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.

项目总结 CCR4-NOT复合体是密码子最优化介导的信使RNA衰变的主要调节因子，a 与翻译率密切相关的机制。共翻译信使核糖核酸衰变事件的中断可以 有重大的生理影响，导致单倍体功能不全或导致癌症。比如CNOT3， CCR4-NOT复合体的一个亚单位，最近被发现是一种肿瘤抑制因子，在7.9%的 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确定了Not5(酵母 人CNOT3的同源物)直接与缓慢翻译的核糖体的E位结合，导致 非最佳信使核糖核酸转录本的优先衰退。这种相互作用最近被发现在 这表明了细胞如何利用信使核糖核酸衰变机制来改变转录水平。 尽管Not5与细长的核糖体相互作用的结构已经解决，但目前还没有解决任何问题 了解Not5如何与CCR4成员相互作用--不是促进mRNA衰退的复合体。因此， 梳理衰变机制如何机械地调节信使核糖核酸的降解将是至关重要的 了解细胞如何调节转录水平以及打开治疗干预的途径 广泛的单倍体功能不全疾病和癌症。为了解决这一知识差距，我将 机械地剖析CCR4-非复合体如何组装到主动翻译的核糖体上并评估 这个复合体的亚基是如何协调去除mRNAs的PolyA尾和5‘-帽的 以芽殖酵母、酿酒酵母为模型系统。我们实验室的初步数据显示，Not5 与核糖体E位点的结合是mRNA衰变因子Dhh1募集到翻译所必需的 核糖体，但这一募集途径的细节仍然不透明。我们还不清楚Not5是否 参与招募剩余的信使核糖核酸衰变因子，以及这种招募可能如何协调 衰变事件。同样，已知损失Dhh1表现出剥离缺陷，最近我们发现 该因子的缺失也显示出PolA尾去除mRNAs的缺陷，这意味着Dhh1的招募可能有助于 弥合这些3‘-5’衰变事件。我假设Not5招募了其余的CCR4-Not亚单位来 积极翻译核糖体，导致mRNA转录本的及时衰退。我会解决这个问题 假设通过以下具体目标：目标1)我将系统地确定CCR4的组装- 将不复杂的亚基添加到翻译的mRNP结构上，并确定该组件是否为Not5- 依附的。目标2)我将描述Dhh1和CCR4之间的接触-不复杂，以确定 它们的相互作用介导了3‘-5’的信使核糖核酸衰变。拟议的研究将加强我们的 对mRNA半衰期是如何调节的机械性洞察，并为详细理解 这在人类疾病中扮演的角色。