Activities of yeast Ccr4-Not transcription factor complex

酵母Ccr4-Not转录因子复合物的活性

• 批准号: 10364658
• 负责人: JOSEPH C REESE
• 金额: $ 38.73万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10364658
• 关键词: Biochemistry; Buffers; Cardiovascular system; Cell Proliferation; Cells; Complex; DNA Damage; DNA Repair; Development; Equilibrium; Eukaryota; Gene Expression; Gene-Modified; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Health; Human; Laboratories; Lead; Maps; Messenger RNA; Modification; Molecular; Molecular Biology; Molecular Genetics; Oxidative Stress; Process; Production; Proteins; Proteomics; RNA Polymerase II; Research; Resistance; Rest; Role; Site; Stimulus; Stress; Syndrome; Translational Repression; Ubiquitin; Work; Yeasts; biological adaptation to stress; flexibility; genetic regulatory protein; human disease; mRNA Decay; novel; programs; protein complex; protein function; reconstitution; response; virtual; willingness

Abstract: The goal of my research program is to understand how multifunctional protein complexes regulate gene expression, especially during stress responses. Our current focus is the yeast Ccr4-Not complex, which has been implicated in virtually all aspects of gene control, including transcription, mRNA decay, translational repression and protein ubiquitylation. The complex is it is highly conserved in all eukaryotes. Our laboratory has been a leader in understanding its role in transcription, especially RNA polymerase II elongation, employing multifaceted approaches including genetics, molecular biology, reconstitution biochemistry and genomics. Characterizing Ccr4-Not requires a flexible strategy and a willingness to move in new directions. This proposal will tackle two disparate processes under its control, namely the role of its ubiquitylation activity in gene expression and the mechanism of the reprogramming of Ccr4-Not mRNA targets during stress responses as a means to balance mRNA synthesis and decay. Protein destruction during transcription and DNA damage responses is an essential process. The Not4 subunit of the complex contains an E3 RING domain and controls ubiquitin-dependent destruction of proteins. We have shown that Ccr4-Not associates with elongation complexes and, recently, that it regulates the destruction of RNAPII after DNA damage. Theme 1 will identify novel targets of Not4 using global proteomics, identify the sites of modification and use molecular genetics and biochemistry to determine the consequences of ubquitylation on the protein’s function. Identifying and characterizing the consequences of Not4 modification of gene regulatory proteins will not only reveal novel functions of the complex, but lead to a greater understanding of the importance of protein ubiquitylation in transcription and DNA repair. Gene expression buffering is a novel conserved phenomenon where reciprocal changes in the rates in mRNA synthesis and degradation occur in response to stimuli to maintain similar levels of mRNAs to “balance” the response. Ccr4-Not has been implicated in this process because it controls both the synthesis and destruction of mRNAs. The molecular underpinnings of buffering are unknown and the mechanism behind it is under intense debate. We have mapped the mRNAs associated with Ccr4-Not under resting and oxidative stress conditions, which revealed extensive redistribution of Ccr4 from constitutive mRNAs to those induced by oxidative stress, which is likely a key component of buffering. Theme 2 will reveal what accounts for the reprogramming of the decay machinery during stress by identifying the cis- and trans- factors that control this response, explore the interplay between the two cellular deadenylases and identify changes in protein composition and localization of mRNAs undergoing buffering during stress. These studies will identify the determinants of mRNA targeting during stress responses and undercover the molecular mechanism behind gene expression buffering.

抽象的： 我的研究计划的目的是了解多功能蛋白复合物如何调节基因 表达，尤其是在压力反应期间。我们目前的重点是酵母CCR4-没有复合物，它具有 在基因控制的几乎所有方面都暗示，包括转录，mRNA衰变，翻译 抑制和蛋白质泛素化。该复合物是在所有真核生物中都高度保守。我们的实验室 一直是理解其在转录中的作用的领导者，尤其是RNA聚合酶II延伸， 采用多方面的方法，包括遗传学，分子生物学，重建生物化学和 基因组学。表征CCR4-不需要灵活的策略，并愿意朝新的方向前进。 该建议将解决其控制下的两个不同过程，即其泛素化活动的作用 在应激期间CCR4-not mRNA靶标重编程的基因表达和机制中 反应是平衡mRNA合成和衰减的手段。 转录和DNA损伤反应过程中的蛋白质破坏是一个必不可少的过程。 NOT4 该复合物的亚基包含一个E3环域，并控制蛋白质的泛素依赖性破坏。 我们已经表明，CCR4与伸长复合物的关联，最近它调节了 DNA损伤后RNAPII的破坏。主题1将使用全局蛋白质组学确定NOT4的新颖目标， 确定修饰的位点，并使用分子遗传学和生物化学来确定后果 蛋白质功能上的Ubquitylation。识别和表征NOT4修改的后果 基因调节蛋白不仅会揭示复合物的新功能，而且会导致更大的功能 了解蛋白质泛素化在转录和DNA修复中的重要性。 基因表达缓冲是一种新颖的现象，其中相互的速率变化 mRNA合成和降解是为了刺激保持相似水平的mRNA以“平衡”而发生的。 反应。 CCR4不参与此过程，因为它同时控制了合成和 破坏mRNA。缓冲的分子基础是未知的，其背后的机制是 在激烈的辩论下。我们绘制了与CCR4相关的mRNA，而不是在静止和氧化下 压力条件表明，CCR4从本构mRNA进行了广泛的重新分布， 氧化应激，这可能是缓冲的关键组成部分。主题2将揭示哪些内容 通过识别控制这一点的顺式和转移因素，在压力期间重新编程了衰减机械 响应，探索两个细胞降苯基酶之间的相互作用并确定蛋白质的变化 在压力期间进行缓冲的mRNA的组成和定位。这些研究将确定 压力反应过程中mRNA靶向的决定因素和秘密分子机制的决定因素 基因表达缓冲。