Nonsense-mediated mRNA decay: Pioneer round of translation

无义介导的 mRNA 衰变：首轮翻译

• 批准号: 7908048
• 负责人: Lynne E Maquat
• 金额: $ 19.27万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-18 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7908048
• 关键词: Binding; Cell Nucleus; Cells; Collaborations; Complex; Data; Deposition; Exons; Fluorescent in Situ Hybridization; Genetic Translation; Grant; Mammalian Cell; Mediating; Medicine; Messenger RNA; Nonsense Codon; Nucleotides; Phosphotransferases; Proteins; Quality Control; RNA; RNA Cap-Binding Proteins; RNA Splicing; Role; Site; Structure; Terminator Codon; Testing; Transcript; Translation Initiation; Translations; Universities; college; interest; mRNA Decay; protein complex; termination factor

DESCRIPTION (provided by applicant): We aim to continue our studies of the pioneer translation initiation complex and the pioneer round of translation. Nonsense-mediated mRNA decay (NMD) is an important quality control mechanism that eliminates transcripts having the potential to generate truncated proteins that could be deleterious to cells. We have found that NMD in mammalian cells generally occurs when translation terminates more than -50- 55 nts upstream of a splicing-generated exon-exon junction during a pioneer round of translation. By definition, this round of translation utilizes mRNA that is bound by the cap binding protein heterodimer CBP80/20. The role of the exon-exon junction in NMD reflects the post-splicing deposition of an exon junction complex (EJC) of proteins that include the Upf NMD factors. We have found that EJCs typify spliced CBP80/20-bound mRNA but not its remodeled product, elF4E-bound mRNA. To date, we have shown that the pioneer translation initiation complex is functionally distinct from but structurally overlaps with the steady- state, i.e., elF4E-bound, translation initiation complex. We have identified degradative activities involved in NMD. We also began to unravel why some mRNAs are subject to nucleus-associated NMD whereas other mRNAs are subject to cytoplasmic NMD. In Aim 1, we will study further the structure of the pioneer translation initiation complex before and during translation, and we will characterize the interacting domains of CBP80-Upf 1, CBP80-SMG1, CBP80-elF4GI, Upf 1 -eRF1 and Upf 1 -eRF3 that we have shown exist. In Aim 2, we will determine why some mRNAs are targeted for nucleus-associated NMD and other mRNAs are targeted for cytoplasmic NMD and, in collaboration with Rob Singer's lab, we will localize the cellular site of nucleus-associated NMD using fluorescent in situ hybridization of single RNA molecules. In Aim 3, we will solidify data obtained in collaboration with Ben Blencowe's lab indicating that there are functional differences among different EJCs or if any EJC that resides sufficiently downstream of a nonsense codon can trigger NMD. These differences will be exploited to determine if NMD is triggered by only the 3'-most EJC or if any EJC that resides more than 50-55 nucleotides downstream of a nonsense codon can trigger NMD. We expect that renewed support of this grant will allow us to continue to make major advances in understanding the mechanism of NMD in mammalian cells.

描述（由申请人提供）：我们旨在继续对先锋翻译启动综合体和先锋式翻译的研究。废话介导的mRNA衰减（NMD）是一种重要的质量控制机制，它消除了具有产生可能对细胞有害的截短蛋白质的潜力的转录本。我们发现，当翻译在剪接生成的外显子外exon连接期间在先锋圈的翻译回合中终止超过-50-55 NTS时，哺乳动物细胞中的NMD通常发生。根据定义，这一轮翻译利用了受盖结合蛋白异二聚体CBP80/20绑定的mRNA。外显子连接在NMD中的作用反映了包括UPF NMD因子的蛋白质外显子连接复合物（EJC）的分解沉积。我们发现EJCS典型地剪接了CBP80/20键mRNA，而不是其重塑产物ELF4E结合的mRNA。迄今为止，我们已经表明，先锋翻译起始复合物在功能上与稳态重叠，即ELF4E结合，翻译起始复合物在结构上与众不同。我们已经确定了NMD涉及的降解活动。我们还开始解开为什么某些mRNA受到核相关的NMD的约束，而其他mRNA则受到细胞质NMD的影响。在AIM 1中，我们将在翻译之前和期间进一步研究先锋翻译启动复合物的结构，我们将表征CBP80-UPF 1，CBP80-SMG1，CBP80-FELP4GI，UPF 1 -ERF1和UPF 1 -erf3的相互作用域。在AIM 2中，我们将确定为什么某些mRNA针对与核相关的NMD，而其他mRNA则针对细胞质NMD，并且与Rob Singer的实验室合作，我们将使用荧光原位杂交单个RNA分子来定位核相关NMD的细胞位点。在AIM 3中，我们将与Ben Blencowe的实验室合作获得获得的数据，表明不同的EJC之间存在功能差异，或者是否有任何驻留在无义密码子下游的EJC是否可以触发NMD。这些差异将被利用以确定NMD是否仅由3'最多的EJC触发，或者是否驻留于无义密码子下游的50-55个核苷酸的任何EJC是否可以触发NMD。我们预计，对该赠款的重新支持将使我们能够继续在理解哺乳动物细胞中NMD机制方面取得重大进步。