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