Nonsense-mediated mRNA decay: Pioneer round of translation

无义介导的 mRNA 衰变：首轮翻译

• 批准号: 7894531
• 负责人: Lynne E Maquat
• 金额: $ 36.59万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7894531
• 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）：我们的目标是继续研究先锋翻译起始复合体和先锋轮翻译。无义介导的 mRNA 衰减 (NMD) 是一种重要的质量控制机制，它消除了可能产生对细胞有害的截短蛋白质的转录本。我们发现，在首轮翻译过程中，当翻译在剪接生成的外显子-外显子连接处上游超过-50-55 nts时终止时，通常会发生哺乳动物细胞中的NMD。根据定义，这一轮翻译利用由帽结合蛋白异二聚体 CBP80/20 结合的 mRNA。外显子-外显子连接在 NMD 中的作用反映了包含 Upf NMD 因子的蛋白质外显子连接复合物 (EJC) 的剪接后沉积。我们发现 EJC 代表剪接的 CBP80/20 结合 mRNA，但不是其重塑产物 eF4E 结合 mRNA。迄今为止，我们已经证明先锋翻译起始复合物在功能上不同于稳态（即 eF4E 结合的）翻译起始复合物，但在结构上与之重叠。我们已经确定了 NMD 涉及的降解活动。我们还开始阐明为什么一些 mRNA 会受到细胞核相关 NMD 的影响，而其他 mRNA 会受到细胞质 NMD 的影响。在目标 1 中，我们将进一步研究翻译前和翻译过程中先锋翻译起始复合体的结构，并且我们将表征 CBP80-Upf 1、CBP80-SMG1、CBP80-elF4GI、Upf 1 -eRF1 和 Upf 1 - 的相互作用域我们已经证明存在 eRF3。在目标 2 中，我们将确定为什么某些 mRNA 靶向细胞核相关 NMD，而其他 mRNA 靶向细胞质 NMD，并且与 Rob Singer 实验室合作，我们将使用荧光原位杂交定位细胞核相关 NMD 的细胞位点单个 RNA 分子。在目标 3 中，我们将巩固与 Ben Blencowe 实验室合作获得的数据，表明不同 EJC 之间存在功能差异，或者位于无义密码子下游的任何 EJC 是否可以触发 NMD。将利用这些差异来确定 NMD 是否仅由最 3'端的 EJC 触发，或者位于无义密码子下游 50-55 个核苷酸以上的任何 EJC 是否可以触发 NMD。我们期望这笔赠款的重新支持将使我们能够在理解哺乳动物细胞中的 NMD 机制方面继续取得重大进展。