulation in RB1 mutant Small Cell Lung Cancer (SCLC) cells, and 3) Determine the role of the Pumilio complex in the Rb1-/-; Trp53-/- mouse SCLC tumorigenesis model. These studies will identify the mechanism(s) that prevent the production of cell death components in highly aggressive and metastatic SCLC.

促凋亡基因的翻译调控 项目摘要 视网膜母细胞瘤1蛋白（pRB）的失活是癌症中最常见的改变之一。这是 因为pRB抑制了三种E2 F转录因子E2 F1 -3的活性，E2 F1 -3控制着 许多关键的RB-E2 F靶基因是癌细胞生长所需的，包括细胞周期和代谢 因素除了肿瘤促进过程之外，E2 F1 -3还调节肿瘤抑制因子的转录。 在细胞凋亡和坏死中起核心作用的基因。这种调节回路将RB-E2 F的表达与 增殖和细胞死亡基因，最终防止不受控制的细胞生长。pRB的广泛缺失 在癌症中的应用强调了该领域的一个重要难题：为什么pRB缺陷细胞不会简单地死亡？ 为了了解RB-E2 F靶基因的调控，我们分析了pRB-1表达后RNA和蛋白质的变化。 耗尽并发现促凋亡基因被转录，但它们不翻译成蛋白质。一 寻找与这些基因结合并阻断其核糖体占据的RNA结合蛋白（RBP） 鉴定了Pumilio复合物作为RB-E2 F促凋亡mRNA的直接翻译调节剂。此外，委员会认为， Pumilio复合物的两个核心成分，pumilio 1和pumilio 2的共缺失引起了细胞死亡。 RB 1-/-癌细胞通过促凋亡mRNA的翻译。人类的普米利奥复合体由 PUM和NANOS（NOS）蛋白，但PUM成员之间存在显着的功能冗余 和NOS蛋白家族的Pumilio复合物的细胞和肿瘤研究有限。为了避免这个问题， 我们已经改造了人类细胞，使其只表达一种我们可以特异性降解的PUM或NOS蛋白， 利用新的生长素诱导降解决定子系统。这项技术的进步使我们能够进行动力学 在不存在PUM或NOS的情况下分析RNA稳定性、定位和翻译起始，并探测PUM或NOS的表达。 不同Pumilio复合物在细胞调节和肿瘤发生中的作用。在这个新的应用程序中，我们建议 1）确定RB-E2 F靶点的Pumilio复合物翻译抑制的机制， 基因，2）定义RB 1突变型小细胞肺癌（SCLC）中Pumilio复合物调控的机制 3）确定Pumilio复合物在Rb 1-/-; Trp 53-/-小鼠SCLC肿瘤发生模型中的作用。 这些研究将确定在高水平的细胞内防止细胞死亡成分产生的机制。 侵袭性和转移性SCLC。