Recognition and degradation of mRNA by nonsense-mediated decay

无义介导的衰变对 mRNA 的识别和降解

• 批准号: 8334474
• 负责人: Kristian Eileen Baker
• 金额: $ 29.83万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8334474
• 关键词: Accounting; Address; Belief; Binding; Binding Proteins; Biological Assay; Cell model; Cells; Core Protein; Data; Discrimination; Disputes; Elements; Ensure; Eukaryotic Cell; Gene Expression; Gene Mutation; Genetic; Goals; Grant; Health; Hereditary Disease; Human; Human Genetics; Individual; Lead; Left; Light; Mediating; Messenger RNA; Metabolism; Modeling; Molecular; Mutation; Nonsense Codon; Nonsense-Mediated Decay; Pathway interactions; Patients; Peptide Initiation Factors; Play; Poly(A) Tail; Process; Protein Binding; Proteins; Quality Control; RNA; RNA Binding; RNA-Binding Proteins; Recruitment Activity; Relative (related person); Ribosomes; Role; Saccharomyces cerevisiae; Signal Transduction; Terminator Codon; Testing; Trans-Activators; Translating; Translations; Untranslated Regions; Yeasts; base; innovation; insight; mRNA Decay; messenger ribonucleoprotein; novel; novel strategies; novel therapeutic intervention; polypeptide; premature; prevent; protein protein interaction; response

DESCRIPTION (provided by applicant): Degradation of mRNA plays an important role in regulating gene expression. In eukaryotic cells a specialized pathway has evolved to stimulate the degradation of aberrant mRNAs containing a premature nonsense codon (PTC), a signal that causes early termination of translation and, if left unchecked, the accumulation of truncated polypeptides. This pathway, referred to as nonsense- mediated mRNA decay (NMD), represents a conserved quality control mechanism that upholds fidelity and protects cells from erroneous gene expression. The widely accepted model for NMD posits that PTC-containing mRNA are recognized as aberrant due an impaired interaction between the terminating ribosome and the mRNA poly(A) tail-bound protein, PAB1, as a consequence of translation termination occurring too far away. Our observations challenge this paradigm and indicate that a model invoking a distance between the terminating ribosome and PAB1 as the critical determinant in NMD substrate recognition cannot sufficiently explain how cells discriminate between normal and premature translation termination. The long term goal of this proposal is to elucidate the molecular mechanisms underlying both recognition and rapid degradation of mRNA by the NMD pathway. We will address the important questions of how nonsense-containing mRNA are both recognized and targeted for rapid degradation under three specific aims. First, we will identify and characterize mRNP elements important for targeting PTC-containing mRNA to NMD. In addition, we propose two innovative and unbiased approaches to identify cis- and trans-acting antagonists of NMD. Second, we will investigate the requirements for assembly of NMD proteins with mRNA substrates. In particular, we will examine the order for protein assembly, whether binding targets are ribosomes, mRNA, or both, and particular mRNA features for their importance in NMD mRNP assembly. Finally we will also assess the functional capabilities of individual NMD proteins independent of their RNA binding capacities. Third, we will elucidate how recognition of a PTC- containing mRNA as aberrant is communicated to the cellular decay machinery and leads to accelerated degradation of the mRNA. Our preliminary data dispute a common belief that the NMD machinery directly recruits the decay machinery to the mRNA through conserved protein- protein interactions. We plan to further challenge this model as well as test our hypothesis that rapid mRNA decay is an indirect consequence of restricting translation initiation factor binding to the mRNA 52 cap.

描述（由申请人提供）：mRNA 的降解在调节基因表达中起着重要作用。在真核细胞中，已经进化出一条专门的途径来刺激含有过早无义密码子 (PTC) 的异常 mRNA 的降解，PTC 是一种导致翻译提前终止的信号，如果不加以控制，则会导致截短多肽的积累。该途径被称为无义介导的 mRNA 衰减 (NMD)，代表了一种保守的质量控制机制，可维持保真度并保护细胞免受错误基因表达的影响。广泛接受的 NMD 模型认为，含有 PTC 的 mRNA 被认为是异常的，因为终止核糖体和 mRNA 聚 (A) 尾结合蛋白 PAB1 之间的相互作用受损，这是翻译终止发生在距离太远的结果。我们的观察结果挑战了这一范式，并表明将终止核糖体和 PAB1 之间的距离作为 NMD 底物识别的关键决定因素的模型无法充分解释细胞如何区分正常翻译终止和过早翻译终止。该提案的长期目标是阐明 NMD 途径识别和快速降解 mRNA 的分子机制。我们将解决以下重要问题：如何在三个特定目标下识别包含无义的 mRNA 并使其快速降解。首先，我们将鉴定和表征对于将含有 PTC 的 mRNA 靶向 NMD 很重要的 mRNP 元件。此外，我们提出了两种创新且公正的方法来识别 NMD 的顺式和反式拮抗剂。其次，我们将研究 NMD 蛋白与 mRNA 底物组装的要求。特别是，我们将检查蛋白质组装的顺序，结合靶标是核糖体、mRNA 还是两者，以及特定 mRNA 特征在 NMD mRNP 组装中的重要性。最后，我们还将评估单个 NMD 蛋白的功能能力，与其 RNA 结合能力无关。第三，我们将阐明如何将含有 PTC 的 mRNA 识别为异常，并传递给细胞衰变机制并导致 mRNA 加速降解。我们的初步数据反驳了一种普遍观点，即 NMD 机制通过保守的蛋白质-蛋白质相互作用直接将衰变机制招募到 mRNA 上。我们计划进一步挑战这个模型，并测试我们的假设，即 mRNA 快速衰减是限制翻译起始因子与 mRNA 52 帽结合的间接结果。