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.

项目概要 Ccr4-Not 复合体是密码子最优性介导的信使 RNA 衰变的主要调节因子， 与翻译率密切相关的机制。共翻译 mRNA 衰变事件的破坏可以 具有重大的生理效应，导致单倍体不足或导致癌症。例如，CNOT3， Ccr4-Not 复合体的一个亚基，最近被鉴定为肿瘤抑制因子，在 7.9% 的肿瘤中发生突变 成人 T 细胞急性淋巴细胞白血病 (T-ALL)。我们实验室最近的工作发现 Not5（酵母 人类 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 转录本的优先衰减。最近发现这种相互作用在 人类，提出了细胞如何利用 mRNA 衰变机制来改变转录水平的机制。 尽管Not5与延长核糖体相互作用的结构已被解析，但目前还没有任何进展。 已知 Not5 如何与 Ccr4-Not 复合体成员相互作用以促进 mRNA 降解。因此， 弄清衰变机制如何机械地调节 mRNA 的降解将是至关重要的 了解细胞如何调节转录水平并为治疗干预开辟途径 广泛的单倍体不足疾病和癌症。为了解决这个知识差距，我将 从机械角度剖析 Ccr4-Not 复合体如何组装到主动翻译核糖体上并评估 该复合物的亚基如何协调去除 mRNA 的多聚腺苷酸尾和 5'-帽 芽殖酵母，酿酒酵母作为模型系统。我们实验室的初步数据表明，Not5 与核糖体 E 位点的结合对于招募 mRNA 衰减因子 Dhh1 进行翻译是必要的 核糖体，但这种招募途径的细节仍然不透明。我们还不知道 Not5 是否是 参与剩余 mRNA 衰减因子的招募以及这种招募如何协调 衰变事件。同样，已知损失 Dhh1 会表现出脱盖缺陷，最近我们发现 该因子的缺失也表现出 pol-A 尾部 mRNA 去除的缺陷，这意味着 Dhh1 募集可能有助于 桥接这些 3'-5' 衰变事件。我假设 Not5 招募剩余的 Ccr4-Not 亚基 积极翻译核糖体，导致 mRNA 转录本及时衰减。我会解决这个问题 通过以下具体目标进行假设： 目标 1) 我将系统地确定 Ccr4- 的组装 将 Not 复杂亚基转移到翻译 mRNP 结构上，并确定该装配体是否为 Not5- 依赖。目标 2) 我将表征 Dhh1 和 Ccr4-Not 复合体之间的接触，以确定是否 它们的相互作用介导 mRNA 衰变的 3'-5' 通讯。拟议的研究将增强我们的 深入了解 mRNA 半衰期如何调节，并为详细了解 这种作用在人类疾病中。