Novel approaches to identify regulators of Bak1 splicing

识别 Bak1 剪接调节因子的新方法

• 批准号: 10040977
• 负责人: Liang Chen
• 金额: $ 45.29万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10040977
• 关键词: Ablation; Alternative Splicing; Apoptosis; Apoptotic; Attenuated; BAK1 gene; Big Data; Biochemical; Brain; CRISPR screen; Cell Death; Cell Line; Cell Survival; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Competence; Computational Biology; Coupled; Cues; Data Set; Development; Disease; Exons; Fluorescence; Gene Expression; Genetic; Genetic Transcription; Goals; Homeostasis; Investigation; Knock-out; Learning; Life; Longevity; Mediating; Memory; Messenger RNA; Methods; MicroRNAs; Mitochondrial Proteins; Molecular; Molecular Genetics; Mus; Natural regeneration; Neurobiology; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Nuclear Pore Complex; Organism; Pathologic; Pathway interactions; Physiological; Positioning Attribute; Predisposition; Protein Isoforms; Proteins; Proteomics; Public Health; RNA; RNA Splicing; RNA-Binding Proteins; Recording of previous events; Regulation; Regulatory Element; Reporter; Research; Resistance; Surveys; Time; Tissues; Transcript; Translations; base; brain tissue; data mining; knock-down; mRNA Decay; mortality; nerve stem cell; neural circuit; neuron apoptosis; neuron loss; neuronal survival; novel; novel strategies; postnatal; pro-apoptotic protein; programs; protein expression; relating to nervous system; screening; stable cell line; tool; transcriptome; transcriptome sequencing; transcriptomics

The objective of this application is to unbiasedly identify regulators essential for programming intrinsic neuronal resistance to apoptosis. Apoptosis is a ubiquitous regulated cell death pathway controlling cell turnover and tissue homeostasis in metazoans. A longstanding issue is how neurons suppress apoptosis in favor of longevity. Previous studies on neuronal regulation of apoptosis have focused on why and how extrinsic survival cues help establish and maintain neural circuits through the control of cell death. While these inspiring studies delineate how neurons compete for survival at the time of circuit formation, important questions remain unresolved, i.e., whether neuronal apoptosis sensitivity is genetically determined. In our preliminary studies, we have found that neuronal resistance to apoptosis is intrinsically programmed before circuit formation by depletion of pro-apoptotic mitochondrial protein BAK1. We further found that BAK1 expression is controlled at the RNA level through robust alternative splicing mechanisms. The critical question is: what factors program Bak1 splicing? Identification of these factors will have significant impacts and enable new investigations of neuronal cell death controls in various settings. The proposed study will generate new tools and develop robust cell-based methods to systematically identify regulators of Bak1 splicing. We will integrate experimental and computational approaches to accelerate discoveries that would otherwise be limited and less sensitive. Our team has demonstrated successful collaborations researching cell death, neuronal survival, and RNA molecular genetics in the brain, and expect the proposed research to be fruitful. By revealing novel regulatory mechanisms of apoptosis and associated genetic factors, our findings may inform strategies for enhancing cell survival and tackling neurodegenerative diseases. Completion of the proposed study will also provide proof-of-principle for our broadly applicable strategy to study any alternative exons.

本申请的目的是无偏地鉴定对编程内源性神经元细胞所必需的调节剂。 抗凋亡。细胞凋亡是一种普遍存在的调节性细胞死亡途径，控制细胞更新， 后生动物的组织内稳态。一个长期存在的问题是神经元如何抑制凋亡， 中心blog以前关于神经元调节细胞凋亡的研究主要集中在为什么和如何外源性存活 线索通过控制细胞死亡来帮助建立和维持神经回路。虽然这些鼓舞人心的研究 描述神经元如何在回路形成时竞争生存，重要的问题仍然存在 未解决的，即，神经元凋亡敏感性是否由遗传决定。在初步研究中，我们 已经发现神经元对凋亡的抵抗在电路形成之前是内在编程的， 促凋亡线粒体蛋白BAK1的耗竭。我们进一步发现，BAK1的表达是受控制的， RNA水平通过强大的选择性剪接机制。关键的问题是：什么因素会导致 Bak1剪接？这些因素的识别将产生重大影响，并使新的调查， 神经元细胞死亡控制在各种设置。拟议的研究将产生新的工具， 以细胞为基础的方法，以系统地确定Bak1剪接的监管机构。我们将整合实验和 计算方法，以加速发现，否则将是有限的和不太敏感的。我们 研究小组已经证明了在研究细胞死亡、神经元存活和RNA方面的成功合作。 大脑中的分子遗传学，并期望拟议的研究是富有成效的。通过揭示新的监管机制， 细胞凋亡和相关遗传因素的机制，我们的研究结果可能为提高细胞凋亡的策略提供信息。 生存和治疗神经退行性疾病。完成拟议的研究还将为我们广泛适用的研究任何替代外显子的策略提供原理证明。