The translation regulation of pro-apoptotic genes

促凋亡基因的翻译调控

• 批准号: 10599664
• 负责人: Wayne Miles
• 金额: $ 8.6万
• 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/10599664
• 关键词: Affect; Alleles; Apoptosis; Apoptotic; Auxins; Award; BRAT gene; Binding; Binding Proteins; Cell Cycle; Cell Death; Cell Proliferation; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; Coupling; Data; Development; Dissection; E2F transcription factors; Engineering; Family; Gene Expression Regulation; Genes; Genetic Transcription; Genetically Engineered Mouse; Genomics; Homologous Protein; Human; Human Genome; Individual; Kinetics; Knock-out; Malignant Neoplasms; Mechanics; Messenger RNA; Metabolic; Methods; MicroRNAs; Necrosis; Neoplasm Metastasis; Neoplasms; Pathologic; Pathway interactions; Patients; Pattern; Plant Growth Regulators; Process; Protein Biosynthesis; Proteins; RNA; RNA Interference; 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; cdc Genes; cell growth; cell growth regulation; cytotoxic; design; experimental study; human disease; in vivo; lung cancer cell; lung small cell carcinoma; mouse model; prevent; protein complex; response; therapy design; tumor; tumorigenesis; uncontrolled cell growth

PROJECT SUMMARY (PARENTAL AWARD) Inactivation of the Retinoblastoma 1 protein (pRB) is found in a substantial fraction of human cancers. This is because pRB inhibits the activity of three important E2F transcription factors (E2F1-3) that control the expression of numerous tumor-promoting genes, which control cell growth including cell cycle genes and metabolic factors. Conversely, the E2Fs also regulate the transcription of tumor-inhibiting processes including important apoptosis and necrosis genes. This Rb-controlled coupled expression of proliferation and cell death genes is designed to prevent uncontrolled cell growth that is a hallmark of cancer. However, the widespread loss of pRB in cancer underlines a major unsolved puzzle: why don’t pRB-deficient cells simply die? To answer this question, we profiled RNA and protein changes following pRB-depletion and found that essential cell death genes were indeed transcribed, but the mRNAs were not translated into functional proteins. We then searched for RNA-binding proteins (RBPs) that bound to and blocked the Ribosome occupancy of these cell- death genes. This analysis identified the PUMILIO (PUM) family of RBPs as potential regulators of cell death RNAs. Importantly, as would be predicted by such a regulatory role, co-depletion of PUM1 and PUM2 resulted in the apoptosis of RB1-deficient cancer cells. However, the high level of functional redundancy has limited functional dissection of PUM1 and PUM2 mechanics in specific translational regulation of cell death mRNAs. To circumvent this technical issue, we have engineered PUM1-/-, PUM2-/- CRISPR-derived knockout human cells that additionally express individually degradable PUM proteins. Following the addition of the plant hormone, AUXIN, PUM1 or PUM2 are rapidly degraded, enabling kinetic analysis of the stability, localization and translation initiation of Rb-regulated mRNA transcripts. Using well-defined candidate cell death genes, we propose to determine the mechanism(s) associated with mRNA regulation by the individual PUM1 or PUM2 proteins. We will then determine which PUM regulatory mechanisms are required in the development of RB1-/- Small Cell Lung Cancer (SCLC) in vivo.

项目概要（母公司奖） 视网膜母细胞瘤1蛋白（pRB）的失活在相当大一部分人类癌症中发现。这是 因为pRB抑制了三种重要的E2 F转录因子（E2 F1 -3）的活性， 许多肿瘤促进基因，控制细胞生长，包括细胞周期基因和代谢因子。 相反，E2 Fs还调节肿瘤抑制过程的转录，包括重要的细胞凋亡 和坏死基因。这种Rb控制的增殖和细胞死亡基因的偶联表达被设计成 防止不受控制的细胞生长，这是癌症的标志。然而，pRB在癌症中的广泛缺失 强调了一个主要的未解之谜：为什么pRB缺陷细胞不会简单地死亡？ 为了回答这个问题，我们分析了pRB耗竭后RNA和蛋白质的变化，发现 细胞死亡基因确实被转录，但mRNA没有被翻译成功能蛋白。然后我们 寻找RNA结合蛋白（RBP），结合并阻止这些细胞的核糖体占据， 死亡基因该分析确定了RBPs的PUM家族作为细胞死亡的潜在调节因子 RNA。重要的是，正如这种调节作用所预测的那样，导致了BMP 1和BMP 2的共同消耗。 RB 1缺陷癌细胞的凋亡。然而，高水平的功能冗余限制了 在细胞死亡mRNAs的特异性翻译调节中，对p53 1和p53 2机制的功能性剖析。到 为了规避这一技术问题，我们已经设计了CRISPR衍生的CRISPR敲除人类细胞， 其另外表达可单独降解的PUM蛋白。加入植物激素后， AUXIN、AUXIN 1或AUXIN 2迅速降解，使得能够进行稳定性、定位和代谢的动力学分析。 Rb调节的mRNA转录物的翻译起始。使用明确的候选细胞死亡基因，我们 我建议确定与mRNA调控相关的机制，通过个体BMP 1或BMP 2 proteins.然后，我们将确定在RB 1-/-的发展中需要哪些PUM调节机制。 小细胞肺癌（SCLC）在体内。