Mechanism of cell lethality following loss of gene expression.

基因表达缺失后细胞致死的机制。

基本信息

批准号：
10751723
负责人：
Nicholas Wade Harper
金额：
$ 3.25万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10751723
关键词：
Accidents Alternative Splicing Apoptosis Apoptotic BCL2 gene Bcl-2 Homology Domain Binding CRISPR screen Cancerous Candidate Disease Gene Cell Death Cell Death Induction Cell Nucleus Cell physiology Cells Cessation of life Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complex Consensus Cytoplasm DNA Polymerase II Data Death Rate Drug Design Experimental Genetics Family member Gene Expression Genes Genetic Transcription Goals Immunoprecipitation Knock-out Knowledge Measures Messenger RNA Methods Mitochondria Modeling Normal Cell Nuclear Nuclear Export Nuclear Protein Patient Selection Patients Pharmaceutical Preparations Phenotype Process Production Protein Isoforms Proteins RNA RNA Splicing RNA immunoprecipitation sequencing RNA-Binding Proteins Regulation Regulator Genes Resistance Role Series Signal Transduction Testing Time Toxic effect Transcript Work cancer cell cell growth cell killing cell type design drug mechanism experimental study genetic regulatory protein genome-wide improved inhibitor inhibitor therapy insight interest live cell imaging live cell microscopy mRNA Expression mRNA Precursor novel novel drug combination pre-clinical response treatment strategy

项目摘要

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.

项目概要该项目的目标是确定转录抑制导致细胞死亡的机制。该领域的共识模型认为，转录抑制后的细胞死亡是由于特定的 mRNA 种类和随后的蛋白质损失。通过靶向这样一个核心细胞过程，转录抑制被认为会压倒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，也不是由使用特定效应器引起的分子。与传统模型相反，我们发现，细胞死亡不会诱导 ACD，而是会导致细胞死亡。转录抑制是由先前未描述的受调节的细胞凋亡信号引起的。此外，我们发现导致细胞死亡的是 RNA Pol II 降解，而不是 mRNA 产生的损失。我们的数据表明新模型，Pol II 的降解诱导信号离开细胞核并被细胞接收线粒体启动细胞凋亡。鉴定转录后调节促凋亡信号的基因抑制，我们进行了全基因组 CRISPR 筛选。全基因组 CRISPR 筛选常常无法识别死亡调节基因，使得阐明细胞死亡机制变得困难。为了克服这个问题，我们开发了一种新颖的实验策略，使我们能够识别出敲除可调节细胞死亡率的基因转录抑制后。根据我们的筛选结果，在目标 1 中，我们将检验以下假设：选择性剪接调节因子 PTBP1 促进调节前 mRNA 的剪接改变和核输出，并且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定 PTBP1 核输出的功能作用。我们将使用 SLAM-seq 和 RIP-seq 来量化 PTBP1 转录抑制后的活性。我们的筛选还确定 BCL2L12 是关键的凋亡效应子转录抑制基因。在目标 2 中，我们将检验 BCL2L12 在以下情况下激活细胞凋亡的假设：以异构体特异性方式进行转录抑制。我们将进行一系列的功能遗传学表征 BCL2L12 在细胞凋亡反应中的作用的实验。通过描述一个新的机制通过转录抑制诱导细胞死亡的模型，我们将加深对如何有效地使用转录抑制剂进行治疗。最终，我们希望我们的工作能够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。