The Translational Regulation of Pro-apoptotic Genes

促凋亡基因的翻译调控 项目总结 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抑制了三个E2F转录因子E2F1-3的活性，这三个转录因子控制着 肿瘤细胞生长所需的许多关键的RB-E2F靶基因，包括细胞周期和代谢 各种因素。除了促进肿瘤的过程外，E2F1-3还调节肿瘤抑制基因的转录 在细胞凋亡和坏死中起中心作用的基因。该调节电路连接Rb-E2F的表达 增殖和细胞死亡基因，最终防止细胞不受控制的生长。公共广播公司的广泛损失 癌症的研究突显了该领域的一个重大难题：为什么缺乏pRb的细胞不会干脆死亡？ 为了了解RB-E2F靶基因的调节，我们分析了PRB-E2F后RNA和蛋白质的变化。 耗尽，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(RBPs) 鉴定Pumilio复合体是Rb-E2F促凋亡mRNAs的直接翻译调节因子。此外， 该蛋白复合体的两个核心成分PUM1和PUM2的共同缺失导致了细胞死亡 Rb1-/-癌细胞通过翻译促凋亡的mRNAs。人类浮标复合体由 PUM和Nanos(NOS)蛋白，但PUM成员之间存在显著的功能冗余 而NOS蛋白家族对Pumilio复合体的细胞和肿瘤研究有限。为了绕过这个问题， 我们已经将人类细胞改造成只表达一种我们可以专门降解的PUM或NOS蛋白 利用一种新的生长素诱导降解系统。这一技术进步使我们能够进行动力学 分析在没有PUM或NOS的情况下RNA的稳定性、定位和翻译启动，并探讨 不同浮石复合体在细胞调控和肿瘤发生中的作用(S)。在这一新的申请中，我们建议 目的：1)确定Pumilio复合体翻译抑制Rb-E2F靶标促凋亡的机制(S) 基因，2)确定RB1突变小细胞肺癌(SCLC)中Pumilio复合体的调控机制 细胞，以及3)确定Pumilio复合体在Rb1-/-；Trp53-/-小鼠SCLC肿瘤发生模型中的作用。 这些研究将确定阻止细胞死亡成分产生的机制(S)。 侵袭性和转移性小细胞肺癌。

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.