Investigating the role of the Ccr4-Not complex in regulating codon optimality-mediated mRNA decay

研究 Ccr4-Not 复合体在调节密码子最优性介导的 mRNA 衰减中的作用

• 批准号: 10749985
• 负责人: Lana Nicole Christensen
• 金额: $ 4.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 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/10749985
• 关键词: Acute T Cell Leukemia; Address; Adult Precursor T Lymphoblastic Leukemia; Affect; Binding; Biological Models; Bone Marrow; Cells; Codon Nucleotides; Communication; Complex; Couples; Cryoelectron Microscopy; Data; Defect; Disease; Eukaryota; Event; Excision; Exhibits; Genetic Transcription; Health; Homologous Gene; Human; Knowledge; Link; Malignant Neoplasms; Mediating; Messenger RNA; Molecular; Monitor; Mutate; N-terminal; Northern Blotting; Open Reading Frames; Pathway interactions; Phenotype; Physiological; Poly(A) Tail; Post-Transcriptional Regulation; Process; Production; Proteins; RNA Decay; Radiolabeled; Reporter; Ribosomal Interaction; Ribosomes; Role; Saccharomyces cerevisiae; Saccharomycetales; Scaffolding Protein; Series; Site; Structure; Tail; Testing; Therapeutic Intervention; Time; Transcript; Translating; Translations; Tumor Suppressor Proteins; Western Blotting; Work; Yeasts; dosage; human disease; insight; mRNA Decay; mRNA Stability; mRNA Transcript Degradation; mRNA capping; mRNA decapping; member; messenger ribonucleoprotein; mutant; nanopore; polysome profiling; recruit

PROJECT SUMMARY The Ccr4-Not complex is the major regulator of codon optimality-mediated messenger RNA decay, a mechanism that is intimately tied to translation rate. The disruption of co-translational mRNA decay events can have major physiological effects, leading to haploinsufficiency or contributing to cancer. For instance, CNOT3, a subunit of the Ccr4-Not complex, was recently identified as a tumor suppressor that is mutated in 7.9% of adult T-cell acute lymphoblastic leukemias (T-ALLs). Recent work in our lab identified that Not5 (yeast homolog of human CNOT3) directly binds to the E-site of slowly translating ribosomes, leading to the preferential decay of non-optimal mRNA transcripts. This interaction was recently found to be conserved in humans, suggesting a mechanism for how cells use the mRNA decay machinery to alter transcript levels. Though the structure of Not5 interacting with elongating ribosomes has been resolved, nothing is currently known about how Not5 interacts with members of the Ccr4-Not complex to facilitate mRNA decay. Thus, teasing apart how the decay machinery mechanistically regulates the degradation of mRNA will be critical in understanding how cells regulate transcript levels as well as open avenues of therapeutic intervention for a broad range of haploinsufficiency diseases and cancer. To address this gap in knowledge, I will mechanistically dissect how the Ccr4-Not complex assembles onto actively translating ribosomes and assess how the subunits of this complex coordinate the removal of the poly-A tail and 5’-cap of mRNAs using the budding yeast, Saccharomyces cerevisiae as a model system. Preliminary data from our lab shows that Not5 binding to the ribosomal E-site is necessary for the recruitment of the mRNA decay factor, Dhh1, to translating ribosomes, but the details of this recruitment pathway remain opaque. We do not yet understand if Not5 is involved in the recruitment of the remaining mRNA decay factors and how this recruitment might coordinate decay events. Likewise, the loss Dhh1 is known to exhibit decapping defects and recently we found that the loss of this factor also exhibits defects in pol-A tail removal of mRNAs, meaning Dhh1 recruitment may serve to bridge these 3’-5’ decay events. I hypothesize that Not5 recruits the remaining Ccr4-Not subunits to actively translating ribosomes resulting in the timely decay of mRNA transcripts. I will address this hypothesis through the following specific aims: Aim 1) I will systematically determine the assembly of the Ccr4- Not complex subunits onto the translating mRNP structure and determine if this assembly is Not5- dependent. Aim 2) I will characterize the contacts between Dhh1 and the Ccr4-Not complex to determine if their interaction mediates the 3’-5’ communication of mRNA decay. The proposed studies will enhance our mechanistic insight into how mRNA half-lives are regulated and provide the basis for detailed understanding of this role in human disease.

项目摘要 CCR4-不加复合物是密码子最佳介导的Messenger RNA Decay的主要调节剂，A 与翻译速率密切相关的机制。共同翻译mRNA衰变事件的破坏可以 具有重大的物理作用，导致单倍薄荷或导致癌症。例如，cnot3， CCR4不复合物的亚基最近被确定为肿瘤抑制因子，在7.9％中突变 成人T细胞急性淋巴细胞白血病（T-Alls）。我们实验室的最新工作确定没有5（酵母 人CNOT3的同源物直接与缓慢翻译核糖体的E位点结合，导致 非最佳mRNA转录本的优先衰变。最近发现这种相互作用是在 人类，提出了一种机制，即细胞如何使用mRNA衰变机械改变转录水平。 尽管Not5与细长核糖体相互作用的结构已经解决，但目前没有什么 知道Not5如何与CCR4复合物的成员相互作用以促进mRNA衰减。那， 挑逗衰减机械如何机械调节mRNA的降解至关重要 了解细胞如何调节转录水平以及开放的治疗干预途径 广泛的单倍疾病疾病和癌症。为了解决这一知识的差距，我将 机械地剖析CCR4-不复合物如何聚集到主动翻译核糖体和评估 该复合物的亚基如何使用该尾巴的去除和5'-cap mRNA使用 萌芽的酵母，酿酒酵母作为模型系统。我们实验室的初步数据表明非5 与核糖体电子点结合对于募集mRNA衰变因子DHH1是必需的 核糖体，但这种招聘途径的细节仍然不透明。我们尚不明白这是否不是 参与募集其余mRNA衰减因素以及该招募如何协调 衰减事件。同样，众所周知，DHH1的损失是宣布缺陷，最近我们发现 该因素的丢失还表现出pol-a尾部去除mRNA的缺陷，这意味着DHH1招募可能有助于 桥接这3'-5'衰减事件。我假设Not 5招募了其余的CCR4，而不是 积极地翻译核糖体，导致mRNA转录的及时衰变。我将解决这个问题 通过以下特定目的假设：目标1）我将系统地确定CCR4-的组装 在翻译MRNP结构上不复杂的亚基，并确定该组件是否不是5- 依赖。目标2）我将表征DHH1和CCR4之间的联系，以确定是否是否 他们的相互作用介导了mRNA衰变的3'-5'通信。拟议的研究将增强我们的 关于如何调节mRNA半衰期的机械洞察力，为详细理解的基础提供了基础 这种在人类疾病中的作用。