The Translational Regulation of Pro-apoptotic Genes

促凋亡基因的翻译调控

• 批准号: 10216207
• 负责人: Wayne Miles
• 金额: $ 35.69万
• 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/10216207
• 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 项目概要 视网膜母细胞瘤 1 蛋白 (pRB) 失活是癌症中最常见的改变之一。这是 因为 pRB 抑制三个 E2F 转录因子 E2F1-3 的活性，E2F1-3 控制 癌细胞生长（包括细胞周期和代谢）所需的许多关键 RB-E2F 靶基因 因素。除了促肿瘤过程外，E2F1-3 还调节肿瘤抑制过程的转录 在细胞凋亡和坏死中起核心作用的基因。该调节电路连接 RB-E2F 的表达 增殖和细胞死亡基因，最终防止不受控制的细胞生长。 pRB的广泛丢失 在癌症中的研究强调了该领域的一个重大难题：为什么缺乏 pRB 的细胞不会直接死亡？ 为了了解 RB-E2F 靶基因的调控，我们分析了 pRB-后 RNA 和蛋白质的变化 耗尽并发现促凋亡基因被转录，但它们没有翻译成蛋白质。一个 寻找与这些基因结合并阻断核糖体占据的 RNA 结合蛋白 (RBP) 鉴定出 Pumilio 复合体是 RB-E2F 促凋亡 mRNA 的直接翻译调节因子。此外， Pumilio 复合体的两个核心成分 PUM1 和 PUM2 的共同缺失引发了细胞死亡 RB1-/- 癌细胞通过促凋亡 mRNA 的翻译。人类的 Pumilio 复合体包括 PUM 和 NANOS (NOS) 蛋白，但 PUM 成员之间存在显着的功能冗余 和 NOS 蛋白家族对 Pumilio 复合物的细胞和肿瘤研究有限。为了规避这个问题， 我们对人类细胞进行了改造，使其仅表达一种我们可以特异性降解的 PUM 或 NOS 蛋白 利用新型生长素诱导降解决定子系统。这项技术进步使我们能够进行动力学 在没有 PUM 或 NOS 的情况下分析 RNA 稳定性、定位和翻译起始，并探测 不同 Pumilio 复合物在细胞调节和肿瘤发生中的作用。在这个新应用中，我们建议 1) 确定 Pumilio 复合物翻译抑制 RB-E2F 靶标促凋亡的机制 基因，2）定义RB1突变型小细胞肺癌（SCLC）中Pumilio复合物的调节机制 细胞，以及 3) 确定 Pumilio 复合物在 Rb1-/- 中的作用； Trp53-/- 小鼠 SCLC 肿瘤发生模型。 这些研究将确定在高度条件下防止细胞死亡成分产生的机制。 侵袭性和转移性 SCLC。