Mechanism of cell lethality following loss of gene expression.

项目总结 这个项目的目标是确定转录抑制导致细胞死亡的机制。 该领域的共识模型假设，转录抑制后的细胞死亡是由于失去 特定的信使核糖核酸种类和随后的蛋白质损失。通过针对这样的核心细胞过程，转录 抑制被认为压倒了细胞控制，并导致不可避免的细胞死亡。这个死亡过程，定义为 作为意外细胞死亡(ACD)，不受细胞控制，也不是由定义的效应器的使用引起的 分子。与传统的模型相反，我们发现，不是诱导ACD，而是细胞死亡之后 转录抑制是由一种先前未被描述的受调控的凋亡信号引起的。此外，我们发现， 导致细胞死亡的是RNA POL II的降解，而不是信使核糖核酸产生的损失。我们的数据表明 新的模型，在此模型中，POL II的降解诱导出一个离开细胞核的信号，并由 线粒体启动细胞凋亡。识别调控转录后促凋亡信号的基因 抑制，我们进行了全基因组CRISPR筛查。全基因组CRISPR筛查往往无法识别 死亡调控基因，使阐明细胞死亡的机制变得困难。为了克服这一点，我们开发了 一种新的实验策略，使我们能够识别其敲除调节细胞死亡率的基因 在转录抑制之后。根据我们的筛选结果，在目标1中，我们将测试假设 选择性剪接调节因子PTBP1促进调节前mRNA的改变剪接和核输出， 这种活性是转录抑制后细胞死亡所必需的。我们将使用活细胞显微镜 确立PTBP1核出口的功能作用。我们将使用SLAM-SEQ和RIP-SEQ来量化PTBP1 转录抑制后的活性。我们的筛查还发现Bcl2L12是关键的细胞凋亡效应因子 转录抑制基因。在目标2中，我们将检验Bcl2L12激活以下细胞凋亡的假设 以异构体特异性的方式进行转录抑制。我们将进行一系列的功能遗传学研究 研究Bcl2L12在细胞凋亡反应中的作用。通过描述一种新的机械论 通过转录抑制诱导细胞死亡的模型，我们将提高我们对如何 在治疗上有效地使用转录抑制物。最终，我们希望我们的工作将提高我们的能力 预测哪些患者对转录抑制剂的反应最好，并帮助确定新的治疗策略。

PROJECT SUMMARY The goal of this project is to determine the mechanism by which cell death results from transcriptional inhibition. The consensus model in the field posits that cell death following transcriptional inhibition results from the loss of specific mRNA species and subsequent loss of protein. By targeting such a core cellular process, transcriptional inhibition is thought to overwhelm cellular control and lead to unavoidable cell death. This death process, defined as Accidental Cell Death (ACD), is not controlled by the cell and does not result from the use of defined effector molecules. Contrary to the conventional model, we found that, rather than induce ACD, cell death following transcriptional inhibition results from a previously undescribed regulated apoptotic signal. Furthermore, we found that RNA Pol II degradation, rather than loss of mRNA production, resulted in cell death. Our data suggests a new model, whereby degradation of Pol II induces a signal that leaves the nucleus and is received by the mitochondria to initiate apoptosis. To identify genes that regulate a pro-apoptotic signal following transcriptional inhibition, we performed a genome-wide CRISPR screen. Genome-wide CRISPR screens often fail to identify death regulatory genes, making it difficult to elucidate mechanisms of cell death. To overcome this, we developed a novel experimental strategy that allowed us to identify genes whose knockout modulated the cell death rate following transcriptional inhibition. Based on the results of our screen, in Aim 1 we will test the hypothesis that the alternative splicing regulator PTBP1 facilitates altered splicing and nuclear export of regulatory pre-mRNA, and that this activity is required for cell death following transcriptional inhibition. We will use live cell microscopy to establish the functional role of PTBP1 nuclear export. We will use SLAM-seq and RIP-seq to quantify PTBP1 activity following transcriptional inhibition. Our screen also identified BCL2L12 as the critical apoptotic effector gene for transcriptional inhibition. In Aim 2, we will test the hypothesis that BCL2L12 activates apoptosis following transcriptional inhibition in an isoform-specific manner. We will perform a series of functional genetics experiments to characterize the role of BCL2L12 in the apoptotic response. By describing a new mechanistic model by which transcriptional inhibition induces cell death, we will improve our understanding of how to effectively use transcriptional inhibitors therapeutically. Ultimately, we hope our work will improve our ability to predict which patients will best respond to transcriptional inhibitors and help identify novel treatment strategies.