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: 10596993
• 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/10596993
• 关键词: Biochemistry; Buffers; Cardiovascular system; Cell Proliferation; Cells; Complex; DNA Damage; DNA Repair; Development; Disparate; 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; Oxidative Stress Induction; 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; 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.

摘要： 我的研究计划的目标是了解多功能蛋白质复合体如何调控基因。 表达，尤其是在应激反应期间。我们目前的重点是酵母菌CCR4-不是复合体，它有 几乎涉及基因控制的方方面面，包括转录、信使核糖核酸衰变、翻译 抑制和蛋白质泛素化。其复杂之处在于，它在所有真核生物中高度保守。我们的实验室 在了解它在转录中的作用，特别是RNA聚合酶II的延伸方面一直处于领先地位， 采用多方面的方法，包括遗传学、分子生物学、重组生物化学和 基因组学。CCR4-NOT的特点需要灵活的战略和朝着新方向前进的意愿。 这一提议将解决其控制下的两个不同的过程，即其泛素化活性的作用 应激状态下CCR4-NOT基因表达及靶基因重编程的机制 反应作为平衡信使核糖核酸合成和衰变的手段。 蛋白质在转录和DNA损伤反应中的破坏是一个必不可少的过程。注意事项4 该复合体的亚基含有一个E3环区，控制泛素依赖的蛋白质破坏。 我们已经证明，CCR4-不与伸长复合体结合，最近，它调节 DNA损伤后RNAPII的破坏。主题1将使用全球蛋白质组学识别Not4的新靶点， 确定修饰的位置，并使用分子遗传学和生物化学来确定后果 蛋白质功能上的泛素化。确定和描述Not4修改的后果 基因调控蛋白的研究不仅揭示了复合体的新功能，而且导致了更大的 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的保守现象，在这种现象中， MRNA的合成和降解是对刺激的反应，以维持类似水平的mRNAs的“平衡” 回应。CCR4-NOT参与了这一过程，因为它控制着合成和 核糖核酸的破坏。缓冲的分子基础是未知的，其背后的机制是 在激烈的辩论中。我们已经绘制了与CCR4相关的mRNAs--不是在静息和氧化状态下 应激条件下，CCR4从构成的mRNAs广泛地重新分布到 氧化应激，这可能是缓冲的关键组成部分。主题2将揭示是什么导致了 通过识别控制这一过程的顺式和反式因子，在应激过程中对腐烂机制重新编程 响应，探索两种细胞死烯基酶之间的相互作用，并确定蛋白质的变化 逆境中缓冲的mRNAs的组成和定位。这些研究将确定 应激反应中信使核糖核酸的决定因素及其背后的分子机制 基因表达缓冲。