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

项目摘要 Ccr 4-Not复合物是密码子优化介导的信使RNA衰变的主要调节因子， 与翻译速度密切相关的机制。共翻译mRNA衰变事件的破坏可以 具有主要的生理作用，导致单倍不足或促成癌症。例如，hocT 3， Ccr 4-Not复合物的一个亚基，最近被鉴定为肿瘤抑制因子，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Not 5（酵母） 人hocT 3的同源物）直接结合缓慢翻译核糖体的E位点，导致 非最佳mRNA转录物的优先衰减。这种相互作用最近被发现是保守的 这表明了细胞如何利用mRNA衰变机制来改变转录水平的机制。 虽然Not 5与延长核糖体相互作用的结构已经被解决，但目前还没有任何研究。 Not 5如何与Ccr 4-Not复合物的成员相互作用以促进mRNA衰变。因此，在本发明中， 解开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，以及为治疗性干预开辟途径， 广泛的单倍性不足疾病和癌症。为了弥补这一知识差距，我将 机械地剖析Ccr 4-Not复合物如何组装到活跃翻译的核糖体上，并评估 该复合物的亚基如何协调使用多聚腺苷酸尾和mRNA的5 '-帽的去除， 芽殖酵母、酿酒酵母作为模型系统。我们实验室的初步数据显示， 与核糖体E位点的结合对于mRNA衰变因子Dhh 1的募集是必需的， 核糖体，但这一招聘途径的细节仍然不透明。我们还不知道Not 5是否 参与剩余mRNA衰变因子的募集以及这种募集如何协调 衰变事件同样，已知损耗Dhh 1表现出去盖缺陷，并且最近我们发现， 该因子的缺失也表现出mRNA的pol-A尾去除缺陷，这意味着Dhh 1募集可能有助于 桥接这些3 '-5'衰变事件。我假设Not 5招募剩余的Ccr 4-Not亚基， 主动翻译核糖体，导致mRNA转录物的及时衰变。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Ccr 4- 不复杂的亚基转移到翻译mRNP结构上，并确定该组装体是否为Not 5- 依赖。目的2）我将表征Dhh 1和Ccr 4之间的接触-不复杂，以确定是否 它们的相互作用介导mRNA衰变的3 ′-5 ′通讯。建议的研究将加强我们的 对mRNA半衰期如何调节的机制性见解，并为详细了解 在人类疾病中的作用。

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.